Transgenic Mice Overexpressing PG1 Display Corneal Opacity and Severe Inflammation in the Eye.

Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice overexpressing protegrin 1 (PG1), a potent porcine cathelicidin. No obvious differences were observed between PG1 transgenic and wild-type mice in terms of growth, development, general behaviour, and the major immune cell population. However, PG1 transgenic mice intranasally infected with Staphylococcus aureus resulted in a reduction in microscopic pulmonary injury, improved clearance of bacteria, and lower proinflammatory cytokine secretion, compared to those of wild-type mice. On the other hand, approximately 25% of PG1 transgenic mice (n = 54/215) showed corneal opacity and developed inflammation in the eye, resulting ultimately in phthisis bulbi. Immunohistochemical analyses revealed that PG1 and its activator, neutrophil elastase, localized to the basal cells of the cornea and glands in eyelids, respectively. In addition, apoptosis indicated by a Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive signal was detected from flat cells of the cornea. Our study suggests that the expression regulation or localization of AMPs such as PG1 is important to prevent their adverse effects. However, our results also showed that the cytotoxic effects of PG1 on cells could be tolerated in animals, except for the eyes.

Genomewide Analysis of the Antimicrobial Peptides in Python bivittatus and Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity.

In this study, we sought to identify novel antimicrobial peptides (AMPs) in Python bivittatus through bioinformatic analyses of publicly available genome information and experimental validation. In our analysis of the python genome, we identified 29 AMP-related candidate sequences. Of these, we selected five cathelicidin-like sequences and subjected them to further in silico analyses. The results showed that these sequences likely have antimicrobial activity. The sequences were named Pb-CATH1 to Pb-CATH5 according to their sequence similarity to previously reported snake cathelicidins. We predicted their molecular structure and then chemically synthesized the mature peptide for three putative cathelicidins and subjected them to biological activity tests. Interestingly, all three peptides showed potent antimicrobial effects against Gram-negative bacteria but very weak activity against Gram-positive bacteria. Remarkably, ΔPb-CATH4 showed potent activity against antibiotic-resistant clinical isolates and also was observed to possess very low hemolytic activity and cytotoxicity. ΔPb-CATH4 also showed considerable serum stability. Electron microscopic analysis indicated that ΔPb-CATH4 exerts its effects via toroidal pore preformation. Structural comparison of the cathelicidins identified in this study to previously reported ones revealed that these Pb-CATHs are representatives of a new group of reptilian cathelicidins lacking the acidic connecting domain. Furthermore, Pb-CATH4 possesses a completely different mature peptide sequence from those of previously described reptilian cathelicidins. These new AMPs may be candidates for the development of alternatives to or complements of antibiotics to control multidrug-resistant pathogens.

Antimicrobial susceptibility profiles and molecular typing of Campylobacter jejuni and Campylobacter coli isolates from ducks in South Korea.

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

Development of a Highly Efficient CRISPR/Cas9-Mediated Herpesvirus of Turkey-Based Vaccine against Novel Variant Infectious Bursal Disease Virus.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is an extremely contagious immunosuppressive disease that causes major losses for the poultry industry worldwide. Recently, the novel variant IBDV (G2d) has been highly prevalent in Korea, but the current vaccines against this very virulent IBDV have limited efficacy against this novel variant. To develop a vaccine against this variant IBDV, a recombinant virus designated rHVT-VP2 was constructed by inserting the IBDV (G2d) VP2 gene into herpesvirus of turkeys (HVT) using CRISPR/Cas9 gene-editing technology. The PCR and sequencing results obtained showed that the recombinant virus rHVT-VP2 was successfully constructed. Vaccination with rHVT-VP2 generated IBDV-specific antibodies in specific pathogen-free chickens starting from 2 weeks post-immunization. Seven days after the challenge, the autopsy results showed that the bursa atrophy rates of the rHVT-VP2, HVT, vaccine A, and positive control groups were 0%, 100%, 60%, and 100%, respectively, and the BBIX values were 1.07 ± 0.22, 0.27 ± 0.05, 0.64 ± 0.33, and 0.32 ± 0.06, respectively. These results indicate that rHVT-VP2 can provide 100% protection against a challenge with the IBDV (G2d), whereas vaccine A only provides partial protection. In conclusion, vaccination with the recombinant virus rHVT-VP2 can provide chickens with effective protection against variant IBDV (G2d).

