【Objective】This study was aimed to investigate the effect of the outer membrane protein OmpA of avian pathogenic Escherichia coli（APEC）on autophagy in DF-1cells and thus provide a basis for understanding whether OmpA helped APEC evade the autophagy mediated clearance in host cells．【Method】The genomic DNA of APEC E058strain was used as a template to recover the ompA gene fragment by PCR amplification,and then cloned into the eukaryotic expression vector pEGFP-N1after double digestion with EcoR Ⅰ and Xho Ⅰ．The positive recombinant plasmid pEGFP-N1-ompA was screened and confirmed by enzyme digestion and sequencing.After transfected into DF-1cells,the expression of OmpA was detected by Western blotting and immunofluorescence assay.The effects of OmpA on autophagy in DF-1cells were detected by transmission electron microscopy,immunofluorescence assay and Western blotting．【Result】After EcoR Ⅰ/Xho Ⅰ enzyme digestion of the sequencing identified recombinant plasmid,two bands with size of 1 038and 4 700bp were obtained,which were consistent with the vector and the target fragment,indicating that the eukaryotic expression plasmid pEGFP-N1-ompA was successfully constructed.After transfection of recombinant plasmid with liposome into DF-1cells,a large amount of green fluorescence could be observed by immunofluorescence assay and 65ku protein band was detected by Western blotting,indicating that pEGFP-N1-ompA was successfully transfected into DF-1cells and ompA-EGFP fusion protein was expressed in large amounts.Meanwhile,Western blotting results showed that overexpression of OmpA caused an increase in the expression of autophagy marker protein LC3Ⅱ．The autophagsomes in DF-1cells were observed by transmission electron microscopy.The single fluorescent GFP-LC3plasmid was transfected into DF-1cells,and the OmpA protein treated group showed aggregation of green fluorescent spots,indicating that OmpA induced autophagy of DF-1cells.Furthermore,OmpA affected the degradation of autophagy marker protein p62,and the transfection of double fluorescent mRFP-GFP-LC3plasmid showed that OmpA could block the oceurrence of autophagic flux．【Conclusion】The APEC outer membrane protein OmpA could cause autophagy in DF-1cells,but it blocked the autophagic flux and induced incomplete autophagy in DF-1cells,which would help to better understand the escape mechanism of APEC from the host cells clearance.

旨在构建高质量的禽源细胞系,应用CRISPR/Cas9技术实现鸡胚成纤维细胞(DF-1)的Toll样受体7(TLR7)编码基因的敲除,构建稳定的细胞培养禽腺病毒4型(FAdV-4)增殖体系.采用荧光定量PCR的方法对FAdV-4感染DF-1细胞后先天性免疫信号通路中效应分子进行检测,选择转录水平显著上调的基因TLR7作为研究对象;构建sgRNA载体与Cas9表达载体共转染DF-1细胞,经绿色荧光蛋白(GFP)阳性特征分选单克隆,PCR验证及增毒试验筛选后获得TLR7编码序列敲除的DF-1-TLR7-KO#3单克隆细胞系,命名为DF-1-TLR7-KO细胞.对敲除前后DF-1细胞的天然免疫系统对FAdV-4感染的拮抗效应和病毒在不同细胞中增殖效率的差异进行了研究.结果表明:成功构建的TLR7编码序列敲除的DF-1细胞系,与原始细胞相比病毒增殖能力提高.试验对细胞的天然免疫系统与FAdV-4感染的互作机理进行了初步探索,为更好地研究宿主对病毒入侵的应答机制,提高疫苗生产效能提供了研究基础和生物材料储备.

为了解广西地区鸡胚中沙门菌的流行情况及耐药现状,本研究对广西某些鸡场送检的病死鸡胚进行沙门菌的分离鉴定.然后利用PCR方法鉴定沙门菌临床分离株的血清型及毒力因子,并检测其耐药性.结果显示,经选择性培养基和PCR鉴定分离到18株沙门菌,分离率为13.95％(18/129).血清型鉴定显示:分离株均为鸡白痢沙门菌.毒力基因检测发现shdA基因的分布率只有22.22％,其余15个毒力基因的分布率均为100％.药敏试验结果显示,沙门菌分离株对卡那霉素、庆大霉素、氯霉素、美罗培南、亚胺培南均敏感;但对林可霉素、克林霉素、罗红霉素、红霉素、利福平的耐药率达100％,表明鸡白痢沙门菌的多重耐药现象严重.本研究提示鸡白痢可能是导致鸡胚死亡及孵化率低的原因,因此应严格进行鸡白痢的防控净化工作.

