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.

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.

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.

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.

Global gene expression in channel catfish after vaccination with an attenuated Edwardsiella ictaluri.

To understand the global gene expression in channel catfish after immersion vaccination with an attenuated Edwardsiella ictaluri (AquaVac-ESC™), microarray analysis of 65,182 UniGene transcripts was performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 2, a total of 52 unique transcripts were found to be upregulated in vaccinated fish at 48 h post vaccination, whereas a total of 129 were downregulated. The 52 upregulated transcripts represent genes with putative functions in the following seven major categories: (1) hypothetical (25%); (2) novel (23%); (3) immune response (17%); (4) signal transduction (15%); (5) cell structure (8%); (6) metabolism (4%); and (7) others (8%). The 129 downregulated transcripts represent genes with putative functions in the following ten major categories: (1) novel (25%); (2) immune response (23%); (3) hypothetical (12%); (4) metabolism (10%); (5) signal transduction (7%); (6) protein synthesis (6.2%); (7) cell structure (5%); (8) apoptosis (3%); (9) transcription/translation (2%); and (10) others (6%). Microarray analysis revealed that apolipoprotein A-I was upregulated the most (8.5 fold, P = 0.011) at 48 h post vaccination whereas a novel protein (accession no. CV995854) was downregulated the most (342 fold, P = 0.001). Differential regulation of several randomly selected transcripts in vaccinated fish was also validated by quantitative PCR. Our results suggest that these differentially regulated genes elicited by the vaccination might play important roles in the protection of channel catfish against E. ictaluri.

Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque).

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 like. Each PRMT nucleic acid sequence has an open reading frame (ORF) and 3'-untranslated regions. Each ORF appears to encode 361, 587 and 458 amino acid residues for PRMT1, PRMT4 and variant, respectively. The partial ORF of PRMT3 and PRMT5 encode 292 and 563 amino acids, respectively. By comparison with the human counterparts, each channel catfish PRMT also has conserved domains. For expression profile, the channel catfish PRMT1 transcript was detected by RT-PCR in spleens, anterior kidneys, livers, intestines, skin and gills of fish examined. Except in liver, the PRMT3 transcript was detected in all catfish tissues examined. However, the PRMT4 cDNA was detected in livers from all three catfish and gills from two fish, but not other tissues. This information will enable us to further elucidate PRMT functions in channel catfish.

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.

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.

Over-expression, purification and immune responses to Aeromonas hydrophila AL09-73 flagellar proteins.

Aeromonas hydrophila is ubiquitous in aquatic environments worldwide and causes many diseases in fish as well as human. Recent outbreaks of aeromonad diseases in channel catfish prompted us to investigate catfish immune responses during infection of A. hydrophila. In this communication, we report to amplify, over-express, purify and characterize 19 A. hydrophila flagellar proteins. All recombinant proteins were confirmed by nucleotide sequencing of expression plasmids, SDS-PAGE analysis and His tag Western blot of induced proteins. Our preliminary result also showed that the purified recombinant FlgK protein reacted strongly to sera from experimentally infected catfish, suggesting that this protein has potential for a novel target for vaccine development. It is also anticipated that these recombinant proteins will provide us with very useful tools to investigate host immune response to this microorganism.

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.

Sequence analysis, characterization and tissue distribution of channel catfish (Ictalurus punctatus Rafinesque, 1818) myeloperoxidase cDNA.

Myeloperoxidase (EC 1.11.1.7), a heme-containing lysosomal glycoprotein, is found predominantly in azurophilic granules of neutrophils. This enzyme upon activation catalyzes hydrogen peroxide in the presence of various halide ions to form hypohalous acids. Subsequently, these reagents are able to kill the invading microorganisms. In this study, we report the identification, characterization and expression analysis of the channel catfish myeloperoxidase transcript. The full-length nucleotide sequence of channel catfish myeloperoxidase cDNA had 3157 nucleotides, including an open reading frame, which appears to encode a putative peptide of 771 amino acid residues with a calculated molecular mass of 87.14 kDa. By comparison with the human counterpart, the channel catfish myeloperoxidase peptide can be divided into domains and has conservative features, including peroxidase catalytic sites, covalent linkage sites for the heme group and all cysteine residues. The channel catfish myeloperoxidase transcript was detected by RT-PCR in anterior kidneys, where the major leukocyte population is neutrophil precursors. Reagent development and the role of this enzyme in Edwardsiella ictaluri infection are under investigation.

Characterization and tissue expression of channel catfish (Ictalurus punctatus Rafinesque, 1818) ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) cDNA.

