Detection and Distribution of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Campylobacter jejuni Isolates from Chicken Livers.

Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide linked to the consumption of undercooked broiler livers. Application of bacteriophages during poultry production has been used as an alternative approach to reduce contamination of poultry meat by Campylobacter. To make this approach effective, understanding the presence of the bacteriophage sequences in the CRISPR spacers in C. jejuni is critical as they may confer bacterial resistance to bacteriophage treatment. Therefore, in this study, we explored the distribution of the CRISPR arrays from 178 C. jejuni isolated from chicken livers between January and July 2018. Genomic DNA of C. jejuni isolates was extracted, and CRISPR type 1 sequences were amplified by PCR. Amplicons were purified and sequenced by the Sanger dideoxy sequencing method. Direct repeats (DRs) and spacers of CRISPR sequences were identified using the CRISPRFinder program. Further, spacer sequences were submitted to the CRISPRTarget to identify potential homology to bacteriophage types. Even though CRISPR-Cas is reportedly not an active system in Campylobacter, a total of 155 (87%) C. jejuni isolates were found to harbor CRISPR sequences; one type of DR was identified in all 155 isolates. The CRISPR loci lengths ranged from 97 to 431 nucleotides. The numbers of spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates that could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases showed that most spacer sequences were homologous to Campylobacter bacteriophage DA10. The results of our study provide important information relative to the development of an effective bacteriophage treatment to mitigate Campylobacter during poultry production.

Genotypic characterization, antimicrobial susceptibility and virulence determinants of Campylobacter jejuni and Campylobacter coli isolated from pastured poultry farms.

Aim: Campylobacter is the leading bacterial pathogen that causes foodborne illnesses worldwide. Pasture farming is regarded as an important source of agricultural production for small farming communities. Consumer preference for pasture-raised animal products has increased; however, there is a paucity of information on the microbiological quality of pasture-raised poultry products. The purpose of this study was to explore genetic relatedness of thermophilic Campylobacter isolates, to assess antibiotic resistance phenotypically and genotypically, and to screen the presence of virulence determinants of Campylobacter isolates from pasture-raised poultry farms from southeastern United States. Methods: Ninety-seven Campylobacter isolates previously identified by Q7 BAX® System Real-Time PCR were genotyped by multilocus sequence typing (MLST). Campylobacter isolates were then evaluated for their phenotypic antimicrobial susceptibility against nine antimicrobial agents using Sensititre plates. Additionally, Campylobacter isolates were tested for the presence of antimicrobial resistance-associated elements. Furthermore, Campylobacter isolates were screened for the presence of 13 genes encoding putative virulence factors by PCR. These included genes involved in motility (flaA and flhA), adhesion and colonization (cadF, docC, racR, and virB11), toxin production (cdtA, cdtB, cdtC, wlaN, and ceuE) and invasion (ciaB and iamA). Results: Among 97 Campylobacter isolates, Campylobacter jejuni (n = 79) and Campylobacter coli (n = 18) were identified. By MLST, C. jejuni isolates were assigned to seven clonal complexes. Among them, ST-353, ST-607 and ST-21 were the most common STs recognized. All C. coli (n = 18) isolates were included in CC-828. Interestingly, eight STs identified were not belonging any previous identified clonal complex. Campylobacter isolates displayed a high resistance rate against tetracycline (81.4%), while a low rate of resistance was observed against macrolides (azithromycin and erythromycin), quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin), aminoglycosides (gentamicin), ketolide (telithromycin), amphenicol (florfenicol) and lincomycin (clindamycin). Thirteen isolates (13.54%) were pan-susceptible to all tested antibiotics, while nine isolates were multi-antimicrobial resistant (MAR; resist to three or more antimicrobial classes). Interestingly, there were no isolates resistant to all antimicrobial classes. Thr86Ile mutation was identified in all quinolones resistant strains. Erythromycin encoding gene (ermB) was identified in 75% of erythromycin resistant isolates. The A2075 mutation was detected in one erythromycin resistant strain, while A2074 could not be identified. The tetO gene was identified in 93.7% of tetracycline resistant isolates and six tetracycline susceptible isolates. In conclusion, the results of this study revealed that Campylobacter isolates from pasture-raised poultry farms showed the ST relatedness to Campylobacter isolates commonly associated with humans, indicating pasture-raised broiler flocks, similar to conventionally-reared broiler flocks, as a potential vector for antibiotic-resistant and pathogenic strains of thermophilic Campylobacter to humans.