2018年4月至2018年10月在江苏省无锡宜兴、常州武进、溧阳和金坛、淮安宝应、宿迁泗洪等6个市(区、县)149个患细菌性败血症的团头鲂养殖场,无菌采集样品肝脏、脾脏、肾脏、腹水进行细菌分离纯化.分离纯化后进行生化鉴定,生化鉴定为嗜水气单胞菌的菌株再用16 S rRNA-PCR进一步鉴定.结果显示,经过分离纯化得到149株细菌,生化鉴定117株为嗜水气单胞菌,16S rRNA-PCR进行验证核对,得到101株嗜水气单胞菌,其余16株可以鉴定到气单胞菌属.对采样数据进行综合分析,在6个采样地区中,嗜水气单胞菌阳性率最低为金坛的57.14％,最高为宝应的75％.在不同规格样品中,0.2 kg～0.3 kg的团头鲂嗜水气单胞菌阳性率最高(76.12％).结果表明,江苏人工养殖团头鲂细菌性败血症的主要病原为嗜水气单胞菌,对其进行防控刻不容缓.

近年来抗生素的不合理使用导致细菌耐药性问题逐渐加剧,开发新型有效的抗菌制剂迫在眉睫.噬菌体及其溶菌酶具有特异性强、抗菌效力强、安全性高、筛选周期短、绿色安全及不易产生耐药等优势,可作为一种天然抗菌剂防控细菌病.为了分析广宿主谱沙门菌噬菌体SHWT1的溶菌酶抑菌活性,本文将沙门菌裂解性噬菌体SHWT1的溶菌酶lys基因克隆至表达载体pET28a(+),然后转化至大肠杆菌BL21(DE3)中进行表达,纯化后获得了大小19 kDa、浓度为2 mg/mL的高纯度融合蛋白LysSHWT1.平板裂解实验显示,溶菌酶重组蛋白LysSHWT1对鸡白痢沙门菌、肠炎沙门菌、鼠伤寒沙门菌、鸡伤寒沙门菌具有较好的裂解活性,裂菌圈直径分别为16、13、14、13 mm,对照组无裂解圈;裂菌谱结果显示,LysSHWT1对101株临床沙门菌株中的60株菌株存在裂解作用,未发现能裂解除沙门菌外的其他菌株;进一步研究发现,溶菌酶在EDTA的协同作用下可以展现更好的抑菌活性,使鸡白痢沙门菌SP01、鸡伤寒沙门菌SG02、鼠伤寒沙门菌SAT52、肠炎沙门菌SE12滴度显著下降(P＜0.05).本研究表明广宿主谱沙门菌噬菌体溶菌酶LysSHWT1可有效裂解沙门菌,在防控沙门菌感染方面具有潜在的应用价值.

某5000头丹系母猪场平均窝产仔数在16头以上,多余仔猪需用奶妈猪来代养.为提升仔猪断奶成活率及母猪利用率,本试验选用5日龄仔猪使用智能哺乳设备代替母猪进行超早期断奶饲养.结果表明,5日龄超早期断奶模式的仔猪断奶重及断奶成活率可以达到猪场断奶要求,提升了母猪利用率.在实际生产中,该模式会大大提升猪场生产效率,值得应用推广.

为分析江苏地区仔猪腹泻性大肠杆菌的致病性、血清型、耐药性及生物被膜(BF)形成能力等生物特性,本研究从2017年～2019年采集江苏地区患腹泻的仔猪粪便、肛拭子等216份病料样品,从中分离培养后经16S rRNA PCR鉴定获得156株大肠杆菌,进一步通过小鼠感染性实验得到118株致病性大肠杆菌.对分离的致病性大肠杆菌采用O血清型玻板凝集试验检测其血清型,结果显示118株致病性大肠杆菌有14种血清型,以O38型、O149型及O109型为主要的优势流行血清型;采用K-B药敏纸片法对分离菌株的耐药性检测结果显示,118株致病性大肠杆菌对氨苄西林、磺胺间甲氧嘧啶、环丙沙星等7种药物的耐药率在47.5％以上,对其他药物耐药率在11.0％～16.1％;结晶紫微孔板法检测分离菌BF的形成能力,结果显示,分离的118株致病性大肠杆菌中BF形成能力强、中等、较弱及不形成BF的菌株分别有48株(40.7％)、36株(30.5％)、24株(20.3％)、10株(8.5％),且对氨苄西林、磺胺间甲氧嘧啶、环丙沙星等7种药物的耐药性与BF形成能力具有一定相关性.本研究为仔猪腹泻性大肠杆菌病的防控提供科学依据.

