Machine learning to attribute the source of Campylobacter infections in the United States: A retrospective analysis of national surveillance data.

OBJECTIVES: Integrating pathogen genomic surveillance with bioinformatics can enhance public health responses by identifying risk and guiding interventions. This study focusses on the two predominant Campylobacter species, which are commonly found in the gut of birds and mammals and often infect humans via contaminated food. Rising incidence and antimicrobial resistance (AMR) are a global concern, and there is an urgent need to quantify the main routes to human infection. METHODS: During routine US national surveillance (2009-2019), 8856 Campylobacter genomes from human infections and 16,703 from possible sources were sequenced. Using machine learning and probabilistic models, we target genetic variation associated with host adaptation to attribute the source of human infections and estimate the importance of different disease reservoirs. RESULTS: Poultry was identified as the primary source of human infections, responsible for an estimated 68% of cases, followed by cattle (28%), and only a small contribution from wild birds (3%) and pork sources (1%). There was also evidence of an increase in multidrug resistance, particularly among isolates attributed to chickens. CONCLUSIONS: National surveillance and source attribution can guide policy, and our study suggests that interventions targeting poultry will yield the greatest reductions in campylobacteriosis and spread of AMR in the US. DATA AVAILABILITY: All sequence reads were uploaded and shared on NCBI's Sequence Read Archive (SRA) associated with BioProjects; PRJNA239251 (CDC / PulseNet surveillance), PRJNA287430 (FSIS surveillance), PRJNA292668 & PRJNA292664 (NARMS) and PRJNA258022 (FDA surveillance). Publicly available genomes, including reference genomes and isolates sampled worldwide from wild birds are associated with BioProject accessions: PRJNA176480, PRJNA177352, PRJNA342755, PRJNA345429, PRJNA312235, PRJNA415188, PRJNA524300, PRJNA528879, PRJNA529798, PRJNA575343, PRJNA524315 and PRJNA689604. Contiguous assemblies of all genome sequences compared are available at Mendeley data (assembled C. coli genomes doi: 10.17632/gxswjvxyh3.1; assembled C. jejuni genomes doi: 10.17632/6ngsz3dtbd.1) and individual project and accession numbers can be found in Supplementary tables S1 and S2, which also includes pubMLST identifiers for assembled genomes. Figshare (10.6084/m9.figshare.20279928). Interactive phylogenies are hosted on microreact separately for C. jejuni (https://microreact.org/project/pascoe-us-cjejuni) and C. coli (https://microreact.org/project/pascoe-us-ccoli).

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

Whole genome characterization of thermophilic Campylobacter species isolated from dairy manure in small specialty crop farms of Northeast Ohio.

Introduction: With more public interest in consuming locally grown produce, small specialty crop farms (SSCF) are a viable and growing segment of the food production chain in the United States. Methods: The goal of this study was to investigate the genomic diversity of Campylobacter isolated from dairy manure (n = 69) collected from 10 SSCF in Northeast Ohio between 2018 and 2020. Results: A total of 56 C. jejuni and 13 C. coli isolates were sequenced. Multi-locus sequence typing (MLST) identified 22 sequence types (STs), with ST-922 (18%) and ST-61 (13%) predominant in C. jejuni and ST-829 (62%) and ST-1068 (38%) predominant in C. coli. Interestingly, isolates with similar genomic and gene contents were detected within and between SSCF over time, suggesting that Campylobacter could be transmitted between farms and may persist in a given SSCF over time. Virulence-associated genes (n = 35) involved in the uptake and utilization of potassium and organic compounds (succinate, gluconate, oxoglutarate, and malate) were detected only in the C. jejuni isolates, while 45 genes associated with increased resistance to environmental stresses (capsule production, cell envelope integrity, and iron uptake) were detected only in the C. coli isolates. Campylobacter coli isolates were also sub-divided into two distinct clusters based on the presence of unique prophages (n = 21) or IncQ conjugative plasmid/type-IV secretion system genes (n = 15). Campylobacter coli isolates harbored genes associated with resistance to streptomycin (aadE-Cc; 54%) and quinolone (gyrA-T86I; 77%), while C. jejuni had resistance genes for kanamycin (aph3'-IIIa; 20%). Both species harbored resistance genes associated with ß-lactam (especially, blaOXA-193; up to 100%) and tetracycline (tetO; up to 59%). Discussion/Conclusion: Our study demonstrated that Campylobacter genome plasticity associated with conjugative transfer might provide resistance to certain antimicrobials and viral infections via the acquisition of protein-encoding genes involved in mechanisms such as ribosomal protection and capsule modification.

Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce.

Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.

Current methodologies and future direction of Campylobacter isolation and detection from food matrices, clinical samples, and the agricultural environment.

