Identification of a New B-Cell Epitope on the Capsid Protein of Avian Leukosis Virus and Its Application.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can impair immunological function, stunt growth and decrease egg production in avian flocks. The capsid protein (P27) is an attractive candidate for ALV diagnostics. In the present study, a new hybridoma cell (1F8) stably secreting an anti-P27 monoclonal antibody (mAb) was developed. The mAb exhibited a high affinity constant (Ka) of 8.65 × 106.0 L/mol, and it could be used for the detection of ALV-A/B/J/K strains. Moreover, a total of eight truncated recombinant proteins and five synthetic polypeptides were utilized for the identification of the B-cell epitopes present on P27. The results revealed that 218IIKYVLDRQK227 was the minimal epitope recognized by 1F8, which had never been reported before. Additionally, the epitopes could strongly react with different ALV subgroup's specific positive serum and had a complete homology among all the ALV subgroups strains. Finally, a new sandwich ELISA method was created for the detection of ALV antigens, demonstrating increased sensitivity compared to a commercially available ELISA kit. These results offer essential knowledge for further characterizing the antigenic composition of ALV P27 and will facilitate the development of diagnostic reagents for ALV.

A Multiplex Quantitative Polymerase Chain Reaction for the Rapid Differential Detection of Subgroups A, B, J, and K Avian Leukosis Viruses.

Avian leukosis (AL), caused by avian leukosis virus (ALV), is a contagious tumor disease that results in significant economic losses for the poultry industry. Currently, ALV-A, B, J, and K subgroups are the most common in commercial poultry and cause possible coinfections. Therefore, close monitoring is necessary to avoid greater economic losses. In this study, a novel multiplex quantitative polymerase chain reaction (qPCR) assay was developed to detect ALV-A, ALV-B, ALV-J, and ALV-K with limits of detection of 40, 11, 13.7, and 96 copies/µL, respectively, and no cross-reactivity with other ALV subtypes and avian pathogens. We detected 852 cell cultures inoculated with clinical samples using this method, showing good consistency with conventional PCR and ELISA. The most prevalent ALV strain in Hubei Province, China, was still ALV-J (11.74%). Although single infections with ALV-A, ALV-B, and ALV-K were not found, coinfections with different subgroup strains were identified: 0.7% for ALV-A/J, 0.35% for ALV-B/J, 0.25% for ALV-J/K, and 0.12% for ALV-A/B/K and ALV-A/B/J. Therefore, our novel multiplex qPCR may be a useful tool for molecular epidemiology, clinical detection of ALV, and ALV eradication programs.

Activity-guided isolation and identification of antiherpesvirus and antineuroinflammatory active terpenoids from Artemisia vulgaris L. based on the LC-MS/MS molecular network.

Seven undescribed terpenoids, comprising two guaiane-type sesquiterpene lactones (1-2), one eucalyptol-type sesquiterpene (3), one monolactone (4), and three triterpenoids (5-7), along with 35 known analogues, were isolated from the leaves of Artemisia vulgaris L. Their structures and configurations were analysed by extensive spectroscopy. Compounds 1, 2, 8-10, 13, 17, 19, and 28 showed antineuroinflammatory activity, and compounds 1 and 2 revealed remarkable antineuroinflammatory effects, with an IC50 value of 2.2 ± 0.1 and 1.6 ± 0.1 µM, more potent than the positive control drug dexamethasone. Furthermore, compounds 1 and 2 could inhibit the expression of BV-2 inflammatory genes (IL-6, TNF-α, IL-1ß) induced by LPS, downregulate the critical inflammatory protein production of iNOS and COX-2. The anti-HSV-1 activity screening revealed that compounds 28, 29 and 38 exhibited inhibitory activity against HSV-1 proliferation. Particularly, compound 28 exhibited a significant anti-HSV-1 effect, inhibiting the proliferation of HSV-1 and acyclovir-resistant strains of HSV-1/153 and HSV-1/Blue. Our research identified compounds 1, 2, and 28 from A. vulgaris., which could potentially serve as lead compounds for antineuroinflammatory and anti-HSV-1 activities.

Virulent Phage vB_EfaS_WH1 Removes Enterococcus faecalis Biofilm and Inhibits Its Growth on the Surface of Chicken Meat.

Enterococcus faecalis is a potential animal and human pathogen. Improper use of antibiotics encourages resistance. Bacteriophages and their derivatives are promising for treating drug-resistant bacterial infections. In this study, phylogenetic and electron microscopy analyses of phage vB_EfaS_WH1 (WH1) isolated from chicken feces revealed it to be a novel phage in the family Siphoviridae. WH1 showed good pH stability (4-11), temperature tolerance (4-60 °C), and broad E. faecalis host range (60% of isolates). Genome sequencing revealed a 56,357 bp double-stranded DNA genome with a G+C content of 39.21%. WH1 effectively destroyed E. faecalis EF01 biofilms, even at low concentrations. When WH1 was applied at 1 × 105 to 1 × 109 PFU/g to chicken breast samples stored at 4 °C, surface growing E. faecalis were appreciably eradicated after 24 h. The phage WH1 showed good antibacterial activity, which could be used as a potential biocontrol agent to reduce the formation of E. faecalis biofilm, and could also be used as an alternative for the control of E. faecalis in chicken products.

