鸭致病性大肠埃希菌的分离鉴定与毒力基因检测及菌蜕制备

Isolation of pathogenic Escherichia coli from duck and detection of virulence gene and preparation of bacterial ghost

从江苏省某水禽场的疑似感染致病性大肠埃希菌的病鸭中分离获得鸭致病性大肠埃希菌野毒株;通过玻板凝集和试管凝集试验确定其血清型,采用PCR进行分群分析和毒力基因检测;扩增φX174噬菌体裂解蛋白E的基因序列,构建温控型表达质粒pBV221–E,并将其电击转化入分离毒株,升温诱导E蛋白表达制备菌蜕;通过测量菌液A600 nm值和在透射电镜下观察细菌形态,评估菌蜕构建效果;用该菌蜕进行灭活处理和无菌检测后,对雏鸭进行免疫,用间接ELISA法检测血清IgG水平。结果表明:该大肠埃希菌的血清型为O24,属于B1群,具有毒力基因fimC、csgA和iroN;获得的鸭致病性大肠埃希菌菌蜕裂解率为99.96%;经透射电镜观察发现,制备获得的菌蜕表面有明显孔道,细胞质从孔道溢出,细胞膜皱缩变形;抗体水平检测结果显示,二免后菌蜕免疫组雏鸭血清IgG水平显著提高。可见,通过将pBV221–E重组质粒转化至鸭致病性大肠埃希菌分离株,调节细菌培养温度诱导E蛋白表达,能制备鸭致病性大肠埃希菌B1群O24型野毒株菌蜕。该菌蜕可诱发机体产生体液免疫。

Wild Strain of duck pathogenic Escherichia coli was isolated from avian pathogenic colibacillosis suspected duck in a waterfowl farm in Jiangsu province. The isolated strain was subject to serotype identification, cluster analysis, virulence gene detection, E protein expression and bacterial ghosts preparing, and morphology observation. After inactivation and sterility test, ducklings were immunized with bacterial ghost and serum Ig G levels were detected. The results showed that the serotype of the strain is O24 and the strain belongs to group B1. It has virulence genes fimC, csgA and iroN. The lysis rate of duck pathogenic Escherichia coli was 99.96%. The results of transmission electron microscopy showed that there were obvious channels on the surface of bacterial ghosts prepared. We found that the cytoplasm overflowed from the channels and the cell membrane was shrank and deformed. The results of antibody level test showed that the serum IgG level of ducklings in bacterial ghost group increased significantly after secondary immunization. It can be seen that by transforming the recombinant plasmid pBV221-E into duck pathogenic Escherichia coli isolate and regulating the temperature of bacterial culture to induce the expression of E protein. The bacterial ghost of wild strain O24 of B1 group of duck pathogenic Escherichia coli were constructed in this study and it owns capacity to induce humoral immunity.