Virus-Like Particles Based on the Novel Goose Parvovirus (NGPV) VP2 Protein Protect Ducks against NGPV Challenge.

Novel goose parvovirus (NGPV), a genetic variant of goose parvovirus, has been spreading throughout China since 2015 and mainly infects ducklings with the symptoms of growth retardation, beak atrophy, and protruding tongue, leading to huge economic losses every year. A safe and effective vaccine is urgently needed to control NGPV infection. In this study, virus-like particles (VLPs) of NPGV were assembled and evaluated for their immunogenicity. The VP2 protein of NGPV was expressed in Spodoptera frugiperda insect cells using baculovirus as vector. The VP2 protein was efficiently expressed in the nucleus of insect cells, and the particles with a circular or hexagonal shape and a diameter of approximately 30 nm, similar to the NGPV virion, were observed using transmission electron microscopy (TEM). The purified particles were confirmed to be composed of VP2 using western blot and TEM, indicating that the VLPs of NGPV were successfully assembled. Furthermore, the immunogenicity of the VLPs of NGPV was evaluated in Cherry Valley ducks. The level of NGPV serum antibodies increased significantly at 1-4 weeks post-immunization. No clinical symptoms or deaths of ducks occurred in all groups after being challenged with NGPV at 4 weeks post-immunization. There was no viral shedding in the immunized group. However, viral shedding was detected at 3-7 days post-challenge in the non-immunized group. Moreover, VLPs can protect ducks from histopathological lesions caused by NGPV and significantly reduce viral load in tissue at 5 days post-challenge. Based on these findings, NGPV VLPs are promising candidates for vaccines against NGPV.

Genetic characteristics, antimicrobial susceptibility, and virulence genes distribution of Campylobacter isolated from local dual-purpose chickens in central China.

Food-borne antibiotic-resistant Campylobacter poses a serious threat to public health. To understand the prevalence and genetic characteristics of Campylobacter in Chinese local dual-purpose (meat and eggs) chickens, the genomes of 30 Campylobacter isolates, including 13 C. jejuni and 17 C. coli from Jianghan-chickens in central China, were sequenced and tested for antibiotic susceptibility. The results showed that CC-354 and CC-828 were the dominant clonal complexes of C. jejuni and C. coli, respectively, and a phylogenetic analysis showed that three unclassified multilocus sequence types of C. coli were more closely genetically related to C. jejuni than to other C. coli in this study. Of the six antibiotics tested, the highest resistance rates were to ciprofloxacin and tetracycline (100%), followed by lincomycin (63.3%), erythromycin (30.0%), amikacin (26.7%), and cefotaxime (20.0%). The antibiotic resistance rate of C. coli was higher than that of C. jejuni. The GyrA T86I mutation and 15 acquired resistance genes were detected with whole-genome sequencing (WGS). Among those, the GyrA T86I mutation and tet(O) were most prevalent (both 96.7%), followed by the blaOXA-type gene (90.0%), ant(6)-Ia (26.7%), aac(6')-aph(3'') (23.3%), erm(B) (13.3%), and other genes (3.3%). The ciprofloxacin and tetracycline resistance phenotypes correlated strongly with the GyrA T86I mutation and tet(O)/tet(L), respectively, but for other antibiotics, the correlation between genes and resistance phenotypes were weak, indicating that there may be resistance mechanisms other than the resistance genes detected in this study. Virulence gene analysis showed that several genes related to adhesion, colonization, and invasion (including cadF, porA, ciaB, and jlpA) and cytolethal distending toxin (cdtABC) were only present in C. jejuni. Overall, this study extends our knowledge of the epidemiology and antibiotic resistance of Campylobacter in local Chinese dual-purpose chickens.

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.

Antibacterial activity mechanism of coptisine against Pasteurella multocida.