Prevalence of Extended-Spectrum ß-Lactamase-Resistant Genes in Escherichia coli Isolates from Central China during 2016-2019.

The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016-2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/µL, 1.10 × 101 copies/µL, and 1.86 × 101 copies/µL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.

Prevalence of colistin resistance gene mcr-1 in Escherichia coli isolated from chickens in central China, 2014 to 2019.

OBJECTIVES: This study investigated the prevalence and characteristics of mcr-1-harbouring Escherichia coli isolated from chickens in central China from 2014 to 2019. METHODS: A total of 1132 E. coli isolated from 1647 chicken swabs were analysed for colistin susceptibility by broth microdilution method and prevalence of mcr-1 gene by PCR. The colistin-resistant E. coli isolates were typed by multi-locus sequence typing (MLST) and tested with 12 antimicrobial agents. The transconjugation assay was conducted for the mcr-1-positive isolates using the transconjugant E. coli C600. RESULTS: Of the 1132 E. coli isolated from chickens, 131 isolates (11.6%) exhibited colistin resistance, and 51 isolates (4.5%) were mcr-1 positive. The mcr-1-positive rate was quite low in 2014 (2.3%) and 2015 (1.7%), increased to peak in 2016 (12.6%) and 2017 (11.4%), and then decreased significantly in 2018 (1.7%) and 2019 (0.9%). The 131 colistin resistant isolates were assigned to 66 unique sequence types (STs), 27 of which contained mcr-1-positive isolates. Compared with mcr-1-negative E. coli, mcr-1-positive E. coli showed higher resistance rates to nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, and tetracycline. Furthermore, 30 of the 51 mcr-1 positive isolates transduced their mcr-1 gene into E. coli C600, and 13 of the 30 transconjugants carried more than one replicon types. CONCLUSION: The mcr-1 positive rate varied enormously during 2014-2019 in central China. The ban on colistin likely decreased the dissemination of mcr-1 in E. coli isolates from chickens. Multidrug-resistant trait is observed in mcr-1 positive E. coli isolates and can be transferred into other transconjugants.

Host innate immune responses of geese infected with goose origin nephrotic astrovirus.

A novel goose astrovirus (GoAstV) outbreak in goslings, characterized by severe articular and visceral gout with high mortality, occurred in China. Although the pathogenesis of GoAstV-infected goslings has been explored in several studies, the host-immune response remains unclear. In this study, a goose astrovirus was isolated from goslings in Xiaogan, and designated as the HBXG strain. The full-length genome of HBXG was 7170 nt. A sequence analysis and phylogenetic trees revealed HBXG belonged to the novel GoAstV. We evaluated the viral distribution systematically and estimated immune related gene expression in HBXG-infected goslings. Results showed that GoAstV replicated quickly in many tissues and the highest titer was observed in the kidney, which reached 109.6 copies. TLR3, RIG-I and MDA5 were involved in the host-immune response to GoAstV, and the expression of IFN types I (IFN-α, IFN-ß), inflammatory cytokines (IL-8, IL-10, TNF-α), antiviral proteins (Mx, OASL, PKR) and MHC-I were also upregulated during the infection. In contrast, the expression of proinflammatory cytokines (IL-1ß, IL-6) and MHC-II were inhibited at 3 dpi. This study suggests that GoAstV is highly pathogenic to goslings, causing multiple systemic infections in tissues and the host-immune response is activated early in infection. However, rapid viral replication, suppression of inflammatory cytokines (IL-1ß, IL-6) and MHC-II expressions were the possible reasons why the host-immune response cannot provide enough protection against GoAstV infection. This study is the first report to illuminate the immune response in goslings infected with GoAstV and offers insight into the pathogenesis of GoAstV.

Role of aspartate ammonia-lyase in Pasteurella multocida.

BACKGROUND: Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. RESULT: Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. CONCLUSION: These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Molecular cloning and functional characterization of duck DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X).

DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X) is demonstrated to have crucial functions in the antiviral immune response. To our knowledge, little information focuses on the function of duck DDX3X. In this study, duck DDX3X (duDDX3X) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated using duck embryo fibroblast (DEF) cells. Full-length duDDX3X cDNA encodes 652 amino acid residues and contains a DEADc domain and a HELICc domain. According to tissue distribution analysis, duDDX3X mRNA was widely expressed in different tissues, especially the spleen and the liver. Overexpression of duDDX3X in DEF cells induced IFN-ß by activating transcription factors IRF1 and NF-κB. Knockdown of duDDX3X in DEF cells with siRNA significantly reduced IFN-ß expression induced by poly(I:C), a double-stranded RNA (dsRNA) analog. Our results demonstrated that duck DDX3X was involved in the dsRNA-mediated type I IFN signaling pathway in DEF cells.