Spray Vaccination with a Newcastle Disease Virus (NDV)-Vectored Infectious Laryngotracheitis (ILT) Vaccine Protects Commercial Chickens from ILT in the Presence of Maternally Derived Antibodies.

Infectious laryngotracheitis (ILT) poses a significant threat to the poultry industry, and vaccines play an important role in protection. However, due to the increasing scale of poultry production, there is an urgent need to develop vaccines that are suitable for convenient immunization methods such as spraying. Previous studies have shown that Newcastle disease virus (NDV)-ILT vaccines administered via intranasal and intraocular routes to commercial chickens carrying maternally derived antibodies (MDAs) are still protective against ILT. In this study, a recombinant NDV (rNDV) was generated to express infectious laryngotracheitis virus (ILTV) glycoprotein B (gB), named rLS-gB, based on a full-length cDNA clone of the LaSota strain. The protective effect of different doses of rLS-gB administered by spray vaccination to commercial chickens at 1 day of age (DOA) was evaluated. The chickens were exposed to 160-µm aerosol particles for 10 min for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs and anti-ILTV MDAs in chickens, the ILTV- and NDV-specific antibody titers were significantly greater in the vaccinated groups than in the unvaccinated group. After challenge with a virulent ILTV strain, no clinical signs were observed in the 107 EID50/ml group compared to the other groups. Furthermore, vaccination with 107 EID50/ml rLS-gB significantly reduced the ILTV viral load and ameliorated gross and microscopic lesions in the trachea of chickens. Overall, these results suggested that rLS-gB is a safe and efficient candidate spray vaccine for ILT and is especially suitable for scaled chicken farms.

BacScan: a novel genome-wide strategy for uncovering broadly immunogenic proteins in bacteria.

In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis, a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance.

The impacts of animal agriculture on One Health-Bacterial zoonosis, antimicrobial resistance, and beyond.

The industrialization of animal agriculture has undoubtedly contributed to the improvement of human well-being by increasing the efficiency of food animal production. At the same time, it has also drastically impacted the natural environment and human society. The One Health initiative emphasizes the interdependency of the health of ecosystems, animals, and humans. In this paper, we discuss some of the most profound consequences of animal agriculture practices from a One Health perspective. More specifically, we focus on impacts to host-microbe interactions by elaborating on how modern animal agriculture affects zoonotic infections, specifically those of bacterial origin, and the concomitant emergence of antimicrobial resistance (AMR). A key question underlying these deeply interconnected issues is how to better prevent, monitor, and manage infections in animal agriculture. To address this, we outline approaches to mitigate the impacts of agricultural bacterial zoonoses and AMR, including the development of novel treatments as well as non-drug approaches comprising integrated surveillance programs and policy and education regarding agricultural practices and antimicrobial stewardship. Finally, we touch upon additional major environmental and health factors impacted by animal agriculture within the One Health context, including animal welfare, food security, food safety, and climate change. Charting how these issues are interwoven to comprise the complex web of animal agriculture's broad impacts on One Health will allow for the development of concerted, multidisciplinary interventions which are truly necessary to tackle these issues from a One Health perspective.

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.

T4 bacteriophage nanoparticles engineered through CRISPR provide a versatile platform for rapid development of flu mucosal vaccines.

Vaccines that trigger mucosal immune responses at the entry portals of pathogens are highly desired. Here, we showed that antigen-decorated nanoparticle generated through CRISPR engineering of T4 bacteriophage can serve as a universal platform for the rapid development of mucosal vaccines. Insertion of Flu viral M2e into phage T4 genome through fusion to Soc (Small Outer Capsid protein) generated a recombinant phage, and the Soc-M2e proteins self-assembled onto phage capsids to form 3M2e-T4 nanoparticles during propagation of T4 in E. coli. Intranasal administration of 3M2e-T4 nanoparticles maintains antigen persistence in the lungs, resulting in increased uptake and presentation by antigen-presenting cells. M2e-specific secretory IgA, effector (TEM), central (TCM), and tissue-resident memory CD4+ T cells (TRM) were efficiently induced in the local mucosal sites, which mediated protections against divergent influenza viruses. Our studies demonstrated the mechanisms of immune protection following 3M2e-T4 nanoparticles vaccination and provide a versatile T4 platform that can be customized to rapidly develop mucosal vaccines against future emerging epidemics.

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.

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.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

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.

Fusion Protein Cleavage Site Containing Three Basic Amino Acids Attenuates Newcastle Disease Virus in Chicken Embryos: Use as an in ovo Vaccine.

In ovo vaccination is an attractive immunization strategy for the poultry industry. However, although most live Newcastle disease virus (NDV) vaccine strains, such as LaSota and V4, can be used after hatching, they are pathogenic to chicken embryos when administered in ovo. We have previously reported that NDV strain TS09-C is a safe in ovo vaccine in specific-pathogen-free and commercial chicken embryos because it is attenuated in chicken embryos. However, the molecular basis of its attenuation is poorly understood. In this study, we firstly evaluated the safety of chimeric NDV strains after exchanging genes between strains TS09-C and LaSota as in ovo vaccines, and demonstrated that the attenuation of NDV in chicken embryos was dependent upon the origin of the fusion (F) protein. Next, by comparing the F protein sequences of TS09-C strain with those of LaSota and V4 strain, the R115 in cleavage site and F379 were found to be unique to TS09-C strain. The mutant viruses were generated by substituting one or two amino acids at position 115 and 379 in the F protein, and their safety as in ovo vaccine was evaluated. Mutation in residue 379 did not affect the viral embryonic pathogenicity. While the mutant virus rTS-2B (R115G mutation based on the backbone of TS09-C strain) with two basic amino acids in F cleavage site, was pathogenic to chicken embryos and similar with rLaSota in its tissue tropism, differing markedly from rTS09-C with three basic amino acids in F cleavage site. Together, these findings indicate that the F protein cleavage site containing three basic amino acids is the crucial determinant of the attenuation of TS09-C in chicken embryos. This study extends our understanding of the pathogenicity of NDV in chicken embryos and should expedite the development of in ovo vaccines against NDV.

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.

Bacteriophage T4 Vaccine Platform for Next-Generation Influenza Vaccine Development.

Developing influenza vaccines that protect against a broad range of viruses is a global health priority. Several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is sufficiently immunogenic to elicit complete protection, and vaccine platforms that can enhance immunogenicity and deliver multiple antigens are desperately needed. Here, we report proof-of-concept studies for the development of next-generation influenza vaccines using the bacteriophage T4 virus-like particle (VLP) platform. Using the extracellular domain of influenza matrix protein 2 (M2e) as a readout, we demonstrate that up to ~1,281 M2e molecules can be assembled on a 120 x 86 nanometer phage capsid to generate M2e-T4 VLPs. These M2e-decorated nanoparticles, without any adjuvant, are highly immunogenic, stimulate robust humoral as well as cellular immune responses, and conferred complete protection against lethal influenza virus challenge. Potentially, additional conserved antigens could be incorporated into the M2e-T4 VLPs and mass-produced in E. coli in a short amount of time to deal with an emerging influenza pandemic.

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.