[目的]研究滑液支原体(Mycoplasma synoviae,MS)脂蛋白P80的免疫反应性及其在MS血清抗体ELISA检测中的应用.[方法]对MS P80的氨基酸序列进行生物信息学分析、原核表达和纯化,并用免疫印迹法分析其与6种不同MS分离株阳性血清的免疫反应性以及与其他禽病原血清的交叉反应性;运用纯化的MS P80表达蛋白作为包被抗原建立了MS血清抗体的间接ELISA检测方法,对其敏感性和重复性进行检测;比较检测了与美国爱德士检测试剂盒对50份临床血清样品的阳性符合率.[结果]生物信息学分析预测MS P80蛋白为脂蛋白且含有信号肽,其在MS种内同源性高达98％-100％,与其他种属P80蛋白同源性在25％-34％之间,成功表达和纯化了MS P80重组蛋白(rMS P80);Western blotting分析表明纯化的rMS P80具有良好的免疫反应性和特异性;运用rMS P80建立的MS血清ELISA抗体检测方法可对不同株MS阳性血清进行抗体效价检测,而对其他禽病原阳性血清均无交叉反应性;该检测方法的批内变异系数小于5％,批间变异系数小于10％,重复性良好;与美国IDEXX检测试剂盒比较,本文建立的ELISA抗体检测方法敏感性更高,阳性符合率为75％,阴性符合率为89.47％,总样本符合率为86％.[结论]MS P80具有较好的免疫反应性、种内保守性和种间特异,并且可用作MS抗体检测的靶标抗原.

由于抗生素的不合理使用,引起了细菌多重耐药性、药物残留、食品安全、环境菌群不平衡等问题,严重威胁人类及动物的健康.因此,寻找新型抗菌药物及治疗方案已成为当前的研究重点.噬菌体是感染细菌、真菌等微生物的病毒,自发现伊始便用于治疗细菌感染.与传统抗生素相比,噬菌体具有分布广、易筛选、高度的宿主专一性、增殖能力强、安全性高、研发成本低等优势.随着多重耐药菌的广泛流行,噬菌体治疗细菌感染重新引起了人们的重视.本文简述了噬菌体的生物学特性、噬菌体疗法在预防细菌感染的应用及噬菌体研究发展方向,以期为噬菌体疗法预防细菌性感染提供参考.

Pigeons were previously thought to be resistant to H5 viruses and to play a minimal role in spreading these viruses. In this study, we evaluated the pathogenicity of two clade 2.3.4.4 H5N6 viruses in pigeons and the potential viral transmissibility to specific-pathogen-free chickens in direct close contact with experimentally infected pigeons. No pigeons from the A/goose/Eastern China/Xin/2015 (GS/Xin) group exhibited clinical signs or mortality, and the virus was only detected in a few organs. However, 3 of 12 pigeons inoculated with the A/goose/Eastern China/0326/2015 (GS/0326) virus died, and 7 of 12 showed neurological symptoms and efficient viral replication in multiple organs. In both groups, viral shedding occurred in only some of the pigeons, the shedding period was relatively short, and the infection was not transmitted to the chickens. We also used chicken, duck, and BALB/c mouse models to evaluate the pathogenicity of the two H5N6 isolates. Both H5N6 isolates showed highly pathogenic to chickens but different degrees of pathogenicity in mice. Interestingly, in ducks, the intravenous pathogenicity index indicated that the GS/Xin isolate was low pathogenic, and the GS/0326 isolate was highly pathogenic, corresponding to the pathogenicity in pigeons. Our results indicated that the pathogenicity of the clade 2.3.4.4 H5N6 virus is diverse in pigeons, and pigeons contribute little to its transmission among poultry. However, pigeons may still be potential healthy reservoirs of the H5N6 highly pathogenic avian influenza virus.

根据肝螺杆菌fla B基因保守区域设计一组特异性引物,经过条件优化、特异性和敏感性分析,成功建立了肝螺杆菌LAMP快速检测方法.结果显示,建立的LAMP扩增方法仅能检测出肝螺杆菌,其他常见细菌未见特异性扩增.检出肝螺杆菌最低模板量为86.9 fg/μL,是普通PCR所需模板量的1/10.本研究建立的LAMP快速检测方法具备操作简单、特异性好、灵敏度高的优点,结果易于观察,对仪器设备要求低,适宜推广应用.

