规模化羊场细菌病的流行病学调查及细菌性病原的分离鉴定和系统进化分析

发布时间：2018-08-11 21:45

【摘要】：近年来,养羊业迅速发展,集约化程度逐渐增高。然而,潜在的问题也初露端倪,疫病发生和传播的频率日益增加,疫病防控体系不完善,饲养管理水平落后,新型疾病层出不穷,导致细菌病始终难以得到有效的控制。2014年,宁阳、汶上、莱芜、滕州等多个规模化羊场出现以呼吸道疾病为主的传染性疫病,给养殖户带来巨大的经济损失。此次疫病发病急,死亡率高,屠宰后多有明显的肺炎病变,其致病原因一时难以断定。为深入探明其致病因子,本研究对多个规模化羊场进行流行病学调查,对致病菌进行分离鉴定和系统发育分析的研究。本研究共分为三部分:第一部分规模化羊场疫病流行病学调查本部分通过对规模化羊场进行的系统流行病学调查,表明规模化羊场中传染病感染率和死亡率都较普通病和寄生虫病要高,传染病发病率和死亡率分别为21.5%和4.5%,寄生虫病发病率和死亡率分别为17.0%和2.5%,而普通病发病率和死亡率分别为10.0%和1.5%。羊场饲养管理水平低下,安全体系不健全是导致传染性疾病感染率升高的重要原因,说明在生产实践中安全防治体系的形成和良好的饲养管理条件非常重要。第二部分规模化羊场细菌性病原的分离鉴定本部分从采集的45头份病料中主要分离出80株、5种病原菌。通过病原的分离培养、染色镜检、生化试验和16SrRNA序列测定发现病原菌主要为链球菌、葡萄球菌、肺炎克雷伯菌、大肠杆菌和溶血曼氏杆菌。通过动物攻毒试验发现,接种12-24小时,各试验组小白鼠均有呼吸困难、活动减少、被毛逆立、采食减少等症状。其中,溶血曼氏杆菌组接种3天后开始陆续全部死亡,肺炎克雷伯菌组接种后第二天开始陆续死亡,两组小白鼠死亡后剖检均能看到肺部严重病变,大肠杆菌组和对照组小白鼠基本没有死亡。溶血曼氏杆菌组、肺炎克雷伯菌组和葡萄球菌组小白鼠死亡率分别为100%、80%、60%,并且从死亡小白鼠体内能分离到相对应的病原菌,说明溶血曼氏杆菌、肺炎克雷伯菌和葡萄球菌为强致病菌。选用15种常用药物,用纸片法对分离的几种菌进行敏感性试验,结果表明,多数菌对氧氟沙星、呋喃妥因和头孢类药物敏感性较强。溶血曼氏杆菌对头孢类、喹诺酮类和呋喃妥因等抗生素敏感性较强,对新霉素、卡那霉素、庆大霉素等氨基糖苷类抗生素产生了一定的耐药性。肺炎克雷伯菌、链球菌、大肠杆菌药敏试验结果一致性较强,对呋喃妥因和庆大霉素不同程度敏感,对头孢类和喹诺酮类药物产生了较强的耐药性。葡萄球菌对多数药物比较敏感,临床上选用药物一定要注意效果和效益的结合。第三部分导致肺炎的病原菌16SrRNA基因序列的测定与系统发育分析本部分将从病变肺脏中分离纯化得到的菌株进行16SrRNA基因序列测序,结果表明导致肺炎的病原菌为溶血曼氏杆菌。将该菌与GenBank中登录的PA标准菌株通过DNAStar进行同源性分析,结果表明分离到的溶血曼氏杆菌与美国、新疆、广东等地的菌株同源性均在99.5%以上,说明目前地区之间和不同动物源性的溶血曼氏杆菌,16SrRNA序列基因突变较少,这有利于规模化羊场运用分子诊断学技术对其进行鉴定,采取科学防治手段对曼氏杆菌病进行有效控制。
[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.
【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】：S858.26

【共引文献】