Protection of Chickens against H9N2 Avian Influenza Isolates with a Live Vector Vaccine Expressing Influenza Hemagglutinin Gene Derived from Y280 Avian Influenza Virus.

Since the outbreak of the H9N2/Y439 avian influenza virus in 1996, the Korean poultry industry has incurred severe economic losses. A novel possibly zoonotic H9N2 virus from the Y280-like lineage (H9N2/Y280) has been prevalent in Korea since June 2020, posing a threat to the poultry sector. Rapid mutation of influenza viruses urges the development of effective vaccines against newly generated strains. Thus, we engineered a recombinant virus rHVT/Y280 to combat H9N2/Y280. We integrated the hemagglutinin (HA) gene of the H9N2/Y280 strain into the US2 region of the herpesvirus of turkeys (HVT) Fc126 vaccine strain, utilizing CRISPR/Cas9 gene-editing technology. The successful construction of rHVT/Y280 was confirmed by polymerase chain reaction and sequencing, followed by efficacy evaluation. Four-day-old specific pathogen-free chickens received the rHVT/Y280 vaccine and were challenged with the H9N2/Y280 strain A21-MRA-003 at 3 weeks post-vaccination. In 5 days, there were no gross lesions among the vaccinated chickens. The rHVT/Y280 vaccine induced strong humoral immunity and markedly reduced virus shedding, achieving 100% inhibition of virus recovery in the cecal tonsil and significantly lowering tissue viral load. Thus, HVT vector vaccines expressing HA can be used for protecting poultry against H9N2/Y280. The induction of humoral immunity by live vaccines is vital in such cases. In summary, the recombinant virus rHVT/Y280 is a promising vaccine candidate for the protection of chickens against the H9N2/Y280.

Simultaneous construction strategy using two types of fluorescent markers for HVT vector vaccine against infectious bursal disease and H9N2 avian influenza virus by NHEJ-CRISPR/Cas9.

Recently, herpesvirus of turkeys (HVT), which was initially employed as a vaccine against Marek's disease (MD), has been shown to be a highly effective viral vector for producing recombinant vaccines that can simultaneously express the protective antigens of multiple poultry diseases. Prior to the development of commercial HVT-vectored dual-insert vaccines, the majority of HVT-vectored vaccines in use only contained a single foreign gene and were often generated using time-consuming and inefficient traditional recombination methods. The development of multivalent HVT-vectored vaccines that induce simultaneous protection against several avian diseases is of great value. In particular, efficacy interference between individual recombinant HVT vaccines can be avoided. Herein, we demonstrated the use of CRISPR/Cas9 gene editing technology for the insertion of an IBDV (G2d) VP2 expression cassette into the UL45/46 region of the recombinant rHVT-HA viral genome to generate the dual insert rHVT-VP2-HA recombinant vaccine. The efficacy of this recombinant virus was also evaluated in specific pathogen-free (SPF) chickens. PCR and sequencing results showed that the recombinant virus rHVT-VP2-HA was successfully constructed. Vaccination with rHVT-VP2-HA produced high levels of specific antibodies against IBDV (G2d) and H9N2/Y280. rHVT-VP2-HA can provide 100% protection against challenges with IBDV (G2d) and H9N2/Y280. These results demonstrate that rHVT-VP2-HA is a safe and highly efficacious vaccine for the simultaneous control of IBDV (G2d) and H9N2/Y280.

Genetic Characterization of Avian Paramyxovirus Isolated from Wild Waterfowl in Korea between 2015 and 2021.