Objective: Pasteurella multocida is a widespread zoonotic pathogen that causes severe damage to the poultry industry. This study focused on the antibacterial effects and mechanism of action of coptisine against P. multocida. Methods: The minimum inhibitory concentration and half maximal inhibitory concentration of coptisine against P. multocida was measured. Additionally, the effect of coptisine on growth, cell wall, activity of respiratory enzymes, soluble protein content and DNA synthesis were also analyzed. Finally, the effect of coptisine on gene transcription was determined using RNA sequencing. Results: We demonstrated that coptisine has a strong antibacterial effect against P. multocida, with a minimum inhibitory concentration of 0.125 mg/mL. Moreover, the measurement of the half maximal inhibitory concentration confirmed that coptisine was safe for the pathogen. The growth curve showed that coptisine inhibited bacterial growth. Measurement of alkaline phosphatase activity in the culture solution showed that coptisine affected cell wall permeability. Transmission electron microscopy revealed that coptisine chloride destroyed the cell structure. In addition, coptisine blocked the respiratory system, as measured by the levels of critical enzymes of the tricarboxylic acid cycle and glycolysis, succinate dehydrogenase and lactate dehydrogenase, respectively. Similarly, coptisine inhibited the synthesis of soluble proteins and genomic DNA. The KEGG pathway analysis of the differentially expressed genes showed that they were associated with cellular, respiratory, and amino acid metabolism, which were downregulated after coptisine treatment. Additionally, genes related to RNA degradation and the aminoacyl-tRNA pathway were upregulated. Conclusion: In this study, we demonstrated that coptisine exerts an antibacterial effect on P. multocida. These findings suggest that coptisine has a multifaceted impact on various pathways, resulting in the inhibition of P. multocida. Thus, coptisine is a potential alternative to antibiotics for the treatment of P. multocida infections in a clinical setting.

C-terminal truncation of the hemagglutinin-neuraminidase (HN) protein enhances the virulence and immunogenicity of Newcastle disease virus (NDV) vaccine strain V4.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein with receptor recognition ability that plays an important role in the infection of cells by NDV. An alignment of NDV HN protein sequences of different genotypes showed that vaccine strains of NDV, such as the LaSota strain, generally have an HN protein of 577 amino acids. In comparison, the HN protein of the V4 strain has 616 amino acids, with 39 more amino acids at the C-terminus. In this study, we generated a recombinant NDV (rNDV) with a 39-amino-acid truncation at the HN C-terminus based on the full-length cDNA clone of the V4 strain. This rNDV, named rV4-HN-tr, displayed thermostability similar to that of the parental V4 strain. However, growth kinetics and pathogenicity analysis suggested that rV4-HN-tr is more virulent than the V4 strain. Notably, the C-terminus of HN affected the ability of the virus to adsorb onto cells. Structural predictions further suggested that the C-terminus of HN may obstruct the sialic acid binding site. Immunization of chickens with rV4-HN-tr induced a 3.5-fold higher level of NDV-specific antibodies than that obtained with the V4 strain and provided 100% immune protection against NDV challenge. Our study suggests that rV4-HN-tr is a thermostable, safe, and highly efficient vaccine candidate against Newcastle disease.

Flos populi (Male Inflorescence of Populus tomentosa Carrière) Aqueous Extract Suppresses Salmonella Pullorum Infection by Affecting T3SS-1.

Pullorum disease, caused by Salmonella Pullorum (S. Pullorum), is one of the most serious infectious diseases in the poultry industry. Flos populi is traditionally used in Eastern Asian countries to treat various intestinal diseases. However, the anti-infection mechanism of Flos populi is not very clear. In this study, we evaluated the anti-infective effects on S. Pullorum of Flos populi aqueous extract (FPAE) in chickens. FPAE significantly reduced S. Pullorum growth in vitro. At the cellular level, FPAE reduced S. Pullorum adhesion and invasion on DF-1 cells but did not affect its intracellular survival or replication in macrophages. Further investigation revealed that FPAE inhibited the transcription of T3SS-1 genes, which is the main virulence factor that mediates S. Pullorum adhesion and invasion in host cells. The results suggest that the anti-infective effect of FPAE likely occurs through the inhibition of S. Pullorum T3SS-1, thereby impairing its ability to adhere to and invade cells. Further, we evaluated its therapeutic effect on animal models (Jianghan domestic chickens) and found that FPAE reduced the bacterial loads in organs and decreased the mortality and weight loss of infected chickens. Our findings provide novel insights into the potential development of FPAE against S. Pullorum as an effective anti-virulence therapeutic substitute for antibiotics.

