A型产气荚膜梭菌家兔感染模型的建立

发布时间：2018-03-25 12:26

本文选题：A型产气荚膜梭菌　切入点：分离鉴定　出处：《山东农业大学》2015年硕士论文

【摘要】：近年来,我国许多兔场频繁爆发兔腹泻性肠炎,主要由A型产气荚膜梭菌引起的一种肠道疾病,给兔养殖户造成很大的经济损失。该病发病迅速,死亡率高,所以对该病的预防工作至关重要。本试验建立的A型产气荚膜梭菌的感染模型,不仅为产气荚膜梭菌病有关疫苗的效力检验;抗毒素血清的治疗效果评价提供了感染模型,还为评估菌株的毒力,产气荚膜梭菌的致病机制研究提供参考。通过细菌的分离培养,染色涂片镜检,分离纯化,生化实验及多重PCR初步诊断临床病例中分离的产气荚膜梭菌基因型为A型。从临床发病兔的肝脏共分离出4株A型产气荚膜梭菌,和参考菌株NCTC 528经过菌种的复苏、增菌和产毒素培养,所产毒素进行卵磷脂酶活性实验和小鼠LD50的测定和比较。在相同的产毒条件下得到产毒效果比较好的菌株(济宁株1)。NCTC 528和济宁株1菌株经过重新复苏、增菌和产毒,所得的培养物用于感染家兔。培养物通过不同处理方式、不同感染方式接种2kg健康家兔,研究结果表明:腹腔注射NCTC528除菌毒素(小鼠LD50:24.25;卵磷脂酶活性:24)可在6-8小时致死家兔;最小致死剂量是3ml;肌肉注射528除菌毒素(小鼠LD50:24.25;卵磷脂酶活性:24)的最小致死剂量是4ml;肌肉注射全菌液(小鼠LD50:25.25;毒素卵磷脂酶活性:25,细菌浓度1.3×109cfu/ml)最小致死量是5ml,对腹腔和肌肉注射的感染死亡兔进行剖检病变和组织病理学病变的观察,病变主要特征是:腹腔和肌肉注射除菌毒素都出现了胃黏膜脱落和小肠黏膜弥散性出血现象;但胃粘膜有溃疡灶,盲肠浆膜面有出血病变少见。分别以培养物(细菌浓度1.3×109cfu/ml,卵磷脂酶活性效价25),30ml,40ml,50ml灌服2kg家兔,并饲喂不同饲料配方饲料,仅见短期腹泻,未见死亡。2Kg家兔回肠注射感染全菌液(细菌浓度1.6×108cfu/ml,卵磷脂酶活性为24)5ml、8ml、10ml。部分兔出现腹泻死亡,不能稳定复制病变,血常规监测发现:攻毒后一周淋巴细胞比率、血小板总数和白细胞总数明显比正常值偏高,证明体内发生炎症反应。感染后3周血清中A型产气荚膜梭菌毒素抗体效价为1:200-1:400。据攻毒后存活家兔的肠道菌群分析发现产气荚膜梭菌在这些肠内容物(空肠、回肠、盲肠)中,并不是优势菌。
[Abstract]:In recent years, many rabbit farms in China frequently outbreak diarrhoeal enteritis, mainly caused by Clostridium perfringens type A, which causes great economic losses to rabbit farmers. The infection model of Clostridium perfringens type A not only provides an infection model for testing the efficacy of the vaccine related to Clostridium perfringens, but also provides an infection model for the evaluation of the therapeutic effect of antitoxin serum. It also provides a reference for evaluating the virulence of the strain and studying the pathogenic mechanism of Clostridium perfringens. The genotypes of Clostridium perfringens isolated from clinical cases diagnosed by biochemical experiments and multiple PCR were genotype A. four strains of Clostridium perfringens type A were isolated from the liver of clinically infected rabbits, and the reference strain NCTC 528 was resuscitated. Lecithin enzyme activity of the toxin and LD50 of mice were tested and compared. Under the same conditions, the strains producing the toxin (Jining strain 1).NCTC 528 and Jining strain 1) were resuscitated after resuscitation, and the strain of Jining strain 1).NCTC 528 and the strain of Jining strain 1 were resuscitated. The culture was used to infect rabbits. The culture was inoculated with 2kg healthy rabbits by different treatment and different infection methods. The results showed that intraperitoneal injection of NCTC528 sterilizing toxin (LD50: 24.25; lecithin activity: 24) could kill rabbits in 6-8 hours. The minimum lethal dose for intramuscular injection of 528 sterilizing toxin (LD50: 24.25; lecithin activity: 24) is 4 ml; the minimum lethal dose for intramuscular injection of 528 sterilizing toxin (LD50: 25.25); the minimum lethal dose for intramuscular injection of bacteriostasis (LD50: 25.25; bacterial concentration 1.3 脳 10 9 cfumlP) is 5 ml. The pathological changes and histopathological changes were observed in the dead rabbits infected by intraperitoneal and intramuscular injection. The main features of the lesion are: intraperitoneal and intramuscular injection of sterilizing toxin has occurred gastric mucosal shedding and small intestinal mucosal diffuse bleeding phenomenon, but gastric mucosal ulcer focus, Bleeding lesions on the serosa of cecum were rare. Culture (bacterial concentration 1.3 脳 10 9 cfur / ml, lecithin enzyme activity titer 25 ~ 30 ml ~ 40 ml ~ (50 ml)) was administered to 2kg rabbits, and fed with different feed formulations, only short-term diarrhea was observed. There was no death. 2Kg rabbit ileum infecting the whole bacterial fluid (bacterial concentration 1.6 脳 10 8 cfur / ml, lecithin enzyme activity 24 5 ml 8 ml 10 ml). Some rabbits died of diarrhea and could not replicate the disease stably. The blood routine monitoring found that the lymphocyte ratio was 1 week after exposure to the virus. The total number of platelets and white blood cells were obviously higher than the normal value. The antibody titer of Clostridium perfringens type A toxin in serum was 1: 200-1: 400 at 3 weeks after infection. According to the intestinal flora analysis of surviving rabbits after infection, it was found that Clostridium perfringens was found in these intestinal contents (jejunum, ileum, cecum). Is not the dominant bacteria.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.291

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