Avian paramyxoviruses (APMVs) are often carried by wild waterfowl, and the wild waterfowl may play an important role in the maintenance and spread of these viruses. In this study, we investigated APMVs in the population of migratory wild waterfowl from 2015 to 2021 in Korea and analyzed their genetic characteristics. Fourteen viruses were isolated and subsequently identified as APMV-1 (n = 13) and APMV-13 (n = 1). Phylogenetic analysis of the full fusion gene of 13 APMV-1 isolates showed that 10 APMV-1 isolates belonged to the class II sub-genotype I.2, which was epidemiologically linked to viruses from the Eurasian continent, and 3 viruses belonged to class I, which linked to viruses from the USA. The APMV-13 isolates from wild geese in this study were highly homology to the virus isolated from China. Sequence analysis of 14 isolates showed that all isolates had a typical lentogenic motif at the cleavage site. In summary, we identified the wild species likely to be infected with APMV and our data suggest possible intercontinental transmission of APMV by wild waterfowl. Our current study also provides the first evidence for the presence of class I of APMV-1 and APMV-13 in wild waterfowl surveyed in Korea.

Analysis of cattle olfactory subgenome: the first detail study on the characteristics of the complete olfactory receptor repertoire of a ruminant.

BACKGROUND: Mammalian olfactory receptors (ORs) are encoded by the largest mammalian multigene family. Understanding the OR gene repertoire in the cattle genome could lead to link the effects of genetic differences in these genes to variations in olfaction in cattle. RESULTS: We report here a whole genome analysis of the olfactory receptor genes of Bos taurus using conserved OR gene-specific motifs and known OR protein sequences from diverse species. Our analysis, using the current cattle genome assembly UMD 3.1 covering 99.9% of the cattle genome, shows that the cattle genome contains 1,071 OR-related sequences including 881 functional, 190 pseudo, and 352 partial OR sequences. The OR genes are located in 49 clusters on 26 cattle chromosomes. We classified them into 18 families consisting of 4 Class I and 14 Class II families and these were further grouped into 272 subfamilies. Comparative analyses of the OR genes of cattle, pigs, humans, mice, and dogs showed that 6.0% (n = 53) of functional OR cattle genes were species-specific. We also showed that significant copy number variations are present in the OR repertoire of the cattle from the analysis of 10 selected OR genes. CONCLUSION: Our analysis revealed the almost complete OR gene repertoire from an individual cattle genome. Though the number of OR genes were lower than in pigs, the analysis of the genetic system of cattle ORs showed close similarities to that of the pig.

Identification and classification of feline endogenous retroviruses in the cat genome using degenerate PCR and in silico data analysis.

The purpose of this study was to identify and classify endogenous retroviruses (ERVs) in the cat genome. Pooled DNA from five domestic cats was subjected to degenerate PCR with primers specific to the conserved retroviral pro/pol region. The 59 amplified retroviral sequences were used for in silico analysis of the cat genome (Felis_catus-6.2). We identified 219 ERV γ and ß elements from cat genome contigs, which were classified into 42 ERV γ and 4 ß families and further analysed. Among them, 99 γ and 5 ß ERV elements contained the complete retroviral structure. Furthermore, we identified 757 spuma-like ERV elements based on the sequence homology to murine (Mu)ERV-L and human (H)ERV-L. To the best of our knowledge, this is the first detailed genome-scale analysis examining Felis catus endogenous retroviruses (FcERV) and providing advanced insights into their structural characteristics, localization in the genome, and diversity.

Chicken embryo lethality assay for determining the virulence of Riemerella anatipestifer isolates.

Riemerella anatipestifer is the causative agent of polyserositis and septicaemia in waterfowl. Twenty-one serotypes have been reported, and there is a strong variation in virulence between strains according to serotype or strain. However, little information is available to assess virulence, such as virulence-associated genes; thus, it is difficult to estimate the risk from field strains. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of R. anatipestifer strains. Three virulent strains (RA T1, RA T7, and V-1) and three avirulent strains (Av-1, Av-2, and Av-3), which were confirmed by duck challenge, were used to perform the ELA. Inoculating 10(2) to 10(4) colony-forming units into the allantoic cavity of 10-day-old embryos discriminated between virulent and avirulent strains based on mortality. Differences in invasion rates into embryonic tissues were found between the RA T1 and Av-1 strains. The maximum colony-forming units of the RA T1 strain were about 1000 times higher than those of the Av-1 strain in the tissue invasion rate for 4 days. We found that the virulent strains killed embryos at mortality rates ≥ 50% during the first 3 days after inoculation and that the avirulent strains had death rates of ≤ 20% over 5 days. These results obtained by repeated testing suggest that the ELA could be used as a first-line screening method to determine the virulence of R. anatipestifer strains.

