Diarrhea and impaired intestinal development are major constraints in newly hatched domestic pigeons raised for meat production, leading to reduced growth performance and increased reliance on antimicrobial interventions. Cecropin AD (CAD) is a broad-spectrum antimicrobial peptide with potential activity against bacterial pathogens and may represent an alternative strategy for improving intestinal health in poultry. In this study, we evaluated the safety and biological effects of a recombinant Newcastle disease virus (NDV) expressing CAD (rTS-CAD3) in one-day-old domestic pigeons. Pigeons were inoculated with rTS-CAD3, parental TS09-C, or PBS and monitored for growth performance, diarrhea incidence, viral RNA persistence, inflammatory responses, intestinal bacterial abundance, gut microbiota composition, intestinal morphology, and epithelial barrier function. rTS-CAD3 caused no mortality, detected only transient viral RNA, and did not induce excessive inflammatory responses. Compared with TS09-C and PBS controls, rTS-CAD3 significantly increased body weight gain and average daily gain while reducing diarrhea incidence. Importantly, rTS-CAD3 decreased intestinal Escherichia coli abundance, reshaped the gut microbiota by reducing Proteobacteria and Escherichia-Shigella while increasing Firmicutes and Lactobacillus, improved villus height and the villus-to-crypt ratio, and enhanced occludin expression. These findings demonstrate that NDV-vectored CAD delivery can reduce intestinal bacterial burden, increase occludin expression, and alleviate diarrhea in domestic pigeons. The rTS-CAD3 represents a promising antimicrobial peptide-based strategy for reducing spontaneous diarrhea and dependence on conventional antibiotics in poultry production.

The O-antigen of Salmonella is a key virulence determinant and serotype-defining structure, yet the regulatory mechanisms underlying its dynamic modification and evolutionary trade-offs remain incompletely understood. Salmonella Pullorum is a pathogen that displays both O-antigen unstable phase variation and stable antigenic conversion between standard (O123) and variant (O122) antigenic types. In this study, we identified the glycosyltransferase encoded by gtrC III as responsible for the variant-specific glucosylation of the O-antigen. The expression of gtrC III is co-regulated by the transcriptional repressor OxyR and methyltransferase Dam, generating stochastic ON‒OFF switching of the O-antigen modification, which is a classic phase variation and promotes immune evasion. Remarkably, a single C-to-A point substitution at position -34 of the gtrABC III promoter disrupts OxyR binding and simultaneously enhances intrinsic promoter activity, resulting in constitutive expression of the variant antigenic type. This monophasic variant strain exhibits enhanced resistance to standard-type antibody-mediated killing, conferring a short-term immune evasion advantage. However, in long-term colonization, it is outcompeted by the phase-variable standard type strain, exemplifying "short-sighted evolution", where an adaptive mutation trades off persistence for transient immune escape. Our findings reveal how a single nucleotide change can subvert a complex epigenetic switch to drive sub-serotype conversion, and provide insights into the evolutionary constraints shaping O-antigen diversity.

Superoxide dismutase (SOD) is an important component of the bacterial antioxidant system. In Salmonella Pullorum, an avian specialist serovar of Salmonella, four SODs are encoded, of which sod1 and sod3 belong to the Cu/ZnSOD family, and sod2 and sod4 belong to the Fe/MnSOD family. However, their respective potential functions in stress responses have remained unclear. In this study, we found that a (p)ppGpp synthetase mutant of S. Pullorum exhibited reduced antibiotic tolerance and SOD activity. To further understand the roles of SODs in antibiotic tolerance and virulence, four sod single mutants (Δsod1, Δsod2, Δsod3, and Δsod4) and two sod double mutants (Δsod1Δsod3 and Δsod2Δsod4) were constructed. We found that all four SODs contributed to total SOD activity and superoxide tolerance of S. Pullorum. However, this contribution was not uniform, as the Δsod2Δsod4 mutant showed the lowest survival rates under paraquat treatment, and the highest reactive oxygen species accumulation and lowest survival rates under antibiotic treatment, suggesting the key roles of sod2 and sod4 in degrading ROS in these processes. We also demonstrated that Δsod2Δsod4 was more easily eliminated in a chicken macrophage cell, as well as in the spleen and liver of infected chicken hosts, in comparison with the wild-type strain and other sod mutants. The Δsod2Δsod4 mutant was also the only strain which was completely nonlethal to one-day-old chickens. Taken together, our results indicate that, of the four sod genes, the Fe/MnSOD (sod2 and sod4) collectively had the most significant impact on antibiotic tolerance and pathogenicity of S. Pullorum.

