奶山羊干酪性淋巴结炎抗体ELISA检测方法研究及血清流行病学初步调查

发布时间：2018-03-18 15:11

本文选题：伪结核棒状杆菌　切入点：分离鉴定　出处：《西北农林科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：山羊干酪性淋巴结炎(又称山羊伪结核)是由伪结核棒状引起的山羊和绵羊的一种慢性的人兽共患病,临床上有体表型、内脏型和混合型三种表现形式,在全世界各地均有流行。患病羊只渐行性消瘦,产毛量、产肉量、产奶量下降,并伴有繁殖功能障碍,给羊业养殖造成了很大的经济损失。尤其近年来随着山羊养殖数量和规模的大幅度增加,本病表现得越来越突出。我国奶山羊的主要养殖地区为陕西、山东、云南等省份,东北三省以及南方浙江、上海、贵州等省份近年来也开始大力发展奶山羊养殖。山羊干酪性淋巴结炎在现在的奶山羊主要养殖省份的流行现状至今并不清楚。本研究从陕西富平采集具有典型伪结核临床症状的山羊脓汁,从中分离得到伪结核棒状杆菌疑似菌株,观察菌落及菌体的形态,对其进行生理生化鉴定并结合协同溶血试验和分子生物学的方法对其进行鉴定;分别运用伪结核棒状杆菌培养上清液中的可溶性蛋白和伪结核棒状杆菌菌体作为包被抗原,从而筛选出建立间接ELISA抗体诊断方法的最佳的包被抗原,并对包被抗原的密度和血清的稀释度进行确定,优化封闭时间,确定阳性血清的临界值,对建立的间接ELISA抗体诊断方法的特异性、敏感性和重复性进行考察,在此基础上,对采集的临床样本进行检测;并对保存的陕西省、山东省和云南省血清样本进行检测,了解伪结核病的流行状况。研究获得如下结果:1.分离得到一株伪结核棒状杆菌,该菌在普通琼脂平板上生长缓慢,24h形成针尖大小的菌落,在5%绵羊血琼脂平板上生长迅速形成白色、干燥、易破碎的菌落,可形成狭窄的β溶血环;细菌菌体革兰氏染色阳性,呈短杆状或者是球状。生理生化鉴定结果与《伯杰细菌鉴定手册》中描述伪结核棒状杆菌生化特性基本一致,药敏试验结果表明,其对头孢唑啉、克拉霉素、红霉素等多种抗生素敏感;协同溶血试验结果表明其与马红球菌有明显的协同溶血现象;分子生物学鉴定结果显示,PCR产物测序结果与Genebank中收录的伪结核棒状杆菌的参考菌株ATCC19410相应序列的同源性可达到99%。2.经筛选,伪结核棒状杆菌细菌菌体作为包被抗原使用时可以取得良好的检测效果;最佳菌体包被抗原浓度为OD600=0.080,血清稀释度为1∶400,封闭时间为2h,阳性血清的临界值为0.329;该间接ELISA抗体检测方法的检测灵敏度高,可特异性的检出伪结核阳性血清,与羊口疮阳性血清、羊痘阳性血清和乳房炎阳性血清没有交叉反应,批内重复性和批间重复性的变异系数均小于10%。该方法对12份临床样本的检测结果与实际患病状况一致。3.陕西、山东、云南的伪结核棒状杆菌抗体阳性率分别为22.3%、6.6%、35.7%。结论:1.成功分离山羊干酪性淋巴结炎病原;2.建立了山羊干酪性淋巴结炎抗体的间接ELISA检测方法;3.明确了我国奶山羊主要养殖地区山羊干酪性淋巴结炎的流行现状。
[Abstract]:Goat caseous lymphadenitis (also known as goat pseudotuberculosis) is a chronic zoonosis of goats and sheep caused by pseudotuberculous rod. There are three clinical manifestations of caseous lymphadenitis, visceral type and mixed type. It is popular all over the world. Sick sheep are gradually wasting, producing wool, producing meat, producing milk, and accompanied by reproductive dysfunction. This disease has become more and more prominent with the large increase in the number and scale of goat breeding in recent years. The main breeding areas of dairy goats in China are Shaanxi, Shandong, Yunnan and other provinces. The three northeastern provinces, as well as the southern provinces of Zhejiang and Shanghai, Guizhou and other provinces have started to develop dairy goat breeding in recent years. The prevalence of goat caseous lymphadenitis in the main breeding provinces of dairy goats is not clear. Tuberculosis clinical symptoms of goat pus, The suspected strains of Corynebacterium tuberculosis were isolated, the colony and the morphology of the bacteria were observed, their physiological and biochemical identification were carried out, and they were identified by means of synergistic hemolysis test and molecular biology. The soluble protein in culture supernatant of Corynebacterium tuberculosis and the bacteria of Corynebacterium pseudotuberculosis were used as coating antigens, respectively, so as to select the best coating antigen for the diagnosis of indirect ELISA antibody. The density of coated antigen and the dilution of serum were determined, the blocking time was optimized, the critical value of positive serum was determined, and the specificity, sensitivity and repeatability of the indirect ELISA antibody diagnostic method were investigated. The clinical samples were tested, and the serum samples of Shaanxi, Shandong and Yunnan provinces were tested to find out the prevalence of pseudotuberculosis. The results were as follows: 1. A strain of pseudotuberculosis corynebacterium was isolated. The bacteria grew slowly on the ordinary Agar plate for 24 hours to form a needle tip size colony, and on the 5% sheep blood Agar plate, it rapidly formed a white, dry and easily broken colony, which could form a narrow 尾 hemolytic ring, and the bacteria were positive for Gram staining. The results of physiological and biochemical identification are basically consistent with the biochemical characteristics of Corynebacterium pseudotuberculosis described in Berger's Manual of bacterial Identification. The results of drug sensitivity test show that it is good for cefazolin and clarithromycin. The results of synergistic hemolysis test showed that Erythromycin had obvious synergistic hemolysis with Erythrococcus. The results of molecular biological identification showed that the homology of the sequencing results of the PCR products with the reference strain ATCC19410 of Corynebacterium tuberculosis included in Genebank was 990.2. When used as coating antigen, Corynebacterium pseudotuberculosis bacteria can obtain good detection effect. The optimal concentration of bacterial coating antigen was OD600,0.080, the dilution of serum was 1: 400, the blocking time was 2 hours, and the critical value of positive serum was 0.329.The indirect ELISA antibody detection method had high sensitivity and could be used to detect pseudotuberculosis positive serum and sheep aphtha positive serum. There was no cross reaction between sheep pox positive serum and mastitis positive serum, and the coefficient of variation of reproducibility within and between batches was less than 10. The results of detection of 12 clinical samples by this method were in agreement with the actual condition of disease. The positive rates of antibodies against Corynebacterium pseudotuberculosis in Yunnan were 22.36.6and 35.70.Conclusion 1. The pathogen of caseous lymphadenitis in goats was successfully isolated 2.The indirect ELISA method for the detection of antibodies against caseous lymphadenitis in goats was established. The prevalence of caseous lymphadenitis in goat in order to be cultured.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.27

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