From 2010, porcine epidemic diarrhea virus (PEDV) variants caused sequential outbreaks of disease in Asia and the United States. In this retrospective study, 49 complete spike (S) gene sequences were obtained from PEDV strains collected in China from 2014 to 2016. We observed that variant PEDV strains with novel insertions, deletions, and multiple S gene recombination types were present in China. In addition, mixed infections involving different variant strains were observed in some areas. Based on phylogenetic and recombination analyses, we determined that the newly emerged PEDV variants potentially originated via recombination between the earliest Chinese G1 genogroup strain, JS-2004-2 and earlier Korean pandemic strains. These findings provide important information for understanding ongoing PEDV outbreaks and suggest that novel variants make it more difficult to prevent PEDV infection.

[Background] Mycoplasma synoviae (MS) can infect chickens and turkeys to cause airsacculitis, joint exudative, synovial bursitis and tendon sheath synovitis. Previous studies have shown that many metabolism-related enzymes in mycoplasma are present not only in the cytoplasm but also on the cell membrane surface, which act as adhesion associated proteins and play roles on the pathogenicity. The fructose-bisphosphate aldolases (FBAs) have been identified on the membrane and cytoplasmic fractions of Mycoplasma bovis and Mycoplasma gallisepticum, however, the MS FBA has not been studied before. [Objective] To explore the biological functions of metabolism related enzymes in MS, the FBA protein was subjected to bioinformatics analysis, prokaryotic expression, as well as immunogenicity and subcellular localization determinations in this study. [Methods] Using softwares of PSORTb, SignalP 4.1 Server and TMHMM Server to predict the subcellular localization, signal peptides and transmembrane regions of MSFBA. Blastn and MEGA 5.0 were used to analyze the homology and construct the phylogenetic tree of MSFBA. The full length of MS fba gene was amplified by overlap PCR and inserted into the expression vector of pET-28a(+). The recombinant MSFBA (rMSFBA) protein was then expressed in Escherichia coli BL21(DE3) and purified. The immunogenicity of rMSFBA was determined by Western blot analysis using MS-positive chicken serum. The rabbit anti-rMSFBA sera were prepared, and used to perform Western blot against the whole-cell, membrane and cytoplasmic fractions of MS to determine the subcellular localization. Suspension immunofluorescence assay further confirmed the non-membrane surface localization of the MSFBA. [Results] The MSFBA is predicted to be a cytoplasmic protein with no signal peptide and no transmembrane region. It is highly conserved protein which shows up to 99％ homology among different MS strains, and 61％ to 78％ homology with different species of mycoplasma. The phylogenetic tree shows that the MSFBA is evolutionally closed to the FBA from Mycoplasma bovirhinis and Mycoplasma cricetuli, but remote to that of Mycoplasma arginini and Mycoplasma hominis. The rMSFBA protein was successfully expressed and purified, and the relative molecular mass was approximately 33 kD. The rMSFBA presented a specific binding to MS-positive chicken serum, which proved that it had good immunogenicity. Western blot analysis showed the rabbit anti-rMSFBA sera can react with the whole-cell and cytoplasmic fractions of MS bacteria, but not the membrane fraction, indicating the MSFBA protein is distributed in the cytoplasma. Suspension immunofluorescence assay further confirmed the FBA was not displayed on the membrane surface of MS cells. [Conclusion] The MSFBA protein is a highly conserved, immunogenic and cytoplasmic localized protein. The results provide a molecular basis for further study of the biological function of MSFBA protein.

沙门菌病是一种严重危害人类健康和养殖业发展的人畜共患病,为了解华东地区沙门菌的分子流行情况,本研究采用选择性培养基和PCR分离鉴定沙门菌,然后利用PCR检测沙门菌临床分离株的血清型及毒力因子,并用药敏纸片法检测其耐药性.结果 显示,本研究分离鉴定出48株沙门菌,分离率为16.84％.血清学结果显示所检测的48株沙门菌中鼠伤寒沙门菌占64.58％,肠炎沙门菌占6.25％,其他血清型占29.17％.毒力基因检测发现除sopE基因分布率为25％外,其他15个毒力基因分布率可达97％以上.药敏试验结果显示沙门菌分离株对常见抗生素具有不同的抗性和敏感性,并且耐药性十分严重,对红霉素、阿奇霉素、克林霉素、新霉素、大观霉素、四环素、利福平、复方新诺明等抗生素的耐药率均超过50％以上.检测结果说明,华东地区沙门菌主要血清型为鼠伤寒沙门菌,且毒力基因分布广泛,多重耐药性菌株十分流行.