Campylobacter spp. are the leading cause of bacterial foodborne infections in both developed and developing countries. The food commodities primarily attributed to campylobacteriosis include raw milk, poultry, seafood, and fresh produce. Furthermore, insects, animal/bird fecal material, and agricultural water have been shown to be the sources of Campylobacter contamination in these commodities. Both established and emerging species of Campylobacter have been recovered from food and environmental sources. Therefore, optimal detection and isolation of Campylobacter spp., including the emerging species, is critical for improved surveillance, prevention, and traceback of Campylobacter outbreaks. This review focuses on the existing variability in Campylobacter enrichment and isolation procedures used by researchers and regulatory agencies worldwide, for various matrices. Additionally, the challenges associated with developing and validating new culture, molecular, and immunological methods for rapid and sensitive Campylobacter detection are discussed.

Draft Genome Sequences of Two Campylobacter jejuni Strains That Show Significantly Different Colonization Potentials in Chickens.

Here, we report the draft genome sequences of robust (A74/C_24-3) and poor (A74/O_2-2) chicken-colonizing Campylobacter jejuni isolates. Whole-genome sequence analyses of these isolates will be helpful in facilitating further studies to identify genetic factors used in chicken colonization.

A loop-mediated isothermal amplification (LAMP) assay for the consensus detection of human pathogenic Campylobacter species.

Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.

A Microbiomic Analysis of a Pasture-Raised Broiler Flock Elucidates Foodborne Pathogen Ecology Along the Farm-To-Fork Continuum.

While conventionally grown poultry continues to dominate the U. S. poultry industry, there is an increasing demand for locally-grown, "all natural" alternatives. The use of next generation sequencing allows for not only the gross (e.g., community structure) but also fine-scale (e.g., taxa abundances) examination of these complex microbial communities. This data provides a better understanding of how a pasture flock's microbiome changes throughout the production life cycle and how that change in microbial ecology changes foodborne pathogens in alternative poultry production systems. In order to understand this ecology better, pooled broiler samples were taken during the entire flock life cycle, from pre-hatch gastrointestinal samples (N = 12) to fecal samples from the brood (N = 5), and pasture (N = 10) periods. Additional samples were taken during processing, including skin and feather rinsates (N = 12), ceca (N = 12), and whole carcass rinses (N = 12), and finally whole carcasss rinsates of final products (N = 3). Genomic DNA was extracted, 16S rDNA microbiome sequencing was conducted (Illumina MiSeq), and microbiomes were analyzed and compared using QIIME 1.9.1 to determine how microbiomes shifted throughout production continuum, as well as what environmental factors may be influencing these shifts. Significant microbiome shifts occurred during the life cycle of the pasture broiler flock, with the brood and pasture fecal samples and cecal samples being very distinct from the other pre-hatch, processing, and final product samples. Throughout these varied microbiomes, there was a stable core microbiome containing 13 taxa. Within this core microbiome, five taxa represented known foodborne pathogens (Salmonella, Campylobacter) or potential/emerging pathogens (Pseudomonas, Enterococcus, Acinetobacter) whose relative abundances varied throughout the farm-to-fork continuum, although all were more prevalent in the fecal samples. Additionally, of the 25 physiochemical and nutrient variables measured from the fecal samples, the carbon to nitrogen ratio was one of the most significant variables to warrant further investigations because it impacted both general fecal microbial ecology and Campylobacter and Enterococcus taxa within the core fecal microbiomes. These findings demonstrate the need for further longitudinal, farm-to-fork studies to understand the ecology of the microbial ecology of pasture production flocks to improve animal, environmental, and public health.

Semi-Quantification of Total Campylobacter and Salmonella During Egg Incubations Using a Combination of 16S rDNA and Specific Pathogen Primers for qPCR.

Rapid molecular techniques that evaluate eggs for the presence of foodborne pathogens is an essential component to poultry food safety monitoring. Interestingly, it is not just table eggs that contribute to outbreaks of foodborne disease. Broiler layer production actively contributes to sustaining of foodborne pathogens within a flock. The surface contamination of production eggs with invasive pathogens such as Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes during embryogenesis results in gastrointestinal tract (GIT) colonization. Pathogens that secure a niche within the GIT during embryonic development are nearly impossible to eradicate from the food chain. Therefore, current monitoring paradigms are not comprehensive because they fail to capture the presence of invasive pathogens within the embryonic GIT rapidly. By developing tools to recognize the pathogens' presence in the GIT during embryogenesis, producers are then able to spot evaluate broiler eggs for their potential risk as carriers of foodborne pathogens. In this study a novel qPCR assay was developed to semi-quantify pathogen load relative to total bacterial burden. Eggs sampled from three independent production broiler flocks of different ages were assayed for S. enterica (invA), C. jejuni (HipO), and L. monocytogenes (HlyA) against total microbial load (16s). The eggs were sampled at 1-day post-set within each flock, 2 weeks post-set, after vaccination (at 2.5 weeks) and 1-day post-hatch. The eggs were washed, and the yolk and embryonic chick GIT were collected. The DNA was extracted and subjected to a qPCR assay. The results confirm a novel technique for pathogen monitoring relative to total bacterial load and a unique method for monitoring the dynamics of foodborne pathogen invasion throughout broiler egg production.

