【作者】 刘伟；

【导师】 王海荣；

【作者基本信息】 山东农业大学 ， 兽医（专业学位）， 2018， 硕士

【摘要】 产气荚膜梭菌(Clostridium perfringens)广泛存在于土壤、人和动物肠道中,可引起人和动物多种疾病。兔产气荚膜梭菌病主要是由A型产气荚膜梭菌引起的一种以剧烈腹泻为特征的急性、致死性传染病。此病发病急、死亡率高,给各国畜牧业带来巨大经济损失。本研究对2016年11月和2017年11月山东省临沂和新泰2个规模化兔场疑似产气荚膜梭菌病疫情进行诊断。采用分离培养鉴定、DAS-ELISA方法与免疫组织化学方法检测临床疑似产气荚膜梭菌病兔样品,运用多种方法对产气荚膜梭菌病进行综合诊断。对由病兔分离的病原菌进行血清型鉴定、耐药性分析,并利用卵磷脂水解试验、DAS-ELISA方法测定分离菌株的最佳产毒时间和产毒能力,筛选出产毒效果好的菌株,为此病的诊断和防控提供参考。临床症状观察和剖检结果显示,疑似自然感染产气荚膜梭菌病兔的临床特征和各个器官的病变与产气荚膜梭菌病变特征相符;通过细菌分离鉴定,分离到产气荚膜梭菌菌株共计9株,其中临沂兔场阳性分离率为66.7%,新泰兔场阳性分离率为83.3%,经多重PCR鉴定均为A型产气荚膜梭菌。DAS-ELISA法检测疑似产气荚膜梭菌病兔盲肠内容物毒素水平,其中临沂兔场和新泰兔场阳性检出率均为83.3%。同时采取临床疑似产气荚膜梭菌病兔的肝脏、肾脏、胃做免疫组化,以至少有一个器官检测到α毒素作为阳性判定依据,其中临沂兔场阳性检出率为83.3%,新泰兔场阳性检出率为100%。药敏结果显示:临沂兔场病兔分离菌株对庆大霉素、四环素耐药,耐药率均达到了100%,对青霉素、氨苄西林、红霉素、诺氟沙星、环丙沙星、林可霉素和杆菌肽敏感;新泰兔场病兔分离菌株对庆大霉素、四环素、青霉素、氨苄西林、红霉素和杆菌肽耐药,耐药率分别为100%、60%、60%、60%、60%和20%,对诺氟沙星、环丙沙星、林可霉素敏感;临沂和新泰两兔场菌株耐药性有显著差异。本试验对β﹣内酰胺类耐药菌株进行了抗性基因检测,仅从一株分离菌株中检测到了CTX-M基因,没有检测到SHV和TEM基因型。利用DAS-ELISA方法、卵磷脂水解试验测定分离菌株不同培养时间产毒能力,结果显示:产毒时间不要超过10 h,最好控制在6-10 h,此时间段不同菌株的产气荚膜梭菌α毒素抗原含量和酶活性均处于较稳定水平。且发现分离菌株T12增殖能力好,产毒能力较为稳定,适合用于制备疫苗。更多还原

【Abstract】 Clostridium perfringens has been widely existed in soil,intestines of humans and animals,which can cause various diseases.Clostridium perfringens disease in rabbits is an acute,lethal diarrhea infectious disease caused by type A Clostridium perfringe ns.Clostridium perfringens disease is acute onset and high mortality,which causes hu ge economic losses to livestock countries.This study was conducted to diagnose the suspected sick rabbits from two scaled rabbit farms in Linyi and Xintai,Shandong Province in November 2016 and November 2017.The suspected sick rabbits were detected by bacterial separation and identification,DAS-ELISA and immunohistochemistry,which was used for the comprehensive diagnosis of Clostridium perfringens disease.The serotype identification and drug resistance analysis were performed on pathogens isolated from diseased rabbits,and the best toxin production time and the ability of toxin production were detected by using lecithin hydrolysis assay and DAS-ELISA method,screening out strains with good toxin production effects,in order to provide reference for the diagnosis and prevention of Clostridium perfringens disease.Clinical symptoms observation and necropsy results showed that the clinical features and the pathological changes of suspected sick rabbits were consistent with the rabbits which are infected with Clostridium perfringens.By means of bacterial separation and identification,a total of 9 isolates were obtained,which the positive isolation rate in Linyi and Xintai rabbit farm was 66.7% and 83.3%,respectively.By using the multiple PCR identification,all of the isolates were identified as Clostridium perfringens type A.DAS-ELISA was used to detect the level of toxins in cecal contents of rabbits with suspected Clostridium perfringens.The positive rates of Linyi and Xintai rabbit farm were 83.3%.The liver,kidney and stomach of sick rabbits were detected useing immunohistochemical method,as a positive criterion,αtoxin should be detected in one organ at least,the positive rate in Linyi and Xintai rabbit farm was 83.3% and 100%,respectively.Antimicrobial susceptibility test results showed that the isolates of sick rabbits fromLinyi rabbit farm were resistant to gentamicin and tetracycline,the drug resistance rate reached 100%,and the isolates were sensitive to penicillin,ampicillin,erythromycin,norfloxacin,ciprofloxacin,lincomycin,and bacitracin;The isolates of sick rabbits from Xintai rabbit farm were resistant to gentamicin,tetracycline,penicillin,ampicillin,erythromycin and bacitracin,the resistance rates were 100%,60%,60%,60%,60% and 20%,respectively;The isolates of sick rabbits from Xintai rabbit farm were sensitive to norfloxacin,ciprofloxacin,and lincomycin.There was a significant difference in the drug resistance between isolates of the two rabbit farms.In this study,three genotypes of CTX-M,SHV and TEM were used for detecting the β-lactam resistance genes.CTX-M gene was detected from only one isolates,and no SHV and TEM genotypes were detected.The DAS-ELISA method and lecithin hydrolysis assay were used to detect the ability of isolates in producing toxin at different culture times.The results showed that the time of toxin production should not exceed 10 h,and it is better to control it between 6-10 h.The antigenic content and enzyme activity of the alpha toxin of Clostridium perfringens in different isolates were at a relatively stable level during that period.The isolated strain T12 had a good proliferation ability and stable toxin production ability,which was suitable for the preparation of vaccines.更多还原