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.

Investigation and development of transient production process for porcine circovirus Type-2 (PCV2) capsid protein in HEK293F cells.

Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model "difficult-to-express" virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90ß, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other "difficult-to-express" virus capsid proteins in the mammalian cell system.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Characterization of a Recombinant Thermostable Newcastle Disease Virus (NDV) Expressing Glycoprotein gB of Infectious Laryngotracheitis Virus (ILTV) Protects Chickens against ILTV Challenge.

Infectious laryngotracheitis (ILT) and Newcastle disease (ND) are two important avian diseases that have caused huge economic losses to the poultry industry worldwide. Newcastle disease virus (NDV) has been used as a vector in the development of vaccines and gene delivery. In the present study, we generated a thermostable recombinant NDV (rNDV) expressing the glycoprotein gB (gB) of infectious laryngotracheitis virus (ITLV) based on the full-length cDNA clone of the thermostable TS09-C strain. This thermostable rNDV, named rTS-gB, displayed similar thermostability, growth kinetics, and pathogenicity compared with the parental TS09-C virus. The immunization data showed that rTS-gB induced effective ILTV- and NDV-specific antibody responses and conferred immunization protection against ILTV challenge in chickens. The efficacy of rTS-gB in alleviating clinical signs was similar to that of the commercial attenuated ILTV K317 strain. Furthermore, rTS-gB could significantly reduce viral shedding in cloacal and tracheal samples. Our study suggested that the rNDV strain rTS-gB is a thermostable, safe, and highly efficient vaccine candidate against ILT and ND.

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.

Quantitative regulation of the thermal stability of enveloped virus vaccines by surface charge engineering to prevent the self-aggregation of attachment glycoproteins.

The development of thermostable vaccines can relieve the bottleneck of existing vaccines caused by thermal instability and subsequent poor efficacy, which is one of the predominant reasons for the millions of deaths caused by vaccine-preventable diseases. Research into the mechanism of viral thermostability may provide strategies for developing thermostable vaccines. Using Newcastle disease virus (NDV) as model, we identified the negative surface charge of attachment glycoprotein as a novel determinant of viral thermostability. It prevented the temperature-induced aggregation of glycoprotein and subsequent detachment from virion surface. Then structural stability of virion surface was improved and virus could bind to and infect cells efficiently after heat-treatment. Employing the approach of surface charge engineering, thermal stability of NDV and influenza A virus (IAV) vaccines was successfully improved. The increase in the level of vaccine thermal stability was determined by the value-added in the negative surface charge of the attachment glycoprotein. The engineered live and inactivated vaccines could be used efficiently after storage at 37°C for at least 10 and 60 days, respectively. Thus, our results revealed a novel surface-charge-mediated link between HN protein and NDV thermostability, which could be used to design thermal stable NDV and IAV vaccines rationally.

Droplet Digital PCR-Based Detection and Quantification of GyrA Thr-86-Ile Mutation Based Fluoroquinolone-Resistant Campylobacter jejuni.