In-Package Inactivation of Pathogenic and Spoilage Bacteria Associated with Poultry Using Dielectric Barrier Discharge-Cold Plasma Treatments.

The goal of this study was to test the efficacy of in-package dielectric barrier discharge-cold plasma (DBD-CP) treatment to inactivate poultry-associated spoilage (Pseudomonas fluorescens) and pathogenic (Salmonella enterica Typhimurium, Campylobacter jejuni) bacteria. Liquid cultures of the bacterial isolates were sealed within packages containing ambient air (Trial 1) or modified air (65% O2:30% CO2:5% N2; Trial 2). The packages were subjected to treatment times ranging from 30 to 180 s, and after 24 h incubation at 4 °C, bacterial titers were determined. The DBD-CP system completely inactivated the four isolates tested, although the in-package gas composition and treatment times were isolate-specific. Both C. jejuni isolates were completely inactivated between 30 s (modified air) and 120 s (ambient air), while modified air was required for the complete inactivation of S. typhimurium (90 s) and P. fluorescens (180 s). This DBD-CP system is effective for inactivating major poultry-associated spoilage and pathogenic bacteria in liquid culture, and through this study, system parameters to optimize inactivation were determined. This study demonstrates the potential for DBD-CP treatment to inactivate major bacteria of economic interest to the poultry industry, thus potentially allowing for reduced spoilage (e.g., longer shelf life) and increased safety of poultry products.

Campylobacter jejuni Isolation/Enumeration from Environmental Samples.

Currently, there is no universally accepted standard media or method for the recovery of Campylobacter species. This is likely due to the ubiquity of the organism in nature, the complex sample matrices from which the organism is often recovered, as well as the fragile/viable-but nonculturable state the organism assumes in response to stress. The use of a sterile filter placed upon a nonselective Brucella Agar Blood Plate (BAB), followed by incubation at 37 °C in a hydrogen-containing atmosphere (Campycheck), is one method to recover stressed and emerging Campylobacter spp. from complex environmental matrices; however, this technique does not currently allow for the enumeration of the recovered organisms. Enumeration is performed using serial dilutions of sample homogenate plated onto modified Campy-Cefex media followed by incubation at either 37 °C or 42 °C in a microaerobic atmosphere.

Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera.

Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis worldwide. The flagellum, composed of more than 35 proteins, is responsible for colonization of C. jejuni in the host gastrointestinal tract as well as inducing protective antibodies against the homologous serotype. In our previous study, we demonstrated that the flagellar capping protein (FliD) is an immunodominant protein that reacted strongly to sera from field chickens. In this communication, we mapped linear immunoreactive epitopes on FliD using a set of 158 synthetic peptides of 15-mer overlapping with 11 amino acid residues on peptide microarrays with sera from field chickens. The results from peptide microarrays showed (1) no cross-reactivity of the immobilized peptides with the secondary anti-chicken antibody in the control incubation, and (2) heterogeneous patterns of sera reacting to the immobilized peptides. The peptides that reacted to more than three chicken sera and had higher averages of fluorescence units were selected for further validation by the peptide ELISA. The results showed peptides 24, 91 and 92 had relatively high reactivity and less variation among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. These peptides were also recognized by sera from chickens immunized with the purified recombinant FliD protein. The findings of the specific shared linear immunodominant epitopes on FliD in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.

Antibiotic Resistance Patterns of Major Zoonotic Pathogens from All-Natural, Antibiotic-Free, Pasture-Raised Broiler Flocks in the Southeastern United States.

