为建立一种快速鉴定O8、O9和O89血清型大肠杆菌的三重PCR方法,根据GenBank登录的3种血清型大肠杆菌O抗原合成基因簇序列,设计3对检测引物,通过优化反应条件建立三重PCR方法.结果:经条件优化后的三重PCR方法可有效鉴别O8、O9和O89血清型的大肠杆菌,具有良好的特异性,对其他肉羊养殖场常见血清型的大肠杆菌和致病菌无特异扩增条带;敏感性检测显示,三重PCR方法对O8和O89血清型的最小检出量为10 pg/μL,对09血清型的最小检出量为100pg/μL;采用该方法对临床分离的大肠杆菌进行检测,结果与传统血清凝集方法一致.综上,本研究建立的三重PCR方法可快速、准确、特异地对O8、O9和O89血清型大肠杆菌进行检测,为养殖及肉品加工环节大肠杆菌的流行病学研究提供了更加快捷的检测手段.

Pathogenic Escherichia coli (E. coli) can cause a variety of intestinal and extra-intestinal infections in humans and animals. Similar with the intestinal disease, respiratory disease is also a major threat to the breeding industry of goats. But the reports on respiratory disease associated E. coli are very limited. In this study, E. coli and other pathogens were examined for the 77 submitted respiratory cases. The serotypes, virulence genes, phylogenetic group and antimicrobial resistance characteristics of the E. coli isolates were identified. The results showed that 34 cases (44.16%) were associated with E. coli and 22 cases showed mixed infections of E. coli with Mycoplasma ovipneumoniae, Mannheimia haemolytica or Pasteurella . Among the 49 E. coli isolates, O8 (32.65%), O9 (20.41%) and O89 (10.20%) were the predominant serotypes (31/49, 63.27%). 22 virulence genes were detected and the most prevalent genes were fimH (100%), yijp (100%), mat (97.96%), ompA (95.92%) ibeB (91.84%) and fyuA (77.55%). In addition, ibeA was detected in 6.12% (3/49) of the strains. Markers of extra-intestinal pathogenic E. coli (ExPEC) were also identified and 14 strains were classified as ExPECs. 14 (28.57%), 25 (51.02%), 3 (6.12%) and 7 (14.29%) strains belonged to phylogenetic group A, B1, B2 and D, respectively, and group A and B1 were the predominant ones. The E. coli strains showed high resistant (48.98%-100%) to the 14 selected antimicrobials and all of them were defined as multiple drug resistant (MDR) strains. This is the first systemically study on E. coli of goats respiratory diseases origin in eastern China. The results suggest that E. coli infection may play an important role in goat respiratory diseases and that goats are reservoir hosts of ExPECs, which needs continuous monitoring in the future.

为明确江苏某羊场外购育肥羊突发呼吸道疾病的病因,本研究结合临床症状、剖检病变观察、病原学检测与分离鉴定等方法对病因进行分析.结果表明:该病是由肠道外致病性大肠杆菌、山羊副流感病毒3型及绵羊肺炎支原体混合感染所致.分离所得的2株大肠杆菌对多黏菌素B、强力霉素敏感,对其他12种常见抗生素均不敏感,检测到其携带有iss、fyuA、iroN、cvaC、fimH、iutA、kpsMT Ⅱ这7种毒力基因,分别属于系统进化群B1群与B2群.通过MDBK细胞成功分离获得1株山羊副流感病毒3型毒株,其M基因片段与流行毒株同源性最高达99.7％.该结果将有助于科学认识和防控山羊呼吸道疾病.

污蝇杆菌(Wohlfahrtiimonas chitiniclastica)是一种新出现的人畜共患病原菌.2020年从安徽某羊厂1只口腔和舌头溃烂羊的口腔中采样并分离细菌,进行药敏试验.结果显示:分离到1株污蝇杆菌,呈β型溶血,该菌对β-内酰胺类、氨基糖苷类、四环素类耐药、大环内酯类、喹诺酮类、磺胺类、氟苯尼考、多黏菌素类药物均出现耐药情况,对美罗培南、替加环素敏感.这是国内首次报道羊源污蝇杆菌,但其耐药性相比之前报道菌株更严重,这对公共卫生安全具有重大挑战.

