一株仔猪产肠毒素大肠杆菌分离鉴定、耐药性分析及其卵黄抗体的制备

Isolation,identification,and drug resistance analysis of a strain of enterotoxigenic Escherichia coli derived from piglet and preparation of its specific yolk antibody

为诊断某规模化养猪场仔猪腹泻病原,并制备其特异性卵黄抗体,采用细菌分离培养、生化试验、16SrRNA PCR鉴定及系统进化树分析、药敏试验等方法对病料进行研究,并利用全菌体灭活和超声波破碎法制备抗原,将其分别与不同佐剂(ISA 71VG、ISA201VG和15AVG)配伍乳化制备疫苗免疫蛋鸡、聚乙二醇法提取高免卵黄抗体、SDS-PAGE电泳和Bradford法鉴定抗体质量及浓度、双向琼脂扩散试验测定抗体效价。结果表明,分离菌株的形状、大小及生化特性均与大肠杆菌相符。分离株的主要毒力基因E.coli-estb和elt基因分别为113、272 bp;分离株elt和estb基因与美国流行毒株如UMNK88处于同一分支,亲缘关系较近;与丹麦株CFSAN018748、瑞士株14OD0056处于不同分支,亲缘关系较远。分离菌株对头孢噻肟、诺氟沙星和复方新诺明敏感,对头孢曲松和多黏菌素B中介,对多西环素、呋喃唑酮以及多种氨基糖苷类和喹诺酮类抗菌药物耐药。各组卵黄抗体的重链(约70 ku)和轻链(约25 ku)均明显,纯度高。首免后56 d 0.2%甲醛处理的菌液与ISA 15AVG配伍所致疫苗的卵黄抗体蛋白含量最高(1.462 mg/g卵黄),超声波破碎处理的菌液与ISA 71VG配合所制疫苗的卵黄抗体蛋白含量次之(1.459 mg/g卵黄)。血清抗体与菌体抗原反应性良好,超声波破碎抗原与ISA 71VG配伍所制疫苗的血清抗体、卵黄抗体效价分别高达1∶16、1∶4。

To identify the pathogen of diarrhea of piglets in a large-scale pig farm and prepare the specific yolk antibody,bacterial iso⁃lation and culture,biochemical test,16SrRNA PCR identification,phylogenetic tree analysis,drug sensitivity test and other methods were used to study the disease materials.Antigen was prepared by whole cell inactivation and ultrasonic fragmentation.It was emulsi⁃fied with different adjuvants(ISA 71VG,ISA201VG and 15AVG)to prepare immunized laying hens,the high immune yolk antibody was extracted with polyethylene glycol,the mass and concentration of the Antibody were identified by SDS-PAGE electrophoresis and Bradford method,and the antibody titer was determined by two-way AGAR diffusion test.Results showed that the shape,size and bio⁃chemical characteristics of the isolates were consistent with escherichia coli.The bands of E.coli-estb and elt were 113 and 272 bp,re⁃spectively.The elt and estb genes of the isolates were in the same branch as those of the American epidemic strains,such as UMNK88.They were in a different branch from the Danish strain CFSAN018748 and the Swiss strain 14OD0056.The isolates were sensitive to cefotaxime,norfloxacin and cotrimoxazole,mediated to ceftriaxone and polymyxin B,and resistant to doxycycline,furazolidone and many aminoglycosides and quinolones.The heavy chain(about 70 ku)and light chain(about 25 ku)of yolk antibody were obvious in each group,and the purity was high.The yolk antibody protein content of the vaccine produced by the combination of bacteria solution treated with 0.2% formaldehyde and ISA 15AVG at 56 d after the first immunization was the highest(1.462 mg/g yolk),followed by the yolk antibody protein content of the vaccine produced by the combination of bacteria solution treated with ultrasonic disruption and ISA 71VG at 56 days after the first immunization(1.459 mg/g yolk).The titers of serum antibody and egg yolk antibody were as high as 1∶16 and 1∶4,respectively,when the ultrasonic broken antigen was combined with ISA 71VG.