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

doi:10.14088/j.cnki.issn0439-8114.2023.04.023

湖北农业科学 ›› 2023, Vol. 62 ›› Issue (4): 127-134.doi: 10.14088/j.cnki.issn0439-8114.2023.04.023

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

尹家银, 喻娇, 唐青海, 滕威, 阳坤, 曹馨, 徐晴, 刘博, 李泽

衡阳师范学院生命科学与环境学院/南岳山区生物资源保护与利用湖南省重点实验室/动物微生物新型检测技术及生物制剂衡阳市重点实验室,湖南衡阳 421008

收稿日期:2022-03-28 出版日期:2023-04-25 发布日期:2023-05-12
通讯作者: 唐青海（1982-）,男,湖南祁阳人,副教授,博士,主要从事动物病毒学与免疫学研究,（电子信箱）qinghaitang109@126.com。
作者简介:尹家银（2000-）,女,湖南衡阳人,本科生,专业方向为微生物学与免疫学,（电话）18773439034（电子信箱）2633124676@qq.com。
基金资助:
湖南省教育厅科学研究项目重点项目（21A0442）; 湖南省重点研发计划（2021NK2026）; 湖南省自然科学基金面上项目（2021JJ30060）; 国家级大学创新创业训练项目（202110546001; 〔2020〕1-7）

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

YIN Jia-yin, YU Jiao, TANG Qing-hai, TENG Wei, YANG Kun, CAO Xin, XU Qing, LIU Bo, LI Ze

Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region/Hengyang Key Laboratory of New Detection Technology and Biological Agents of Animal Microorganism/College of Life Sciences and Environment, Hengyang Normal University, Hengyang 421008, Hunan, China

Received:2022-03-28 Online:2023-04-25 Published:2023-05-12

摘要/Abstract

摘要： 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。

关键词: 猪致病性大肠杆菌（Escherichia coli）, 分离培养, 进化分析, 耐药性分析, 卵黄抗体

Abstract: To identify the pathogen of diarrhea of piglets in a large-scale pig farm and prepare the specific yolk antibody, bacterial isolation 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 emulsified 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 biochemical characteristics of the isolates were consistent with escherichia coli. The bands of E. coli-estb and elt were 113 and 272 bp, respectively. 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.

Key words: swine pathogenic Escherichia coli, separate culture, evolutionary analysis, drug resistance analysis, egg yolk antibody

中图分类号:

S852.4⁺3

尹家银, 喻娇, 唐青海, 滕威, 阳坤, 曹馨, 徐晴, 刘博, 李泽. 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[J]. 湖北农业科学, 2023, 62(4): 127-134.

YIN Jia-yin, YU Jiao, TANG Qing-hai, TENG Wei, YANG Kun, CAO Xin, XU Qing, LIU Bo, LI Ze. Isolation, identification, and drug resistance analysis of a strain of enterotoxigenic Escherichia coli derived from piglet and preparation of its specific yolk antibody[J]. HUBEI AGRICULTURAL SCIENCES, 2023, 62(4): 127-134.

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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

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