Alpha (1,2)-fucosyltransferase M307A polymorphism improves piglet survival.

To confirm the beneficial effects of alpha (1,2)-fucosyltransferase (FUT1) M307 (A) on piglet survival on commercial farms, we performed PCR-RFLP analysis of FUT1 M307 in successfully marketed (n = 245) and disease affected/deceased pigs during weaning (n = 252) at a commercial farm. We also evaluated the FUT1 genotypes of 190 healthy pigs from three different genetic backgrounds. The distribution of genotypes differed between the successfully marketed and disease affected/deceased pig groups. The frequency of the A allele, associated with resistance to edema and post-weaning diarrhea, was higher in the post-weaning survival group (0.21) than in the non-survival group (0.16, P < 0.05). The odds ratio for piglet survival between AA and GG genotypes was 1.98; thus, piglet survival for individuals with the AA genotype was almost two-fold greater than for GG individuals. The FUT1 gene polymorphism can be used as an effective marker for selection programs to improve post-weaning piglet survival.

Immunomodulatory effect of a proanthocyanidin-rich extract from Pinus radiata bark by dosing period in chickens.

We evaluated the immunomodulatory effects of a proanthocyanidin-rich extract (PAE) from Pinus radiata bark in Korean native chickens. In experiment 1, proliferation of peripheral blood mononuclear cells was significantly enhanced in chickens administered 1.25, 2.5, 5, and 10 mg/kg of PAE for 2 wk once daily by oral gavage compared with no administration of PAE in vitro. Proliferation of splenocyte and thymocyte cells was also significantly enhanced in chickens administered 1.25, 2.5, 5, and 10 mg/kg of PAE for 5 wk. In experiment 2, proliferation of peripheral blood mononuclear cells, splenocytes, and thymocytes in chickens administered PAE for 6 wk was continuously higher than that in other groups. Additionally, increasing cell populations for which CD4(+)CD8(-)(Th cells) and Bu-1(+) (B cells) protein was expressed on the cell surface were significantly different after 4 wk of PAE administration in chickens. Thus, the immunomodulatory effects of a PAE from P. radiata bark differed by dosing period in chickens.

Comparative Studies of Antimicrobial Resistance in Escherichia coli, Salmonella, and Campylobacter Isolates from Broiler Chickens with and without Use of Enrofloxacin.

This study investigated the effect of enrofloxacin (ENR) administration on the prevalence and antimicrobial resistance of E. coli, Salmonella, and Campylobacter isolated from broiler chickens under field conditions. The isolation rate of Salmonella was significantly lower (p < 0.05) on farms that administered ENR (6.4%) than on farms that did not (11.6%). The Campylobacter isolation rate was significantly higher (p < 0.05) in farms that administered ENR (6.7%) than in farms that did not (3.3%). The ratio of resistance to ENR was significantly higher (p < 0.05) in E. coli isolates from farms that used ENR (88.1%) than farms that did not (78.0%). The respective ratio of resistance to ampicillin (40.5% vs. 17.9%), chloramphenicol (38.0% vs. 12.5%), tetracycline (63.3% vs. 23.2%), and trimethoprim/sulfamethoxazole (48.1% vs. 28.6%) and the ratio of intermediate resistance to ENR (67.1% vs. 48.2%) were significantly higher (p < 0.05) in Salmonella isolates from the farms that used ENR than farms that did not. In conclusion, the use of ENR at broiler farms was an important factor in decreasing the prevalence of Salmonella but not Campylobacter and caused ENR resistance among E. coli and Salmonella but not Campylobacter. Exposure to ENR could have a co-selective effect on antimicrobial resistance in enteric bacteria in the field.