Use of automated capillary immunoassays for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins.

The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In the present study, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations was also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. The chicken immunoglobin G (IgG) antibody response to the FliD protein from the immunized group was 1110- and 51,400-fold higher than that from the un-immunized chickens both two- and three-weeks post-vaccination, respectively. It was also observed that IgM antibody against the FliD protein from the immunized chickens was 1030-fold higher than that from the un-immunized chickens two weeks post-vaccination, but the IgM response declined to 120-fold between two groups from two weeks to three weeks after immunization. The IgM antibody response to the FimA protein from the immunized group was 1.84- and 1.12-fold higher than that from the un-immunized group, respectively, both two- and three-weeks post-vaccination, while the IgG antibody response from the immunized group was 8.07- and 27.6-fold higher than that from the un-immunized group, respectively, during the same period. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

Prevalence and Characterization of Quinolone Resistance in Campylobacter spp. Isolates in Chicken Livers from Retail Stores in Georgia, USA.

ABSTRACT: Campylobacter is a bacterial pathogen that causes human foodborne illnesses worldwide, and outbreaks have been associated with consumption of undercooked chicken livers. The objectives of this study were to compare two PCR assays of 250 Campylobacter isolates for identification to species, to assess antibiotic resistance of the isolates, and to analyze genetic diversity of the quinolone resistance determining regions (QRDRs) of the isolates. A double-blind design was used to identify the species of Campylobacter; 181 (72%) of the isolates were identified as Campylobacter jejuni, and 69 (28%) isolates were identified as Campylobacter coli by both PCR assays. A total of 93 (37.2%) isolates were resistant to at least one antibiotic. Among 88 C. jejuni isolates, 33 (18%) were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), 25 (14%) were resistant to tetracycline (TET), and 18 (10%) were resistant to NAL and TET. Two C. jejuni isolates were resistant to four of the tested antibiotics, and one isolate was resistant to five antibiotics. Two C. coli isolates were resistant to TET, and two were resistant to NAL, CIP, and TET. The amino acid sequences of the QRDRs for the isolates had eight point mutations and could be classified into 12 groups. Thirty-eight C. jejuni isolates resistant to NAL and CIP had a point mutation at residue 86 (substitution from threonine to isoleucine). However, six isolates without this substitution were resistant to NAL and/or CIP. Ten isolates with a point mutation at residue 86 were susceptible to NAL and CIP. This observation suggests that in addition to the substitution at residue 86 other mechanisms may confer resistance to quinolones. Further studies are needed to elucidate mechanisms for quinolone resistance in Campylobacter. The Campylobacter spp. isolated from chicken livers in this study were resistant to quinolones and other classes of antibiotics.

Detection of Campylobacter jejuni diversity by clustered regularly interspaced short palindromic repeats (CRISPR) from an animal farm.

BACKGROUND: Campylobacter jejuni is the leading bacterial pathogen that causes foodborne illness worldwide. Because of genetic diversity and sophisticated growth requirements of C. jejuni, several genotyping methods have been investigated to classify this bacterium during the outbreaks. One of such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). OBJECTIVES: The goal of this study was to explore the diversity of C. jejuni isolates with CRISPR from an animal farm. METHODS: Seventy-seven C. jejuni isolates from an animal farm were used in this study. The day-old broilers were reared with other poultry and farm animals, including layer hens, guinea hens, dairy goats and sheep. A small swine herd was also present on an adjacent, but separate plot of land. Isolation and identification of C. jejuni were performed according to the standard procedures. The CRISPR type 1 was PCR amplified from genomic DNA, and the amplicons were sequenced by the Sanger dideoxy method. The direct repeats (DRs) and spacers of the CRISPR sequences were identified using the CRISPRFinder. RESULTS: The CRISPR sequences were detected in all 77 isolates. One type of DRs was identified in these 77 isolates. The lengths of the CRISPR locus ranged from 100 to 560 nucleotides, whereas the number of spacers ranged from one to eight. The distributions of the numbers of CRISPR spacers from different sources seemed to be random. Overall, 17 out of 77 (22%) C. jejuni isolates had two and five spacers, whereas 14 out of 77 (18%) isolates had three spaces in their genomes. By further analysis of spacer sequences, a total of 266 spacer sequences were identified in 77 C. jejuni isolates. By comparison with known published spacer sequences, we observed that 49 sequences were unique in this study. The CRISPR sequence combination of Nos. 16, 19, 48 and 57 was found among a total of 15 C. jejuni isolates containing various multi-locus sequence typing (MLST) types (ST-50, ST-607, ST-2231 and ST-5602). No. 57 spacer sequence was unique from this study, whereas the other three (Nos. 16, 19 and 48) sequences were found in previous reports. Combination of Nos. 5, 9, 15, 30 and 45 was associated with ST-353. To compare the CRISPR genotyping with other methods, the MLST was selected due to its high discriminatory power to differentiate isolates. Based on calculation of the Simpson's index of diversity, a combination of both methods had higher Simpson's index value than those for CRISPR or MLST, respectively. CONCLUSIONS: Our results suggest that the MLST from C. jejuni isolates can be discriminated based on the CRISPR unique spacer sequences and the numbers of spacers. In the future, investigation on the CRISPR resolution for C. jejuni identification in outbreaks is needed. A database that integrates both MLST sequences and CRISPR sequences and is searchable is greatly in demand for tracking outbreaks and evolution of this bacterium.