Cecropin AD (CAD), a hybrid antimicrobial peptide composed of the first 11 residues of cecropin A and last 26 residues of cecropin D, is a promising antibiotic candidate. Therefore, an efficient and convenient method for producing CAD is necessary for commercial applications. The Newcastle disease virus (NDV) has been widely used as a platform for gene delivery and exogenous protein expression. In this study, we constructed a recombinant NDV that expresses CAD. To obtain high expression of the CAD peptide, tandem repeats of the cad gene were inserted into the genomes of the thermostable NDV strain TS09-C using reverse genetic technology. The thermostable recombinant NDV, namely rTS-CAD3, showed thermostability and growth kinetics similar to those of their parental strain. A bacteriostatic test showed that rTS-CAD3 inhibited Staphylococcus aureus (gram-positive bacteria) and Escherichia coli (gram-negative bacteria) in vitro. We further determined the bacteriostatic effects of rTS-CAD3 expressed CAD against S. aureus in skin wound infections. The results showed that rTS-CAD3 subcutaneously injection improved wound healing and reduced S. aureus decolonization. In summary, our results indicate that the rTS-CAD3 expressing CAD peptide is a potent antimicrobial agent against S. aureus and E. coli and may be applied to accelerate wound healing in farm animals.

Since the 1980s, despite vaccination, the infectious bronchitis virus (IBV) infection rate in commercial broilers and layers in China has continued to rise significantly, causing substantial economic losses to the poultry industry. In this study, an IBV strain was isolated from a layer farm in northeast China and named CK/CH/LN/2302. The whole genome sequence analysis revealed that CK/CH/LN/2302 shared a high level of homology (96.41 %) with the GI-19 strain SC/SDL/19. The phylogenetic tree based on the S1 gene indicates that CK/CH/LN/2302 belongs to the GI-19 lineage. Notably, recombination analysis using RDP5 and SimPlot software suggested that the GI-19 strain and a 4/91-like strain likely contributed to four recombination events in the CK/CH/LN/2302 genome. Phylogenetic analysis of these four regions further supported this conclusion. Protein structure analysis revealed that most of the nonstructural protein 2 (nsp2), main protease (Mpro), S1, and 5a protein regions were replaced by sequences from the 4/91-like strain. After infecting 1-day-old SPF chickens, CK/CH/LN/2302 presented a mortality rate as high as 60 %. Higher viral loads were detected in tissues such as the larynx, trachea, lungs, duodenum, jejunum and kidneys, indicating the multitissue tropism of this strain. Neutralization assay results revealed that the serum from 28-day-old commercial chickens immunized with the H120 vaccine was unable to effectively neutralize CK/CH/LN/2302. Compared with the S1 subunit of H120, CK/CH/LN/2302 demonstrated conformational changes, particularly in the hypervariable regions (HVRs), which may facilitate immune evasion. The genetic characteristics and pathogenicity of CK/CH/LN/2302 highlight the ongoing evolution of GI-19 IBV strains in China, emphasizing the urgent need for appropriate control strategies.

Infectious bronchitis is a severe disease caused by the infectious bronchitis virus (IBV), resulting in significant economic losses to the poultry industry. We isolated an IBV strain from H120-vaccinated layers in Northeast China called CK/CH/LN/2301. Phylogenetic analysis of the S1 gene classified the isolate within the GVI-1 genotype. Further analysis revealed that CK/CH/LN/2301 is a recombinant strain derived from the GVI-1, GI-19, and 4/91 genotypes, with recombination events primarily located in the open reading frame (ORF) 1a region. Experimental infection of 1-day-old specific pathogen-free (SPF) chickens induced clinical signs, including depression and head shaking, with 10% mortality rate. Necropsy revealed characteristic tracheal hemorrhages, and quantitative viral load analysis demonstrated significantly higher viral titers in tracheal tissues than in other organs. Histopathological examination confirmed lesions in the trachea, lungs, kidneys, and duodenum. The neutralization assay showed that CK/CH/LN/2301 exhibited limited cross-neutralization with existing vaccine strains such as H120. Predicted structural comparison of the S protein revealed substantial conformational differences in the hypervariable regions (HVRs) and the S1 subunit between CK/CH/LN/2301 and the vaccine strains, which may contribute to the reduced level of cross-protection provided by current vaccines. Our findings demonstrate that GVI-1 IBV strains are undergoing evolution, highlighting the urgent need for updated vaccine strategies to control emerging variants.

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.

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 d 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 titres 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.