Isolation and Genomic Characterization of Avian Reovirus From Wild Birds in South Korea.

Avian reoviruses (ARVs) cause severe arthritis, tenosynovitis, pericarditis, and depressed growth in chickens, and these conditions have become increasingly frequent in recent years. Studies on the role of wild birds in the epidemiology of ARVs are insufficient. This study provides information about currently circulating ARVs in wild birds by gene detection using diagnostic RT-PCR, virus isolation, and genomic characterization. In this study, we isolated and identified 10 ARV isolates from 7,390 wild birds' fecal samples, including migratory bird species (bean goose, Eurasian teal, Indian spot-billed duck, and mallard duck) from 2015 to 2019 in South Korea. On comparing the amino acid sequences of the σC-encoding gene, most isolates, except A18-13, shared higher sequence similarity with the commercial vaccine isolate S1133 and Chinese isolates. However, the A18-13 isolate is similar to live attenuated vaccine av-S1133 and vaccine break isolates (SD09-1, LN09-1, and GX110116). For the p10- and p17-encoding genes, all isolates have identical fusion associated small transmembrane (FAST) protein and nuclear localization signal (SNL) motif to chicken-origin ARVs. Phylogenetic analysis of the amino acid sequences of the σC-encoding gene revealed that all isolates were belonged to genotypic cluster I. For the p10- and p17-encoding genes, the nucleotide sequences of all isolates indicated close relationship with commercial vaccine isolate S1133 and Chinese isolates. For the σNS-encoding gene, the nucleotide sequences of all isolates indicated close relationship with the Californian chicken-origin isolate K1600657 and belonged to chicken-origin ARV cluster. Our data indicates that wild birds ARVs were derived from the chicken farms. This finding suggests that wild birds serve as natural carriers of such viruses for domestic poultry.

Avian Reoviruses From Wild Birds Exhibit Pathogenicity to Specific Pathogen Free Chickens by Footpad Route.

Avian reoviruses (ARVs) are ubiquitous in domestic poultry with 80% of them being non-pathogenic and they are frequently found in clinically healthy birds. ARVs have also been known to be the etiological agents of viral arthritis (VA), tenosynovitis, myocarditis, runting-stunting syndrome (RSS), and respiratory and enteric disease in chickens. Significant economic losses during the process of poultry husbandry are due, in part, to unmitigated ARV infections throughout the poultry industry. Recently, many isolates shared genetic similarities between those recovered from wild birds and those recovered from poultry. One explanation may be that there is a degree of spillover and spillback of ARVs between the two groups. However, studies on the role of wild birds in the epidemiology and pathogenicity of ARVs are insufficient. Here, we describe the pathogenicity in specific pathogen-free (SPF) chickens of ARV originating from wild birds. The challenge experiment was conducted in six groups including a negative control group, a positive control group (reference strain of S1133), and four groups (A15-157, A18-13, A18-205, A19-106) infected with ARVs from wild birds. The 7-day-old SPF chickens were inoculated with 106TCID50 ARV to evaluate the clinical signs, changes in weight gain, gross lesions, histological changes, virus replication, and serum antibody levels. The peak of clinical signs was from 3 to 5 days post infection (dpi). In addition, the death of one chicken was found in the group infected with the A18-13 isolate. Reduced body weight was also found in chickens infected with ARVs from wild birds compared to the negative control group. All the ARVs infection groups showed noticeable swelling of the footpad. In addition, ARVs were detected in the bursa, tendon, and hock joint by reverse transcription-polymerase chain reaction (RT-PCR) in all infected groups at 5 and 15 dpi. Histopathological observations revealed acute inflammatory responses on the synovium covering the joint surfaces (arthritis) and tendon sheaths (tenosynovitis), as well as bursa atrophy and lymphocyte depletion. The analysis of the humoral response was performed by ELISA assay, and chickens infected with ARVs showed seroconverted. In conclusion, this study described the typical severe disease of acute VA and tenosynovitis in SPF chickens infected with ARVs derived from wild birds. This study confirmed the pathogenicity of ARVs infection in SPF chickens for the first time, and these results enrich our understanding of the pathogenicity of ARVs derived from wild birds.