Bacterial Community Assessed by Utilization of Single Carbon Sources in Broiler Ground Meat after Treatment with an Antioxidant, Carnosine, and Cold Plasma.

ABSTRACT: Contaminated poultry meat is a major source of human foodborne illnesses. Many interventions have been developed to reduce and/or eliminate human foodborne pathogens in poultry products; however, treatments with cold plasma or carnosine or their combination have not been extensively investigated. In this study, the bacterial microflora of poultry meat samples after treatments with cold plasma and carnosine were characterized with EcoPlates in the OmniLog system. The plates were incubated at 25°C for 7 days in the OmniLog chamber, and bacterial growth was monitored by recording formazan production every 30 min at an optical density of 590 nm. The kinetics of lag, log, and stationary phases of bacterial growth followed the Gompertz sigmoidal model but with different inflection times and asymptotes at the log phase and the stationary phase, respectively. Results indicated that treatment of poultry meat samples with cold plasma technology and carnosine could inhibit growth of the bacteria in the treated meat samples. Of 31 chemicals tested, phenylethylamine, α-d-lactose, d,l-α-glycerol phosphate, 2-hydroxybenzoic acid, γ-hydroxybutyric acid, α-ketobutyric acid, and d-malic acid could not be metabolized by bacteria in the meat samples. Future research is required to determine whether these seven chemicals that inhibited growth of bacteria in these meat samples can be used as food preservatives for extending the shelf life of these products. Whether the bacterial flora can be an indicator of effectiveness for meat samples treated with cold plasma, carnosine, or both needs further study.

Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Campylobacter jejuni is the leading bacterial foodborne pathogen that causes human acute gastrointestinal illness worldwide. Due to its genetic diversity, fastidious growth and sophisticated biochemical requirements, classification of Campylobacter by traditional techniques is problematic. Several molecular typing methods have been explored in this bacterium. One such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). These CRISPRs consist of a direct repeat interspaced with nonrepetitive spacer sequences. In this study, we applied this genotyping method to explore the genetic diversity of C. jejuni isolated from poultry sources. Ninety-nine C. jejuni isolates from poultry environments in four different US states were used. Genomic DNA of the isolates were extracted from cultures using a commercial kit. PCR primers and conditions for CRISPR type 1 amplification were described previously. The amplicons were purified and sequenced by the Sanger dideoxy sequencing method. The direct repeats (DR) and spacers of the CRISPR sequences were identified using the CRISPRFinder. The results show there were 21% isolates no detectable, 30% isolates questionable, and 49% isolates confirmed CRISPR, respectively. The lengths of CRISPR range from 100 to 695 nucleotides. One type of DR was found in CRISPR of these isolates. The number of spacers in CRISPR ranges from 1 to 10 with various sequences. A total of 55 distinctive spacer sequences were identified in 78 isolates. Among them, 33 sequences were found unique in this study. In addition, the CRISPR genotyping had higher the Simpson's index of diversity value than that from flaA nucleotide typing. The results of our study show the CRISPR genotyping on C. jejuni may be complementary to the other genotyping methods.

The effect of rosemary Extract and cold plasma treatments on bacterial community diversity in poultry ground meats.