Fluoroquinolone (FQ)-resistant Campylobacter jejuni is a serious problem worldwide that limits effective treatment of infections. The traditional detection method depends on bacterial isolation and MIC testing, or traditional PCR, which is time-consuming and hard to identify the FQ-resistant C. jejuni in a high abundance wild-type background. This study aimed to develop a rapid and accurate ddPCR assay to detect FQ-resistant C. jejuni mutants based on the crucial resistance mutation C257T (Thr-86-Ile) in gyrA. Our ddPCR gyrA assay showed high specificity and accuracy. Sanger sequencing and the qPCR assay could only recognize gyrA mutant sequences when the ratios of wild-type/mutant were 1:1 or 10:1, respectively. Our ddPCR gyrA assay was able to detect gyrA mutant sequences in the mixtures with up to at least 1000:1 wild-type/mutant ratios, which suggested a significant advantage to distinguish the low mutant signal from the wild-type background. We further monitored the occurrence of gyrA mutations under ciprofloxacin pressure using our ddPCR gyrA assay, and clearly showed that the transition of a dominant C. jejuni subpopulation from wild-type to gyrA C257T mutant, resulting in FQ-resistance. We tested 52 samples from live chickens and retail chicken meat and showed that four samples contained wild-type/mutant mixtures comprising 1.7%, 28.6%, 53.3%, and 87.0% gyrA C257T mutants, respectively. These results demonstrated that the ddPCR gyrA assay was a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that could help guide the appropriate use of FQs in clinical practice. IMPORTANCE Campylobacter jejuni is considered to be the leading cause of human bacterial gastroenteritis worldwide, and fluoroquinolones (FQs) are the main choices for the treatment of bacterial gastroenteritis in clinical practice. In theory, antimicrobial susceptibility testing should help us to choose the most appropriate drugs for the treatment. However, to test the susceptibility of C. jejuni to FQs, the standardized method is bacteria isolation and MIC measurement, which will take more than 4 days. In addition, a low abundance of FQ-resistant C. jejuni is also hardly distinguished from a high abundance of wild-type background in the mixed infection. Therefore, the development of rapid and accurate detection technology for FQ-resistant C. jejuni is very important. This study provided a ddPCR gyrA assay, which is a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that may help guide the appropriate use of FQs both in veterinary and human clinical practice.

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.

Genetic diversity and multidrug resistance of phylogenic groups B2 and D in InPEC and ExPEC isolated from chickens in Central China.

BACKGROUND: Avian colibacillosis is an infectious bacterial disease caused by avian pathogenic Escherichia coli (APEC). APEC causes a wide variety of intestinal and extraintestinal infections, including InPEC and ExPEC, which result in enormous losses in the poultry industry. In this study, we investigated the prevalence of InPEC and ExPEC in Central China, and the isolates were characterized using molecular approaches and tested for virulence factors and antibiotic resistance. RESULTS: A total of 200 chicken-derived E. coli isolates were collected for study from 2019 and 2020. The prevalence of B2 and D phylogenic groups in the 200 chicken-derived E. coli was verified by triplex PCR, which accounted for 50.53% (48/95) and 9.52% (10/105) in ExPEC and InPEC, respectively. Additionally, multilocus sequence typing method was used to examine the genetic diversity of these E. coli isolates, which showed that the dominant STs of ExPEC included ST117 (n = 10, 20.83%), ST297 (n = 5, 10.42%), ST93 (n = 4, 8.33%), ST1426 (n = 4, 8.33%) and ST10 (n = 3, 6.25%), while the dominant ST of InPEC was ST117 (n = 2, 20%). Furthermore, antimicrobial susceptibility tests of 16 antibiotics for those strains were conducted. The result showed that more than 60% of the ExPEC and InPEC were resistant to streptomycin and nalidixic acid. Among these streptomycin resistant isolates (n = 49), 99.76% harbored aminoglycoside resistance gene strA, and 63.27% harbored strB. Among these nalidixic acid resistant isolates (n = 38), 94.74% harbored a S83L mutation in gyrA, and 44.74% harbored a D87N mutation in gyrA. Moreover, the prevalence of multidrug-resistant (MDR) in the isolates of ExPEC and InPEC was 31.25% (15/48) and 20% (2/10), respectively. Alarmingly, 8.33% (4/48) of the ExPEC and 20% (2/10) of the InPEC were extensively drug-resistant (XDR). Finally, the presence of 13 virulence-associated genes was checked in these isolates, which over 95% of the ExPEC and InPEC strains harbored irp2, feoB, fimH, ompT, ompA. 10.42% of the ExPEC and 10% of the InPEC were positive for kpsM. Only ExPEC isolates carried ibeA gene, and the rate was 4.17%. All tested strains were negative to LT and cnf genes. The carrying rate of iss and iutA were significantly different between the InPEC and ExPEC isolates (P < 0.01). CONCLUSIONS: To the best of our knowledge, this is the first report on the highly pathogenic groups of InPEC and ExPEC in Central China. We find that 50.53% (48/95) of the ExPEC belong to the D/B2 phylogenic group. The emergence of XDR and MDR strains and potential virulence genes may indicate the complicated treatment of the infections caused by APEC. This study will improve our understanding of the prevalence and pathogenicity of APEC.