Conjugative Plasmid-Mediated Extended Spectrum Cephalosporin Resistance in Genetically Diverse Escherichia coli from a Chicken Slaughterhouse.

ESC-resistant E. coli isolates were collected from broiler chickens, a slaughterhouse, and retail meat to assess their dispersion and their involvement in cross-contamination. ESBL-/AmpC-producing E. coli were isolated during the slaughter process of all six investigated chicken flocks from scalding, feather removal, first conveyor, evisceration, second washing, third conveyor, and third washing areas, and from handling workers in the slaughterhouse. ESC-resistant E. coli isolates with the same pulsed-field gel electrophoresis type were found in the same site (scalding) on different sampling days. ESBL/AmpC-producing E. coli isolates were absent in the lairage area in the slaughterhouse, but present in the retail markets in 36.8% (7/19) of the chicken flocks. The blaCTX-M genes and blaCMY-2 were conjugated to recipient E. coli J53 in 67.5% (27/40) and 56.1% (23/41) of ESBL-producing and AmpC-producing E. coli isolates, respectively. The presence of the same conjugative plasmids was found in genetic diversity ESC-resistant E. coli colonies collected on different sampling days. Our study emphasizes that cross-contamination of ESBL/AmpC-producing E. coli in slaughterhouse has a crucial impact on the occurrence of ESC resistance in retail chicken meat.

Clonal dissemination of Salmonella enterica serovar albany with concurrent resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, and nalidixic acid in broiler chicken in Korea.

The aim of this study was to determine the prevalence, serovar distribution, antimicrobial resistance, and genotypic analyses of the dominating serovars of Salmonella in chickens from a national study in Korea. Between 2017 and 2018, a total of 550 chicken samples were collected from the top 12 integrated broiler chicken operations in Korea. Salmonella was isolated from 117 (32.5%) chicken feces and 19 (10.0%) retail chicken meat sources. Ten serovars were identified, and the most common Salmonella serovar was Salmonella ser. Albany (50 isolates, 36.8%), followed by S. Enteritidis (38 isolates, 27.9%), and S. Montevideo (23 isolates, 16.9%) isolated from 6, 10, and 6 operations, respectively. A total of 35 (25.7%) isolates were with the ACSSuTN (ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, and nalidixic acid) resistance pattern, with high prevalence of this resistance pattern in S. Albany (29 isolates, 58.0%). A total of 35 PFGE types were identified among Salmonella isolates of the serovars Albany, Enteritidis, Virchow, Montevideo, and Senftenberg, while 11 distinct types of PFGE patterns were found among S. Albany isolates, which showed an overall homology similarity of higher than 85%. Among these 35 PFGE types, 22 PFGE types corresponded to 32 isolates from samples limited to one operation, and the other 13 PFGE types corresponded to 72 isolates from samples widely distributed among different operations. These results highlighted rapid colony dissemination of multidrug-resistant S. Albany in chicken all over Korea after it first appeared in 2016; furthermore, the spread of Salmonella colonies between various integrated operations was common, and several operations played an important role in Salmonella carriage and transmission in Korea.

Genetic diversity of extended-spectrum cephalosporin resistance in Salmonella enterica and E. coli isolates in a single broiler chicken.