To provide safer food, many technologies have been used to preserve food. One such technology is cold plasma, which can reduce viable bacterial counts in various food matrices. However, bacterial communities in food matrices before and after cold plasma treatment have not been investigated. In this communication, the EcoPlates™ were used to physiologically profile bacterial communities from poultry ground meat treated with rosemary, cold plasma or both. The cultures in the plates were incubated at 25 °C for seven days in an OmniLog® system. Responses of the bacterial communities to 31 chemicals were measured on formazan production. The results show that the three parameters of the Gompertz growth curves were observed in all samples, 2-hydroxybenzoic acid could not be used, while pyruvic acid methyl ester was used for a carbon source by the bacterial communities from all meat samples, each bacterial community metabolized different numbers of chemical compounds at different rates, and reduction of bacterial functional diversity was observed in the poultry meat samples treated with cold plasma and rosemary. In the future, investigations on whether the physiological profiling in bacterial communities be used as an indicator for effectiveness of cold plasma treatment of meat samples.

Community-Level Physiological Profiling for Microbial Community Function in Broiler Ceca.

Poultry production is a major agricultural output worldwide. It is known that the gut health of broilers is essential for their growth and for providing wholesome products for human consumption. Previously, the microbial diversity of broiler ceca was studied at the genetic level. However, the functional diversity and metabolic activity of broiler cecal bacterial communities are not fully investigated. Recently, the EcoPlates™ from Biolog, Inc. have been used for characterizing bacterial communities from various environments. In this study, we applied these plates to physiologically profile cecal bacterial communities in broilers. The ceca were aseptically excised from 6-week-old broilers, and their contents were suspended in phosphate buffered saline. The cultures in the EcoPlates™ were incubated at 42 °C for 5 days in an OmniLog® system. Responses of the bacterial communities to the various chemicals as carbon sources were measured on formazan production. The results show sigmoidal growth curves with three phases in all 12 cecal samples. Cecal bacterial communities could not use 11 carbon substrates for carbon sources; instead, they used pyruvic acid methyl ester, glycogen, glucose-1-phosphate and N-acetyl-D-glucosamine most frequently. Each bacterial community metabolized various numbers of the substrates at different rates among broilers. In the future, modification of the culture conditions to mimic the gut environment is needed. More investigations on the effects of nutrients, Salmonella or Campylobacter on physiological functions of cecal bacterial communities will provide insights into the improvement of animal well-being, saving production expenditures for producers and providing safer poultry products for human consumption.

Epitope mapping of Salmonella flagellar hook-associated protein, FlgK, with mass spectrometry-based immuno-capture proteomics using chicken (Gallus gallus domesticus) sera.

Salmonella, a Gram-negative rod, is the leading foodborne pathogen associated with human acute bacterial gastroenteritis worldwide. The Salmonella flagellum is responsible for bacterial movement, colonization and invasion in the host gastrointestinal tract. The flagellum has a complex structure, composed of more than 35 proteins. Among them, we were interested in the flagellar hook-associated protein (FlgK), which is an immunodominant protein in chickens. In this communication, we applied mass spectrometry-based proteomics in conjunction with chicken immunized sera to map the linear immunoepitopes in the FlgK protein, validated the epitopes with peptide ELISA, and determined serum reactivity to the epitopes from commercial chickens. We previously demonstrated the FlgK proteins are highly conserved among Salmonella serovars. The rFlgK protein was produced by the recombinant technique, and was able to induce immune response in chickens. Further, this study identified four peptides (AEG, GAQ, TAD and LEI) in the rFlgK protein that were captured by sera from chickens immunized with the rFlgK protein. These four peptides were also reacted to 64 individual serum samples collected from 44 - 52 weeks old chickens, suggesting that these peptides may represent the shared immuno-epitopes on the FlgK protein. The findings of the specific shared linear immuno-epitopes on the FlgK protein 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.

Molecular Analysis, Biochemical Characterization, Antimicrobial Activity, and Immunological Analysis of Proteus mirabilis Isolated from Broilers.