随着规模化养羊业的发展,肉羊呼吸道疾病流行普遍、发病严重、防治困难,给养殖业及养殖户造成较大的经济损失.为明确安徽某肉羊养殖场呼吸道疾病的病因,结合流行病学、临床症状、剖检病变观察、细菌学及病毒学检测与分离鉴定以及血清学检测对病因进行分析,结果表明该病是由山羊副流感病毒3型与巴氏杆菌混合感染所致.分离菌株对丁胺卡那霉素、氟苯尼考、阿奇霉素、恩诺沙星和头孢曲松敏感,对青霉素、克林霉素和强力霉素不敏感.通过MDBK细胞成功分离获得山羊副流感病毒3型毒株,其M基因片段与已有毒株同源性达99％.血清学检测也进一步证实病毒的感染.这为临床上科学防控山羊呼吸道疾病提供了指导和借鉴.

裂谷热(Rift Valley fever,RVF)是由裂谷热病毒(Rift Valley fever virus,RVFV)引起的一种烈性人兽共患传染病.RVFV囊膜蛋白Gn可诱导产生中和抗体,是RVFV检测方法和疫苗研究的重要抗原靶标.本研究通过分析蛋白抗原位点信息,构建包含Gn蛋白两个主要抗原区域的重组表达载体,随后将质粒转化至BL21感受态细胞,以IPTG诱导重组蛋白表达并优化蛋白表达条件,通过Western Blot鉴定重组蛋白;将重组蛋白免疫BALB/c小鼠,制备多克隆抗体,并以ELISA、Western Blot、IFA检测多克隆抗体的反应性.结果显示:诱导表达的Gn重组蛋白分子质量约为45 kDa;蛋白表达条件优化为IPTG终浓度0.25 mmol/L,诱导时间5 h;Western Blot鉴定发现蛋白成功表达.通过ELISA测定小鼠三免后血清抗体,结果发现抗体效价大于1:51200;Western Blot检测显示,制备的多抗血清能与重组蛋白发生反应;进一步的IFA检测结果表明,制备的多克隆抗体可与真核质粒转染细胞中表达的Gn蛋白反应.本研究获得的Gn重组蛋白及制备的多克隆抗体为后续RVFV检测方法的建立奠定了基础.

旨在建立一种检测绵羊肺炎支原体(Mo)血清抗体的间接ELISA方法,笔者构建了 Mo Enolase基因原核表达载体,诱导表达后,纯化的重组蛋白用Western-blot分析其反应原性.以重组蛋白为包被抗原,建立了 Mo间接ELISA抗体检测方法.结果显示,重组蛋白得到可溶性表达,Western-blot证实具有良好的反应原性.间接ELISA反应条件优化结果显示,包被抗原浓度为2mg/L,37℃2h,封闭条件为含10g/L BSA的PBS,4℃过夜,待检血清稀释度为1∶50,37℃1 h,酶标二抗最佳稀释度为1∶4 000,37℃1 h,底物最佳显色条件为37℃避光10min.分别利用38份阴性血清、38份阳性血清确定临界值为S/P=0.365.用该方法与间接血凝法对180份血清进行检测,两者符合率为81.11％.结果表明,本研究建立的间接ELISA方法敏感、特异,具有良好的临床应用前景.

为了解我国主要的规模化养羊区规模化羊场的细菌耐药性情况,2018年8月至2020年11月,从江苏、安徽、浙江、江西、河北等地规模化羊场采集临床发病羊的呼吸道、消化道和其他样品,分离鉴定细菌,用药敏纸片法测定耐药性.结果,共分离到细菌147株;药敏结果显示,革兰阳性菌对大环内酯类耐药严重,对β内酰胺类中度耐药,对氨基糖苷类、喹诺酮类整体耐药率低于40％;革兰阴性菌对大环内酯类耐药率在60％～100％,对青霉素类的耐药率在80％以上,仅对大观霉素和阿米卡星的耐药率低于30％.对24种抗生素药敏试验结果显示,45株细菌耐药20种以上(占30.6％),主要为肠杆菌属菌;50株细菌耐药10～20种(占34％).从2018到2020年总耐药率从80.1％下降到56.9％,江苏、河北等省耐药严重,春夏季耐药率高于秋冬季.这些结果提示,规模化羊场细菌耐药性严重.本研究结果为进一步开展细菌耐药性分析和制定合理的安全用药措施奠定了基础.