新疆地区致犊牛腹泻大肠杆菌的分离鉴定及部分特性研究

发布时间：2018-06-24 23:19

本文选题：犊牛腹泻 + 产肠毒素大肠杆菌　；参考：《石河子大学》2016年硕士论文

【摘要】：产肠毒素大肠杆菌(ETEC)是一种可引起人畜腹泻的致病性细菌,尤其对初生犊牛、羔羊、仔猪等幼畜具有较大危害。2015年3~6月,新疆石河子、沙湾、阿克苏、博乐四个地区的6个规模化牛场先后发生初生犊牛腹泻,发病率达40%~60%,死亡率达50%~70%,导致犊牛死亡千余头,造成了严重的经济损失,为查明这些牛场犊牛腹泻是否与大肠杆菌有关,在流行病学、临床检查以及剖解的基础上,无菌采取6个规模化牛场因腹泻死亡犊牛的肠系膜淋巴结等病料分别进行了细菌的分离鉴定、血清型鉴定、肠毒素基因及菌毛基因的PCR检测、溶血性测定、小鼠致病性试验、药物敏感性测定。研究结果如下:(1)采集了新疆南北疆4个地区的6个规模化牛场因腹泻死亡犊牛的肠系膜淋巴结、肝脏及肠内容物等病料60份,分离获得53株培养特性、染色特性及生化鉴定均符合大肠埃希菌的菌株,绵羊血平板上均为β溶血,表明溶血性大肠杆菌是引起犊牛腹泻死亡的主要病原之一。(2)采用O抗原凝集试验对新疆南北疆4个地区6个规模化牛场的29株代表株进行O血清型鉴定,其中10株被定型,共覆盖8种血清型,分别是O101、O7、O11、O20、O30、O44、O1、O76,且存在明显的地区性差异。(3)采用黏附素和肠毒素基因多重PCR检测的方法从53株分离株中检出49株同时含有黏附素基因K99、F41和肠毒素基因STa的菌株,阳性率为92.5%,表明多数分离株携带K99、F41和Sta毒力因子,为产肠毒素性大肠杆菌。(4)药敏结果显示:53株分离株对抗生素的敏感程度不同,且存在区域差异,多数分离株对阿莫西林、氨苄西林、阿莫西林/克拉维酸、头孢拉啶、头孢噻吩、庆大霉素、环丙沙星等耐药;对阿米卡星、头孢西丁现较高敏感性。(5)10株不同地区来源代表株的纯培养物经腹腔注射小鼠后,感染小鼠均在12~36h死亡,其中9株可致小鼠在12h内死亡,而10只生理盐水对照组小鼠健活。无菌采取死亡小鼠肝脏,进行细菌分离鉴定,获得形态、生化鉴定均符合大肠杆菌的菌株,表明分离株具有较强的致病性。综上研究结果表明:携带K99、F41菌毛及Sta的产肠毒性大肠杆菌是引起这6个牛场初生犊牛腹泻并造成死亡的主要病原之一,同时也初步揭示了该地区牛源大肠杆菌的耐药情况及毒力基因的流行情况,可为该地区犊牛大肠杆菌性腹泻的药物和免疫防治提供科学参考。
[Abstract]:Enterotoxigenic Escherichia coli (ETEC) is a pathogenic bacterium that can cause diarrhea in human and animal, especially to newborn calves, lambs, piglets and other young animals. In six large-scale cattle farms in four districts of Bole, diarrhea occurred successively in newborn calves, with an incidence of 400.60 and a mortality of 500.700.This resulted in the death of more than 1,000 calves, resulting in serious economic losses. In order to find out whether diarrhea in these calves is related to E. coli, On the basis of epidemiology, clinical examination and dissection, bacterial isolation and serotype identification were carried out in 6 large scale calf calves died of diarrhea, such as mesenteric lymph nodes. The detection of enterotoxin gene and fimbriae gene by PCR, hemolytic test, pathogenicity test in mice and drug sensitivity test. The results were as follows: (1) 60 samples of mesenteric lymph nodes, liver and intestinal contents were collected from 6 large scale cattle farms in four areas of Xinjiang, Xinjiang. The staining characteristics and biochemical identification were consistent with Escherichia coli strains, and 尾 -hemolysis was found on sheep blood plate. The results showed that hemolytic Escherichia coli was one of the main pathogens leading to the death of calf diarrhea. (2) using O antigen agglutination test, 29 representative strains of 6 large-scale cattle farms in 4 regions of Xinjiang were identified with O serotype, of which 10 strains were identified. A total of 8 serotypes, O101, O7, O11, O20, O30, O4, O1, O76, were isolated, and there were significant regional differences. (3) A total of 49 strains containing adhesion gene K99F41 and enterotoxin gene STa were detected from 53 isolates by multi-weight PCR. The positive rate was 92.5, indicating that most of the isolates carried K99F41 and Sta virulence factors, which were enterotoxigenic Escherichia coli. (4) the drug sensitivity results showed that the sensitivity of 53 strains to antibiotics was different, and there were regional differences, and most of the isolates were sensitive to amoxicillin. Ampicillin, amoxicillin / clavulanic acid, cefradine, cefthiophene, gentamicin, ciprofloxacin, etc. Cefoxitin was highly sensitive. (5) 10 strains of pure cultures from different regions were injected intraperitoneally into mice, the infected mice died within 12 ~ 36 hours, 9 of them could cause mice to die within 12 hours, while 10 normal saline control mice were healthy. The bacteria were isolated and identified in the liver of dead mice. The morphology and biochemical identification of the bacteria were in accordance with the strains of Escherichia coli, which indicated that the isolates had strong pathogenicity. The results showed that enterotoxigenic Escherichia coli carrying K99F41 fimbriae and Sta was one of the main pathogens causing diarrhea and death in the six calves. At the same time, the drug resistance and virulence gene prevalence of bovine Escherichia coli in this area were preliminarily revealed, which could provide a scientific reference for drug and immune prevention and control of Escherichia coli diarrhea in calves in this area.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.23

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