【摘要】：近年來,養(yǎng)羊業(yè)迅速發(fā)展,集約化程度逐漸增高。然而,潛在的問題也初露端倪,疫病發(fā)生和傳播的頻率日益增加,疫病防控體系不完善,飼養(yǎng)管理水平落后,新型疾病層出不窮,導致細菌病始終難以得到有效的控制。2014年,寧陽、汶上、萊蕪、滕州等多個規(guī)�；驁龀霈F(xiàn)以呼吸道疾病為主的傳染性疫病,給養(yǎng)殖戶帶來巨大的經(jīng)濟損失。此次疫病發(fā)病急,死亡率高,屠宰后多有明顯的肺炎病變,其致病原因一時難以斷定。為深入探明其致病因子,本研究對多個規(guī)�；驁鲞M行流行病學調(diào)查,對致病菌進行分離鑒定和系統(tǒng)發(fā)育分析的研究。本研究共分為三部分:第一部分規(guī)�；驁鲆卟×餍胁W調(diào)查本部分通過對規(guī)模化羊場進行的系統(tǒng)流行病學調(diào)查,表明規(guī)�；驁鲋袀魅静「腥韭屎退劳雎识驾^普通病和寄生蟲病要高,傳染病發(fā)病率和死亡率分別為21.5%和4.5%,寄生蟲病發(fā)病率和死亡率分別為17.0%和2.5%,而普通病發(fā)病率和死亡率分別為10.0%和1.5%。羊場飼養(yǎng)管理水平低下,安全體系不健全是導致傳染性疾病感染率升高的重要原因,說明在生產(chǎn)實踐中安全防治體系的形成和良好的飼養(yǎng)管理條件非常重要。第二部分規(guī)�；驁黾毦圆≡姆蛛x鑒定本部分從采集的45頭份病料中主要分離出80株、5種病原菌。通過病原的分離培養(yǎng)、染色鏡檢、生化試驗和16SrRNA序列測定發(fā)現(xiàn)病原菌主要為鏈球菌、葡萄球菌、肺炎克雷伯菌、大腸桿菌和溶血曼氏桿菌。通過動物攻毒試驗發(fā)現(xiàn),接種12-24小時,各試驗組小白鼠均有呼吸困難、活動減少、被毛逆立、采食減少等癥狀。其中,溶血曼氏桿菌組接種3天后開始陸續(xù)全部死亡,肺炎克雷伯菌組接種后第二天開始陸續(xù)死亡,兩組小白鼠死亡后剖檢均能看到肺部嚴重病變,大腸桿菌組和對照組小白鼠基本沒有死亡。溶血曼氏桿菌組、肺炎克雷伯菌組和葡萄球菌組小白鼠死亡率分別為100%、80%、60%,并且從死亡小白鼠體內(nèi)能分離到相對應的病原菌,說明溶血曼氏桿菌、肺炎克雷伯菌和葡萄球菌為強致病菌。選用15種常用藥物,用紙片法對分離的幾種菌進行敏感性試驗,結(jié)果表明,多數(shù)菌對氧氟沙星、呋喃妥因和頭孢類藥物敏感性較強。溶血曼氏桿菌對頭孢類、喹諾酮類和呋喃妥因等抗生素敏感性較強,對新霉素、卡那霉素、慶大霉素等氨基糖苷類抗生素產(chǎn)生了一定的耐藥性。肺炎克雷伯菌、鏈球菌、大腸桿菌藥敏試驗結(jié)果一致性較強,對呋喃妥因和慶大霉素不同程度敏感,對頭孢類和喹諾酮類藥物產(chǎn)生了較強的耐藥性。葡萄球菌對多數(shù)藥物比較敏感,臨床上選用藥物一定要注意效果和效益的結(jié)合。第三部分導致肺炎的病原菌16SrRNA基因序列的測定與系統(tǒng)發(fā)育分析本部分將從病變肺臟中分離純化得到的菌株進行16SrRNA基因序列測序,結(jié)果表明導致肺炎的病原菌為溶血曼氏桿菌。將該菌與GenBank中登錄的PA標準菌株通過DNAStar進行同源性分析,結(jié)果表明分離到的溶血曼氏桿菌與美國、新疆、廣東等地的菌株同源性均在99.5%以上,說明目前地區(qū)之間和不同動物源性的溶血曼氏桿菌,16SrRNA序列基因突變較少,這有利于規(guī)模化羊場運用分子診斷學技術(shù)對其進行鑒定,采取科學防治手段對曼氏桿菌病進行有效控制。
[Abstract]:In recent years, sheep farming has developed rapidly and intensified gradually. However, potential problems have emerged, the incidence and spread of epidemic diseases are increasing, the epidemic prevention and control system is imperfect, the level of feeding and management is backward, and new diseases emerge in endlessly, which makes it difficult to effectively control bacterial diseases. Infectious diseases, mainly respiratory diseases, occurred in many large-scale sheep farms, such as Tengzhou, which brought huge economic losses to farmers. The epidemic occurred in an emergency, with a high mortality rate. After slaughtering, there were many obvious pneumonia lesions. The pathogenesis was difficult to determine for a while. In order to further explore the pathogenic factors, this study carried out flow in many large-scale sheep farms. This study is divided into three parts: the first part is the epidemiological investigation of epidemic diseases in large-scale sheep farms. Through the systematic epidemiological investigation on large-scale sheep farms, it shows that the infection rate and mortality of infectious diseases in large-scale sheep farms are higher than that of common diseases and mailing. The morbidity