东北地区致犊牛腹泻大肠杆菌的分离鉴定与HPI毒力岛相关序列分析

发布时间：2018-04-01 13:07

本文选题：犊牛腹泻　切入点：大肠杆菌　出处：《吉林农业大学》2016年硕士论文

【摘要】：犊牛腹泻性大肠杆菌病是由致病性大肠杆菌所引起的一种初生犊牛的消化道疾病,该病的主要临床症状是腹泻。大肠杆菌的毒力因子种类多,致病作用复杂,菌株的致病力主要是多种毒力因子综合作用的结果。近年来,犊牛大肠杆菌病的死亡率和发病率逐渐升高,致病性大肠杆菌作为人畜共患病的重要病原,对人类公共卫生健康造成了严重的危害。为了研究东北地区犊牛腹泻病的病原,本试验于2013年8月-12月采用灭菌棉拭子无菌采集东北地区部分地区奶牛场发生疑似腹泻症状犊牛直肠拭子样品,共153份,进行病原菌的分离,理化、16s rDNA鉴定、O因子血清型鉴定、致病性分析和HPI毒力岛核心功能区的六种毒力基因irp2、irp3、irp5、irp8、asn_tRNA_intB、fyuA、ybtA基因携带情况进行检测。试验对分离株进行培养特性、一系列生化鉴定、16SrRNA鉴定以及分子生物学鉴定,分离得到的细菌中大肠杆菌共74株,同时分离得到1株伤口埃希氏菌、3株肺炎克雷伯菌、7株肠杆菌科(未鉴定到种属)和1株阪崎肠杆菌。本次试验鉴定出8种单一血清型,分别是O2、O9、O14、O15、O36、O78、O101、O137,2种混合血清型包括O14-O15、O14-O137。其中O14、O36、O14-O15、O14-O137为优势血清型。74株大肠杆菌均具有一定的致病力,其中28株大肠杆菌具有高度致病力。最后,对确定的大肠杆菌菌株进行HPI毒力岛的检测,irp2基因阳性率74%(55/74),即55株大肠杆菌HPI阳性;fyuA、irp3、irp5、irp8、asn_tRNA_intB和ybtA基因阳性检出率分别为32%(18/55)、23%(13/55)、12%(7/55)、27%(15/55)、10%(6/55)、23%(13/55)。综上,本实验初步了解了东北地区牛源大肠杆菌的优势血清型以及HPI毒力岛中毒力因子的分布情况,为临床的合理用药以及对大肠杆菌病的预治提供了有利的科学依据。
[Abstract]:Diarrhoeal Escherichia coli is a kind of digestive tract disease caused by pathogenic Escherichia coli in calves. The main clinical symptom of the disease is diarrhea. There are many kinds of virulence factors and complex pathogenicity of Escherichia coli. In recent years, the mortality and morbidity of Escherichia coli in calves have increased gradually, and pathogenic Escherichia coli is an important pathogen of zoonosis. In order to study the pathogen of calf diarrhea disease in Northeast China, A total of 153 samples of rectal swabs from calves with suspected diarrhea symptoms were collected from dairy farms in northeast China from August to December 2013. The pathogenic bacteria were isolated, and the serotype of factor O was identified by 16s rDNA. Pathogenicity analysis and the detection of six virulence genes irp2rp3rp3rp3rp3rp8asntRNAin intBfyuAybtA gene carried out in the core functional region of HPI virulence island. The culturing characteristics, a series of biochemical identification, 16s rRNA identification and molecular biological identification of the isolated strains were studied. A total of 74 strains of Escherichia coli were isolated, 3 strains of Klebsiella pneumoniae and 1 strain of Enterobacter sakazakii were isolated. The two mixed serotypes, O14-O15, O14-O36-O78-O101O137and O14O36-O14O15O14-O137, have some virulence, and 28 of them have high virulence. Finally, O14O36-O14O15O14-O137 is the dominant serotype of Escherichia coli, and 28 of them have high virulence. Finally, O14O36-O14O15O14-O137 is the predominant serotype of Escherichia coli, and 28 of them are highly virulent. The positive rate of the HPI virulence island of E. coli strains was 7455% 74%, that is 55 strains of Escherichia coli HPI positive Fyu Airp5irp5 irp8asntRNAintB and ybtA gene positive rates were 32 ~ 18 / 55 / 55 ~ 12755 ~ 25 ~ 12755 ~ 25 ~ 12755 ~ (5)% ~ (27) ~ 15 ~ 15 ~ 55 ~ 10 / 10 / 10 / 55 / 10 / 10 ~ 55 / 10 / 10 / 10 ~ 55 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10. This experiment preliminarily understood the predominant serotype of bovine Escherichia coli and the distribution of toxic factor of HPI virulence island in Northeast China, which provided a favorable scientific basis for the rational use of drugs in clinic and the pre-treatment of Escherichia coli disease.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61

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