苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

Isolation, identification and evaluation of inactivated vaccine of enterotoxigenic Escherichia coli (ETEC) in large-scale pig farms in northern Jiangsu

摘要:

[目的]产肠毒素大肠杆菌（Enterotoxigenic Escherichia coli，ETEC）是引起仔猪腹泻的重要病原菌，本研究通过调查苏北地区规模化猪场ETEC的流行情况，分析其生物学特性，研制具有免疫保护效果的优势血清型菌株的灭活疫苗，以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3–30日龄的仔猪新鲜粪样、肛拭子及小肠组织样，分离出ETEC，对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定；最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份，通过PCR鉴定及测序得到141株ETEC；血清凝集试验鉴定出85株菌的O抗原血清型，其中O8、O101和O128为优势血清型，占定型菌株的61.2%（52/85），其他血清型包括O9、O3、O20、O148、O149等；分析141株ETEC对14种常见抗生素的耐药情况，得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药，耐药率均高达80%以上；对恩诺沙星敏感性较高，敏感率达50.4%（71/141）；对多粘菌素B和头孢噻肟中介耐药，占比分别为66%（93/141）和51.8%（73/141）；多重耐药现象严重，其中10重耐药的菌株占比最大，为19%（27/141）；小鼠攻毒试验测得O8血清型强毒株YC-6的半数致死量（medianlethal dose，LD₅₀）为1.4×10⁷CFU/只，最低致死量（minimum lethal dose，MLD）为3×10⁷CFU/只；O8血清型强毒株YC-6和O101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%，因此利用O8血清型强毒株YC-6和O101血清型强毒株LYG-3研制二价灭活疫苗，结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查，得出其优势血清型，并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗，给临床ETEC的监测和防控提供参考。

关键词:产肠毒素大肠杆菌;分离鉴定;血清型;耐药性;灭活疫苗

Abstract:

[Objective] Enterotoxigenic Escherichia coli (ETEC) is an important pathogen causing diarrhea of piglets. In this study, the prevalence and biological characteristics of ETEC in large-scale pig farms in northern Jiangsu was investigated, and protective and inactivated vaccines of predominant serotype were developed to provide guidance and suggestions for the prevention and control of ETEC in northern Jiangsu. [Methods] Fresh fecal samples, anal swabs and small intestinal tissue samples of piglets aged 3-30 days were collected from large-scale pig farms in northern Jiangsu province, ETEC was isolated, and serotypes, antibiotic resistance, pathogenicity on mice of these isolates were determined. Finally, the inactivated vaccine of predominant serotype was developed and the immune protection effect of mice was evaluated. [Results] We collected 562 samples from 21 large-scale pig farms. We identified 141 ETEC strains containing ST or LT virulence genes by PCR. O serotypes of 85 isolates were determined by serum agglutination tests. O8, O101 and O128 were dominant serotypes, accounting for 61.2% of the confirmed strains, besides, O9, O3, O20, O148, O149 and the like were identified. The drug resistance of 141 ETEC isolates to 14 common antibiotics was analyzed. The isolates were highly resistant to neomycin, erythromycin, tetracycline, gentamicin, doxycycline, amoxicillin, and trimethoprim/sulfamethoxazole, with resistance rate higher than 80%. Strains were most sensitive to enrofloxacin, with the rate of 50.4% (71/141). 66% (93/141) and 51.8% (73/141) strains were intermediate to polymyxin B and cefotaxime. Multiple drug resistance is serious. 19% (27/141) of strains were resistant to 10 drugs. The virulence of the dominant serotype strains was obtained by mice challenge test. LD₅₀ of one strong strain YC-6 of O8 serotype was measured as 1.4×10⁷ CFU, while the MLD₅₀ was 3×10⁷ CFU. The monovalent inactivated vaccines prepared by O8 virulent strain YC-6 and O101 virulent strain LYG-3 both have a protection rate of 100% for mice. The bivalent inactivated vaccine of YC-6 and LYG-3 were developed as well, the protection rate of which in mice was more than 83%. [Conclusion] Based on the epidemiological investigation of ETEC in northern Jiangsu, dominant serotypes and drug resistance were tested, and bivalent inactivated vaccine with superior immune protection effect against isolated strains were developed, which will provide reference for monitoring and prevention in pig industry.

Key words:enterotoxigenic Escherichia coli(ETEC);identification;serotype;antibiotic resistance;inactivated vaccine