Extended-spectrum cephalosporin (ESC) resistance investigated in Salmonella and E. coli from the same chicken was to improve the understanding of the inter-species transmission of ESC resistance determinants in Salmonella and E. coli from a single chicken individual. Fifteen (13.6%) farms and 44 (8.0%) chicken individuals were positive for ESC-resistant E. coli and/or Salmonella, 8 farms (7.3%) and 12 (2.2%) individuals were simultaneously positive for ESC-resistant E. coli and Salmonella. The genetic diversity of ESC resistance determinants in E. coli and Salmonella was observed. Most E. coli isolates (67.6%) produced CTX-M-type of blaCTX-M-55, and 9 isolates (24.3%) produced CMY-type of blaCMY-2. Most Salmonella isolates (94.1%) produced blaCTX-M-15. Two broiler chicken farms were simultaneously positive for blaCMY-2- and blaCTX-M-15-harboring E. coli and Salmonella isolates. Whole-plasmid sequence for the transferable plasmid harboring blaCMY-2 showed genomic diversity of the plasmids from Salmonella and E. coli sourced from the same chicken. The genetic arrangement of blaCMY-2 in Salmonella was IS1294b-ΔISEcp1-blaCMY-2-blc-sugE and ISEcp1-blaCMY-2-blc-sugE in E. coli located on multi-host plasmids of IncI1-pST-2 and IncI1-pST-12. In conclusion, the study illustrates the genetic diversity of ESC resistance determinants in E. coli and Salmonella in a single chicken. Considering the possibility of transmission of antimicrobial resistance to humans through the food chain, a large reservoir of ESC resistance in chicken which could be co-infected with ESC-resistant E. coli and Salmonella poses a serious risk of potential transmission of ESC-resistant E. coli and Salmonella, and their transferable ESC resistant gene, to human simultaneously.

Evaluation of Safety and Protective Efficacy of a waaJ and spiC Double Deletion Korean Epidemic Strain of Salmonella enterica Serovar Gallinarum.

With an aim to develop a highly attenuated and strongly immunogenic distinguishable vaccine candidate, a waaJ (a gene involved in the synthesis of lipopolysaccharide) and spiC (a virulence gene) double deletion Korean epidemic strain of S. enterica ser. Gallinarum (SG005) was constructed. Our results showed that the growth and biochemical characteristics were not altered by this double deletion. The double deletion strain contained dual markers. One was a bacteriological marker (rough phenotype) and the other was a serological marker helping distinguish infected chickens from vaccinated chickens. The double deletion strain showed good genetic stability and reduced resistance to environmental stresses in vitro; furthermore, it was extremely safe and highly avirulent in broilers. Single intramuscular or oral immunization of 7-day-old broilers with the double deletion strain could stimulate the body to produce antibody levels similar to the conventional vaccine strain SG9R. In addition, against a lethal wild-type challenge, it conferred effective protection that was comparable to that seen in the group vaccinated with SG9R. In conclusion, this double deletion strain may be an effective vaccine candidate for controlling S. enterica ser. Gallinarum infection in broilers.

Longitudinal Study of the Distribution of Antimicrobial-Resistant Campylobacter Isolates from an Integrated Broiler Chicken Operation.

The aim of this study was to analyze the prevalence, antimicrobial resistance, and genetic diversity of Campylobacter isolates that were obtained from whole chicken production stages in Korea. A total of 1348 samples were collected from 10 production lines. The prevalence of Campylobacter in breeder farm, broiler farm, slaughterhouse, and retail meat products was 50.0%, 3.3%, 13.4%, and 68.4%, respectively, and Campylobacter was not detected at the hatchery stage. Resistance to quinolones/fluoroquinolones was the most prevalent at all stages. Among the multidrug-resistant isolates, 16 isolates (19.8%) from breeder farm were resistant to both azithromycin and ciprofloxacin. A total of 182 isolates were subdivided into 82 pulsed-field gel electrophoresis (PFGE) genotypes with 100% similarity. Diverse genotypes were presented with discontinuous patterns along the whole production chain. Thirty percent of Campylobacter-free flocks became positive after slaughtering. An identical genotype was simultaneously detected from both breeder farm and retail meat, even from different production lines. This study reveals that antimicrobial-resistant Campylobacter contamination can occur at all stages of the chicken supply chain. In particular, the breeder farm and slaughterhouse should be the main control points, as they are the potential stages at which antimicrobial-resistant Campylobacter could spread to retail meat products by horizontal transmission.