The ubiquitin-proteasome cycle is a complex, non-lysosomal biochemical process for intracellular protein degradation. This process involves many enzymes. One enzyme involved in this process is ubiquitin carboxyl-terminal hydrolase L5 (UCHL5), which deubiquitylates the polyubiquitin chain into ubiquitin. In this report, we isolated, sequenced, and characterized the channel catfish UCHL5 cDNA. The complete nucleic acid sequence of the channel catfish UCHL5 cDNA is comprised of 1,357 nucleotides, including an open reading frame, which appears to encode a putative peptide of 329 amino acid residues. The estimated molecular mass and pI of this peptide are 37.6 kDa and 4.84 at pH 7.0, respectively. The degree of conservation of the channel catfish UCHL5 amino acid sequence in comparison to other species ranged from 85% (vs. mouse) to 92% (vs. zebrafish and spotted green pufferfish). The channel catfish UCHL5 transcript was detected by RT-PCR in spleen, head kidney, liver, intestine, skin and gill, suggesting the UCHL5 transcript is constitutively expressed. This research provides important information for further elucidating UCHL5 in the channel catfish ubiquitin-proteasome pathway.

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.

Channel catfish, Ictalurus punctatus, cysteine proteinases: cloning, characterisation and expression of cathepsin H and L.

The antigen recognition by the host immune system is a complex biochemical process that requires a battery of enzymes. Cathepsins are one of the enzyme superfamilies involved in antigen degradation. We observed the up-regulation of catfish cathepsin H and L transcripts during the early stage of Edwardsiella ictaluri infection in preliminary studies, and speculated that cathepsin H and L may play roles in infection. We identified, sequenced and characterized the complete channel catfish cathepsin H and L cDNAs, which comprised 1415 and 1639 nucleotides, respectively. The open reading frames of cathepsin H appeared to encode a protein of 326-amino acid residues, which that of cathepsin L encoded a protein of 336 amino acids. The degree of conservation of the channel catfish cathepsin H and L amino acid sequences in comparison to other species ranged from 61% to 77%, and 67% to 85%, respectively. The catalytic triad and substrate binding sites are conserved in cathepsin H and L amino acid sequences. The cathepsin L transcript was expressed in all tissues examined, while the cathepsin H was expressed in restricted tissues. These results provide important information for further exploring the roles of channel catfish cathepsins in antigen processing.

Channel catfish, Ictalurus punctatus Rafinesque 1818, tetraspanin membrane protein family: characterization and expression analysis of CD81 cDNA.

CD81, also known as the target of an antiproliferative antibody 1 (TAPA-1) in human, is a member of tetraspanin integral membrane protein family. This protein plays many important roles in immune and other physiological functions. In this report, we characterized and analyzed expression of the channel catfish CD81 transcript. The full-length of channel catfish CD81 cDNA comprised of 1130 nucleotides, including an open reading frame which appears to encode a putative peptide of 234 amino acid residues. By comparison with the human counterpart, the channel catfish CD81 peptide could be divided into domains, including four transmembrane domains, three intracellular domains, and one of each small and large extracellular loops. The degree of conservation of the channel catfish CD81 amino acid sequence to that of mammalian counterparts ranged from 65% to 67%. The large extracellular domain shows the least conservation between fish and mammals. However, the characteristic Cys(159)-Cys(160)-Gly(161) motif and Cys(176/188) in this domain were conserved. The channel catfish CD81 transcript was detected by RT-PCR in spleen, head kidney, liver, intestine, skin and gill. This result provides important information for further elucidating CD81 functions in channel catfish.

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.

Channel catfish, Ictalurus punctatus, cyclophilin A and B cDNA characterization and expression analysis.

The preliminary observation of up-regulation of cyclophilin transcripts during Edwardsiella ictaluri infection prompted us to speculate on the potential importance of cyclophilins in the early stage of infection. To provide a framework for answering these questions, two cyclophilin cDNA of channel catfish, Ictalurus punctatus, were identified, sequenced and characterized. The complete nucleotide sequences of cyclophilin A and cyclophilin B cDNA consisted of 1170 and 996 bases, respectively. Analyses of the sequences revealed each had one open reading frame potentially encoding 164 amino acids with calculated molecular mass of 17,450Da and 216 amino acids with calculated molecular mass of 23,852Da for cyclophilin A and cyclophilin B, respectively. The degrees of conservation of channel catfish cyclophilin A and cyclophilin B amino acid sequences to counterparts of other species ranged from 74 to 84% and 80 to 92%, respectively. Both cyclophilin A and cyclophilin B transcripts were constitutively expressed in all tissues of channel catfish examined in this study. These results provide valuable information not only for further exploring the roles of cyclophilins in fish immune responses to infection, but also for production of polyclonal/monoclonal antibodies for channel catfish cyclophilins.