Evaluation of a Virus-like Nanoparticle Porcine Circovirus Type-2 (PCV2) Capsid Protein Fused with the Pig Immunoglobulin Fc Fragment as a Novel Vaccine Candidate against PCV2 in Mice.

Porcine circovirus Type 2 (PCV2) is a primary etiological pathogen of post-weaning multi-systemic wasting syndrome (PMWS). The capsid protein of PCV2 is the crucial immunogenic protein which can induce antibody generation and immune responses. However, there is still a lack of efficient PCV2 vaccines with high immunogenicity. In the current study, we developed a novel engineered PCV2 capsid (∆1-41aa)-pFc fusion protein (PCFP), which comprised a truncated capsid protein of PCV2 and a porcine IgG Fc fragment, fused to the capsid protein of PCV2 at the C-terminus. We found that this novel fusion protein could auto-assemble into virus-like nanoparticles with an estimated mean diameter of 22.6 nm, characterized by transmission electron microscopy. Immunization of BALB/c mice with this fusion protein significantly increased the production levels of anti-PCV2-capsid protein antibody in serum. Besides, the virus-like nanoparticles, PCFP was demonstrated to induce efficient cellular immune responses in mice, as evident by the high specific T cell reactivity to the PCFP fusion protein and the high production of the immune cytokines IFN-γ and IL-10 in an ex vivo re-stimulation system. Collectively, these findings demonstrate that the PCV2 truncated capsid subunit Fc-fusion protein can induce both cellular and humoral immune responses, and it displays great application potential.

Molecular characterization and functional analysis of duck CCCH-type zinc finger antiviral protein (ZAP).

This is the first study to clone duck CCCH-type zinc finger antiviral protein (duZAP) from Jingjiang duck (Anas platyrhynchos). Full-length duZAP cDNA was 2154 bp and encoded a 717-amino acid polypeptide containing four highly conserved CCCH-type finger motifs, a WWE domain and a poly (ADP-ribose) polymerase (PARP) domain. duZAP was expressed in multiple duck tissues, with the highest mRNA expression in the spleen. Overexpression of duZAP in duck embryo fibroblast cells (DEFs) led to activation of the transcription factors IRF1 and NF-κB, and induction of IFN-ß. Analysis of deletion mutants revealed that both the WWE and PARP domains of duZAP were essential for activating the IFN-ß promoter. Knockdown of duZAP in DEFs significantly reduced poly (I:C)- and duck Tembusu virus (DTMUV)-induced IFN-ß activation. Our findings further the understanding of the role of duZAP in the duck innate immune response.

(p)ppGpp synthetases are required for the pathogenicity of Salmonella Pullorum in chickens.

Salmonella Pullorum is a pathogen specific to birds that can cause Pullorum disease in young chickens and lead to considerable economic losses in the poultry industry. During transmission and infection, S. Pullorum will encounter various environmental stresses and host defenses. The stringent response is an important adaptation response induced by (p)ppGpp, and in Salmonella, (p)ppGpp is synthesized by two (p)ppGpp synthetases, RelA and SpoT. To investigate the role of (p)ppGpp synthetases in the adaptation and pathogenicity of S. Pullorum, a (p)ppGpp synthetases mutant (ΔrelAΔspoT) was constructed, and its physiological phenotypes and pathogenicity, as well as transcription profiling, were compared with the parent strain. The ΔrelAΔspoT mutant showed decreased ability to form biofilms, and reduced resistance to acidic, alkaline, high osmolarity and H2O2 conditions. The internalization of the ΔrelAΔspoT mutant into host cells in vitro and its lethality and colonization abilities within young chickens were also significantly reduced. RNA sequencing showed that the (p)ppGpp synthetases did not only affect the classic stringent response, such as inhibition of DNA replication and protein synthesis, but also controlled the expression of many virulence factors, in particular, the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs), and adhesion factors. These results suggest that the (p)ppGpp synthetases are required for the pathogenicity of S. Pullorum by affecting its stress response and the expression of the virulence factors.

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.