【背景】鼠伤寒沙门菌（Salmonella typhimurium）是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。

Two reassortant H5N2 viruses in which hemagglutinin (HA) was clustered into Glade 2.3.4.4, were isolated from apparently healthy waterfowl in live poultry markets in Eastern China in 2016. We used specific pathogen-free chickens, mallard ducks, and BALB/c mice to evaluate the isolates' biological characteristics in different animal models. The newly isolated reassortant H5N2 viruses were able to cause severe disease in chickens and effective contact transmission, only at high doses. Our pathogenicity studies in ducks yielded an interesting result: the intravenous pathogenicity index (IVPI) indicated that isolate A/goose/Eastern China/1106/2016(1106) was low pathogenic and the other isolate A/duck/Eastern China/YD1516/2016(YD1516) was of highly pathogenicity in ducks. However, our 50% duck lethal dose (DLD50) experiment demonstrated that these viruses were all of low pathogenicity (DLD50 > 10(7.0) EID50) in ducks. Additionally, despite the fact that reassortant H5N2 were of low pathogenicity in mice, they could bind to both avian-type (SA alpha-2,3 Gal) and human-type (SA alpha-2,6 Gal) receptors, suggesting that these isolates still present a high risk for human infection. Therefore, it is of great importance to implement continual surveillance of avian influenza virus (AIV) to protect both veterinary and public health.

以四氯化碳(CCl4)构建建鲤体内急性肝损伤模型,对姜黄素、甘草次酸和香菇多糖联合护肝作用进行研究.分别采用含有姜黄素、甘草次酸、香菇多糖和低、中、高剂量姜黄素+甘草次酸+香菇多糖联合中草药的饲料投喂60 d后,各组建鲤腹腔注射30％CCl4,注射剂量为每1 g鱼体注射0.005 ml,禁食72 h,以诱导建鲤急性肝损伤.通过测定各组建鲤生长指标、血清和肝脏生化指标及肝组织炎性细胞因子mRNA的表达情况,观察姜黄素、甘草次酸和香菇多糖联合用药的保肝效果.结果显示:甘草次酸单味用药及中、高剂量姜黄素+甘草次酸+香菇多糖联合用药能显著提高建鲤的相对增重率和特定生长率,显著降低饵料系数;与模型组相比,各用药组能显著抑制CCl4导致的血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)和乳酸脱氢酶(LDH)活性的升高,显著恢复肝组织中谷胱甘肽(GSH)和超氧化物歧化酶(SOD)水平,抑制丙二醛(MDA)生成,能显著抑制C-Rel、iNOS、IL-1β、TNF-α、IL-6和IL-8表达量的升高;与姜黄素、甘草次酸或香菇多糖单味用药相比较,姜黄素+甘草次酸+香菇多糖联合用药的保肝效果更明显,随着联合用药剂量的升高,作用愈加显著.说明姜黄素、甘草次酸和香菇多糖联合用药,对CCl4诱导的建鲤肝损伤具有协同保护作用.

The objective of the present study was to investigate the immunological injury and mucosal response induced by attenuated Newcastle disease virus strains JS10(Class Ⅰ)and La Sota (Class Ⅱ). In this experiment, 60 SPF chicks were randomly divided into group A, B and C. At the age of 7 days, chicks in group A and B were respectively immunized with JS10 strain and La Sota strain and chicks in group C received PBS as controls. 5 chicks in each group were randomly terminated at d1, 4, 7 or 14 post immunization. Saliva, trachea, lung and intestinal samples were collected for detection of mucosal antibodies SIg A, Ig M and Ig Y using double sandwich ELISA kits. The results showed that these two NDV strains did not cause any immune injury. The JS10 strain induced higher levels of mucosal antibodies as compared with La Sota strain and significant differences (p <0.05) were revealed at d 7 and d 14 post immunization.

Objective To study the role of capsule polysaccharide in pathogenesis of extraintesinal pathogenic Escherichia coli (ExPEC). Methods By using λ Red recombination system, we generated the capsular polysaccharide transport associated genes kpsE and kpsD double knockout mutants E058ΔkpsED and U17ΔkpsED. We then compared and analyzed the characteristics of the mutant strains and wild-type strains. Results The growth curves in Luria Bertani showed that the deletion of kpsED did not affect growth kinetics of the mutants. The abilities of resistance to serum and killing by chicken macrophages were significantly impaired. LD50 results showed that the double mutants completely abolished the virulence, whereas the complementation strains restored the virulence to resemble that of wild-type strains, and the colonization and coinfection model demonstrated that the deletion of kpsED led to attenuation of virulence, because the double mutant showed significantly decreased colonization compared with the wild-type strains in all organs tested in chickens. Conclusion These results indicated that the virulence factors encoded by capsule genes were important for the pathogenesis of ExPEC.