本文選題：偽結(jié)核棒狀桿菌　切入點(diǎn)：分離鑒定　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：山羊干酪性淋巴結(jié)炎(又稱山羊偽結(jié)核)是由偽結(jié)核棒狀引起的山羊和綿羊的一種慢性的人獸共患病,臨床上有體表型、內(nèi)臟型和混合型三種表現(xiàn)形式,在全世界各地均有流行�；疾⊙蛑粷u行性消瘦,產(chǎn)毛量、產(chǎn)肉量、產(chǎn)奶量下降,并伴有繁殖功能障礙,給羊業(yè)養(yǎng)殖造成了很大的經(jīng)濟(jì)損失。尤其近年來隨著山羊養(yǎng)殖數(shù)量和規(guī)模的大幅度增加,本病表現(xiàn)得越來越突出。我國奶山羊的主要養(yǎng)殖地區(qū)為陜西、山東、云南等省份,東北三省以及南方浙江、上海、貴州等省份近年來也開始大力發(fā)展奶山羊養(yǎng)殖。山羊干酪性淋巴結(jié)炎在現(xiàn)在的奶山羊主要養(yǎng)殖省份的流行現(xiàn)狀至今并不清楚。本研究從陜西富平采集具有典型偽結(jié)核臨床癥狀的山羊膿汁,從中分離得到偽結(jié)核棒狀桿菌疑似菌株,觀察菌落及菌體的形態(tài),對(duì)其進(jìn)行生理生化鑒定并結(jié)合協(xié)同溶血試驗(yàn)和分子生物學(xué)的方法對(duì)其進(jìn)行鑒定;分別運(yùn)用偽結(jié)核棒狀桿菌培養(yǎng)上清液中的可溶性蛋白和偽結(jié)核棒狀桿菌菌體作為包被抗原,從而篩選出建立間接ELISA抗體診斷方法的最佳的包被抗原,并對(duì)包被抗原的密度和血清的稀釋度進(jìn)行確定,優(yōu)化封閉時(shí)間,確定陽性血清的臨界值,對(duì)建立的間接ELISA抗體診斷方法的特異性、敏感性和重復(fù)性進(jìn)行考察,在此基礎(chǔ)上,對(duì)采集的臨床樣本進(jìn)行檢測(cè);并對(duì)保存的陜西省、山東省和云南省血清樣本進(jìn)行檢測(cè),了解偽結(jié)核病的流行狀況。研究獲得如下結(jié)果:1.分離得到一株偽結(jié)核棒狀桿菌,該菌在普通瓊脂平板上生長緩慢,24h形成針尖大小的菌落,在5%綿羊血瓊脂平板上生長迅速形成白色、干燥、易破碎的菌落,可形成狹窄的β溶血環(huán);細(xì)菌菌體革蘭氏染色陽性,呈短桿狀或者是球狀。生理生化鑒定結(jié)果與《伯杰細(xì)菌鑒定手冊(cè)》中描述偽結(jié)核棒狀桿菌生化特性基本一致,藥敏試驗(yàn)結(jié)果表明,其對(duì)頭孢唑啉、克拉霉素、紅霉素等多種抗生素敏感;協(xié)同溶血試驗(yàn)結(jié)果表明其與馬紅球菌有明顯的協(xié)同溶血現(xiàn)象;分子生物學(xué)鑒定結(jié)果顯示,PCR產(chǎn)物測(cè)序結(jié)果與Genebank中收錄的偽結(jié)核棒狀桿菌的參考菌株ATCC19410相應(yīng)序列的同源性可達(dá)到99%。2.經(jīng)篩選,偽結(jié)核棒狀桿菌細(xì)菌菌體作為包被抗原使用時(shí)可以取得良好的檢測(cè)效果;最佳菌體包被抗原濃度為OD600=0.080,血清稀釋度為1∶400,封閉時(shí)間為2h,陽性血清的臨界值為0.329;該間接ELISA抗體檢測(cè)方法的檢測(cè)靈敏度高,可特異性的檢出偽結(jié)核陽性血清,與羊口瘡陽性血清、羊痘陽性血清和乳房炎陽性血清沒有交叉反應(yīng),批內(nèi)重復(fù)性和批間重復(fù)性的變異系數(shù)均小于10%。該方法對(duì)12份臨床樣本的檢測(cè)結(jié)果與實(shí)際患病狀況一致。3.陜西、山東、云南的偽結(jié)核棒狀桿菌抗體陽性率分別為22.3%、6.6%、35.7%。結(jié)論:1.成功分離山羊干酪性淋巴結(jié)炎病原;2.建立了山羊干酪性淋巴結(jié)炎抗體的間接ELISA檢測(cè)方法;3.明確了我國奶山羊主要養(yǎng)殖地區(qū)山羊干酪性淋巴結(jié)炎的流行現(xiàn)狀。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.27

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