and mortality of infectious diseases were 21.5% and 4.5% respectively, the morbidity and mortality of parasitic diseases were 17.0% and 2.5% respectively, while the morbidity and mortality of common diseases were 10.0% and 1.5% respectively. The second part is the isolation and identification of bacterial pathogens in sheep farms. In this part, 80 strains and 5 kinds of pathogens were isolated from 45 samples. Pathogens were found by isolation and culture, staining microscopy, biochemical test and 16S rRNA sequencing. The main bacteria were Streptococcus, Staphylococcus, Klebsiella pneumoniae, Escherichia coli and Mansonella haemolyticus. It was found that after 12-24 hours of inoculation, all the mice in each experimental group had symptoms of dyspnea, decreased activity, reversed coat, and reduced feeding intake. Klebsiella pneumoniae group began to die the next day after inoculation. Severe lung lesions were observed in both groups of mice after death. Escherichia coli group and control group mice did not die. The mortality rates of mice in hemolytic Mansoni group, Klebsiella pneumoniae group and Staphylococcus group were 100%, 80%, 60% respectively, and from the dead mice. The results showed that most of the bacteria were susceptible to ofloxacin, furantoin and cephalosporins. Hemolytic bacteria were susceptible to cephalosporins. Quinolones and furantoins are highly sensitive to aminoglycosides such as neomycin, kanamycin and gentamicin. Klebsiella pneumoniae, Streptococcus and Escherichia coli have high consistency in drug susceptibility test results. They are sensitive to furantoin and gentamicin to varying degrees, and to cephalosporins and quinolones. Staphylococcus aureus is sensitive to most of the drugs, so we must pay attention to the combination of effect and benefit when we choose the drugs in clinic. Part 3: Determination of 16S rRNA gene sequence and phylogenetic analysis of pathogenic bacteria causing pneumonia The result of sequencing showed that the pathogenic bacteria causing pneumonia was M. haemolyticus. The homology analysis between M. haemolyticus and the PA standard strain registered in GenBank by DNA Star showed that the isolates of M. haemolyticus had more than 99.5% homology with the strains from the United States, Xinjiang, Guangdong and other places, indicating that the current region and different activities. The genetic mutation of 16S rRNA sequence in the material-derived M. haemolyticus is less than that in the other strains, which is helpful for the identification of M. haemolyticus by molecular diagnostic techniques and the effective control of M. haemolyticus disease by scientific means.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.26

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