Pigeon has become popular as a food recently, due to their highly nutritive value. Colibacillosis, which is caused by pathogenic E. coli, is one of the most important bacterial diseases in pigeon breeding. However, data on E. coli isolates from pigeons are currently limited. In order to understand the prevalence and genetic characteristics of pathogenic E. coli in pigeon farms, a total of 199 E. coli strains were isolated from domestic pigeons in central China. Among them, 30.15% isolates (60/199) were identified to belong to phylogroups B2 and D, which were recognized as pathogenic E. coli. To further characterize these 60 pathogenic E. coli, whole genome sequencing and antimicrobial susceptibility testing were carried out. These isolates covered 12 serotypes, and the dominant serotypes were O166 (30/60), O17 (7/60) and O7 (7/60). Eleven sequence types (ST) were identified, and ST646, ST38 and ST2001 were the dominant genotypes. Among these 60 pathogenic E. coli strains, high resistance rates to florfenicol (96.7%), tetracycline (96.7%), ampicillin (98.3%) and trimethoprim (96.7%) were observed. Twenty-six resistance genes were identified, and the most popular resistance genes included floR (58/60), tetA (58/60), blaTEM-1 (58/60), dfrA (57/60) and sul2 (53/60). Virulence gene analysis revealed five different iron uptake systems in these strains, and all the O166 serotype strains contained three iron uptake systems. In addition, diarrhoeagenic E. coli-associated gene eaeH was detected in 96.67% of the pathogenic isolates, highlighting their foodborne threat. Overall, this study extends our knowledge of the epidemiology of pathogenic E. coli in domestic pigeons.

The unreasonable use of antibiotics is one of the important causes of antimicrobial resistance (AMR) that poses a huge public health threat. Magnolol is a traditional Chinese medicine exhibiting antibacterial-, antifungal-, anti-inflammatory-, and antioxidant activities. However, it is unclear whether magnolol has an inhibitory effect on mycoplasma. This study found that magnolol showed excellent inhibitory activity against various mycoplasmas. Magnolol showed dose-dependent inhibition of Mycoplasma synoviae growth and biofilm formation in vitro. Magnolol caused severely sunken and wrinkled M. synoviae cell membranes at the minimum inhibitory concentration, and an enlarged cell diameter. The chicken embryo infection model showed that magnolol significantly reduced M. synoviae pathogenicity in vivo. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism were significantly disturbed at the minimum inhibitory concentration of magnolol. Interestingly, 41% of differential metabolites were in the categories of lipids and lipid-like molecules. Protegenin A was up-regulated 58752-fold after magnolol treatment. It belongs to fatty acyls, and destroys cell membrane integrity and cell activity. Ghosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine related to membrane maintenance and stress response were widely down-regulated. Collectively, our results illustrate the feasibility of magnolol as a phytochemical compound to treat mycoplasma infection.

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.

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.

ObjectivePasteurella 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.MethodsThe 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.ResultsWe 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.ConclusionIn 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.

[目的]试验旨在构建一种基因Ⅶ型新城疫病毒(Newcastle disease virus,NDV)嵌合疫苗,并对其免疫效力进行评估.[方法]利用反向遗传学技术,以含有禽偏禽腮腺炎病毒2型(Avian metaavulavirus-2,AMAV-2)Y2株基因组的重组质粒pT7-Y2为模板,将Y2株的F和HN蛋白的胞外区替换为基因Ⅶ型NDV HB0901株的F和HN蛋白的胞外区,将HB0901株的F蛋白裂解位点突变为LaSota弱毒株的F蛋白裂解位点,构建嵌合重组病毒rY2-FHNR株.对rY2-FHNR株的增殖特性、致病力及遗传稳定性等生物学特性进行检测,并通过接种2周龄SPF鸡评估rY2-FHNR株的免疫原性及其免疫血清与Y2株的交叉反应性,利用NDV NP蛋白的间接ELISA方法对免疫血清进行检测,验证其鉴别诊断效果.[结果]试验成功获得了嵌合重组病毒rY2-FHNR株,生物学特性检测结果显示,rY2-FHNR株在鸡胚中的增殖滴度和致病性符合弱毒特征,其鸡胚传代的遗传稳定性良好.rY2-FHNR株可诱导机体产生针对NDV的抗体,且免疫血清不与rY2株抗原发生交叉反应.通过NDV NP ELISA抗体检测方法可以实现区分疫苗免疫与野毒感染.[结论]本试验研发了一种基因Ⅶ型NDV嵌合候选疫苗,为基因Ⅶ型NDV的监测、防控和净化提供了技术支撑.

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. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

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.

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 an d 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.

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.