Proteus mirabilis, a Gram-negative bacterium, is ubiquitous in the environment and is considered as the normal microflora in the human gastrointestinal tract. However, this bacterium is an opportunistic pathogen in humans, often causing urinary tract infections. Moreover, Proteus has been frequently isolated from food animals, including poultry. Whether this bacterium contributes to the foodborne illness in humans is unclear. In this report, P. mirabilis isolates recovered from broilers during housing in the units were characterized, their antimicrobial activity was assayed, and broiler immune response to the soluble proteins was determined. Cecal contents and fecal droppings were treated according to the standard protocol for isolation. Speciation based on biochemical reactions and the antimicrobial activity of the isolates were carried out using commercial kits. Immunoblot was assayed to determine immune status of broilers against P. mirabilis. A total of 10 isolates of P. mirabilis were selected for further characterization. These isolates could grow in pH 6.0 and 1% NaCl conditions. They were resistant to sodium lactate, troleandomycin, rifamycin SV, vancomycin, but sensitive to nalidixic acid, cefotaxime and novobiocin. Moreover, the CTX, ACC, CMY-1, BIC, NDM, VEB, qnrB and qnrD genes were detected by PCR amplification in all isolates. Sera from broilers harboring this bacterium reacted to the P. mirabilis soluble proteins, but not from litter- and age-matched P. mirabilis negative and SPF chickens, indicating that this bacterium infected chickens that could have humoral immune response against P. mirabilis. This study provides a rationale for further monitoring P. mirabilis during poultry production to determine whether this bacterium poses potential threats to public health.

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.

Production of recombinant Salmonella flagellar protein, FlgK, and its uses in detection of anti-Salmonella antibodies in chickens by automated capillary immunoassay.

Conventional immunoblot assays are a useful tool for specific protein identification, but tedious, labor-intensive and time-consuming. A capillary electrophoresis-based immunoblot assay so-called "Simple Western" was developed to enable the protein identification in an automatic manner. This communication describes the use of Simple Western for detecting anti-Salmonella FlgK antibodies from chicken sera.

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.

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.

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.

Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins.

Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni.

Channel catfish, Ictalurus punctatus (Rafinesque), tetraspanin membrane protein family: identification, characterization and phylogenetic analysis of tetraspanin 3 and tetraspanin 7 (CD231) transcripts.

Tetraspanins, a large cell surface protein superfamily characterized by having four transmembrane domains, play many critical roles in physiological and pathological processes. In this study, we report the identification, characterization and phylogenetic analysis of the channel catfish tetraspanin 3 and tetraspanin 7 (CD231) transcripts. The full-length nucleotide sequences of tetraspanin 3 and tetraspanin 7 cDNA have 1,453 and 1,842 base pairs, respectively. Analysis of the nucleotide sequences reveals that each has one open reading frame (ORF). The ORF of tetraspanin 3 appears to encode 241 amino acids with calculated molecular mass of 26.8 kDa, while the ORF of tetraspanin 7 potentially encodes 251 amino acids with calculated molecular mass of 27.9 kDa. By comparison with the human counterparts, the channel catfish tetraspanin 3 and tetraspanin 7 peptides have four transmembrane domains, three intracellular domains and two (small and large) extracellular domains. In addition, several characteristic features critical for structure and functions in mammalian tetraspanins are also conserved in channel catfish tetraspanin 3 and tetraspanin 7. The transcripts were detected by RT-PCR in restrictive organs. These results with those from our previous studies on other channel catfish tetraspanins provide important information for further investigating the roles of various tetraspanins in channel catfish infection with microorganisms.

Global transcription analysis of vaccinated channel catfish following challenge with virulent Edwardsiella ictaluri.

To determine the identities of genes involved in either innate or adaptive immunity, microarray analysis of 65,182 UniGene transcripts were performed to compare gene expression in vaccinated channel catfish after challenge with a virulent Edwardsiella ictaluri compared to that in sham-vaccinated fish without challenge. With a filter of false-discovery rate less than 0.05 and fold change greater than 2, a total of 167 functionally known unique transcripts were found to be up-regulated, whereas 40 were down-regulated. The 167 up-regulated transcripts represent genes with putative functions in the following eight major categories: (1) immunity (30%); (2) metabolism and energy production (22%); (3) transcription or translation (12%); (4) protein degradation (11%); (5) signal transduction (6%); (6) traffic and transport (6%); (7) cell structure or cell cycle (8%); and (8) others (5%). The 40 down-regulated transcripts represent genes with putative functions in the following six major categories: (1) metabolism (27.5%); (2) immunity (17.5%); (3) cell structure (17.5%); (4) cell motility (10%); (5) signal transduction (15%); and (6) others (12.5%). Microarray analysis revealed that lysozyme c was up-regulated the most (70-fold) in vaccinated fish at 48 h post challenge of virulent E. ictaluri whereas myotubularin related protein 1a and cytochrome P450 2J27 were down-regulated the most (8.1 fold). Differential regulation of eight randomly selected transcripts in vaccinated fish after challenge with virulent E. ictaluri was also validated by quantitative PCR. Our results suggest that these differentially regulated genes might play important roles in channel catfish immunity against E. ictaluri.