The use of antibiotics in agroecosystems has been implicated in the rise in antibiotic resistance (AR), which can affect environmental, animal, and human health. To determine the environmental impact of antibiotic use in agroecosystems, appropriate background levels of AR in agricultural environments in the absence of antibiotic application must be determined. Therefore, to determine background levels of AR in broiler production, four target microbes (, , , and ) were isolated from 15 all-natural, antibiotic-free, pasture-raised broiler flocks from six farms within the southeastern United States. The AR profiles of these isolates were characterized using the CDC National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and these resistance patterns were compared across target microbes and farms and throughout the life cycle of the flocks along the farm-to-fork continuum. Antibiotic resistances were most prevalent in and and least prevalent in . Although and were isolated from the same farms and characterized using the same NARMS plates, they exhibited distinct AR profiles, with demonstrating clear farm-specific resistance patterns. Multidrug resistance rates (three or more antibiotics), in order of prevalence, were (63.9%), (36.0%), (12.7%), and (1.4%). The results of this study demonstrate the variability in background AR among major food safety-related microbes, even when isolated from similar production and processing samples from the same farms, and indicate the need for the proper design of future broiler production studies to account for this highly dynamic background AR.

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain.

The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n=45) and group B S. Heidelberg (n=11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.

Reactions of chicken sera to recombinant Campylobacter jejuni flagellar proteins.

Campylobacter jejuni is a Gram-negative spiral rod bacterium and is the leading but underreported bacterial food-borne pathogen that causes human campylobacteriosis worldwide. Raw or undercooked poultry products are regarded as a major source for human infection. C. jejuni flagella have been implicated in colonization and adhesion to the mucosal surface of chicken gastrointestinal tracts. Therefore, flagellar proteins would be the excellent targets for further investigation. In this report, we used the recombinant technology to generate a battery of C. jejuni flagellar proteins, which were purified by His tag affinity chromatography and determined antigenic profiles of these recombinant flagellar proteins using sera from chickens older than 6 weeks of age. The immunoblot results demonstrate that each chicken serum reacted to various numbers of recombinant flagellar proteins. Among these recombinant proteins, chicken sera reacted predominantly to the FlgE1, FlgK, FlhF, FliG and FliY proteins. These antibody screening results provide a rationale for further evaluation of these recombinant flagellar proteins as potential vaccines for chickens to improve food safety as well as investigation of host immune response to C. jejuni.

A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

Characterization and reactivity of broiler chicken sera to selected recombinant Campylobacter jejuni chemotactic proteins.

Campylobacter jejuni, a Gram-negative rod bacterium, is the leading causative agent of human acute bacterial gastroenteritis worldwide. Consumption and handling of raw or undercooked poultry are regarded as a major source for human infection. Because bacterial chemotaxis guides microorganisms to colonization and invasion in the host cells, proteins involved in chemotactic processes can be novel targets for vaccine development. In this communication, we report amplification, cloning and expression of the C. jejuni chemotactic proteins in an Escherichia coli expression system. A total of 15 chemotactic protein genes were successfully expressed. These recombinant proteins were confirmed by nucleotide sequencing, SDS-PAGE analysis and immunoblot analysis of six-His and hemagglutinin tags. Twelve recombinant chemotactic proteins were further tested whether they were antigenic using sera from broiler chickens older than 4 weeks. The immunoblot results show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product (annotated as a methyl-accepting chemotaxis protein), suggesting that anti-Campylobacter antibodies may be prevalent in the poultry population. These antibody screening results provide a rationale for further evaluation of the Cjj0473 protein as a potential vaccine for broilers to improve human food safety.

Characterization and antigenicity of recombinant Campylobacter jejuni flagellar capping protein FliD.

Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population. These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate for broiler chickens to improve food safety for poultry.

Characterization of the Campylobacter jejuni cryptic plasmid pTIW94 recovered from wild birds in the southeastern United States.

The complete nucleotide sequence was determined for a cryptic plasmid, pTIW94, recovered from several Campylobacter jejuni isolates from wild birds in the southeastern United States. pTIW94 is a circular molecule of 3860 nucleotides, with a G+C content (31.0%) similar to that of many Campylobacter spp. genomes. A typical origin of replication, with iteron sequences, was identified upstream of DNA sequences that demonstrated similarity to replication initiation proteins. A total of five open reading frames (ORFs) were identified; two of the five ORFs demonstrated significant similarity to plasmid pCC2228-2 found within Campylobacter coli. These two ORFs were similar to essential replication proteins RepA (100%; 26/26 aa identity) and RepB (95%; 327/346 aa identity). A third identified ORF demonstrated significant similarity (99%; 421/424 aa identity) to the MOB protein from C. coli 67-8, originally recovered from swine. The other two identified ORFs were either similar to hypothetical proteins from other Campylobacter spp., or exhibited no significant similarity to any DNA or protein sequence in the GenBank database. Promoter regions (-35 and -10 signal sites), ribosomal binding sites upstream of ORFs, and stem-loop structures were also identified within the plasmid. These results demonstrate that pTIW94 represents a previously un-reported small cryptic plasmid with unique sequences as well as highly similar sequences to other small plasmids found within Campylobacter spp., and that this cryptic plasmid is present among Campylobacter spp. recovered from different genera of wild birds.