奶山羊干酪性淋巴结炎病原的分离鉴定及血清学调查

发布时间：2018-09-18 19:11

【摘要】：奶山羊规模化养殖已成为我国奶山羊产业的主要发展方向,由于羊奶价格的竞争优势,养殖奶山羊的地区也越来越多,如河南、黑龙江、福建、贵州等省份。在我国奶山羊主要养殖省份陕西和云南,规模化养殖已经被养殖者普遍接受。但奶山羊传染病至今仍是严重制约我国奶山羊产业规模化健康发展的主要因素之一,尤其是难以治愈的奶山羊干酪性淋巴结炎(又名山羊伪结核病,Caseous lymphadenitis(CLA))。患病奶山羊不仅出现体重下降、生产性能差、繁殖功能障碍等疾病症状。尤为严重的是,泌乳羊乳汁中含有一定数量的病原菌,从而对喜食生羊乳的消费者健康造成巨大隐患。奶山羊干酪性淋巴结炎在我国各地很多养殖场都可看到,但更为准确地流行病学调查数据仍是空白,这也是本研究要解决的主要问题之一。本研究选取陕西富、陇县和云南泸西的奶山羊养殖场,采集疑似山羊干酪性淋巴结炎病羊脓肿脓汁,采用细菌分离培养、显微镜观察、生化试验、协同溶血试验、PCR、动物致病性试验等方法对病原菌进行分离鉴定;采用间接ELISA对采集的奶山羊血清进行CLA抗体测定。研究结果如下:1.从陕西富平、陇县和云南泸西奶山羊羊群采集的样品中共分离到3株菌株。分离菌株在普通琼脂平板上生长贫瘠,形成灰白色、圆形、较干燥小菌落;麦康凯琼脂平板上不生长。鲜血琼脂平板上可见灰白色、不透明、同心圆状、边缘不齐、有狭窄β溶血环菌落,传代后溶血现象消失。LB液体培养基中表面形成白色菌膜,管底有颗粒状、絮状沉淀。细菌镜检革兰氏染色阳性,无荚膜,无芽孢,不运动,多呈棒状或球杆状。细菌生化特性与《伯杰细菌鉴定手册》所述伪结核棒状杆菌生化特性基本一致。2.在绵羊鲜血平板上,分离菌株能与马红球菌呈现特征性协同溶血现象。3.分子生物学检测显示3株菌株与Gen Bank中已收录的伪结核棒状杆菌参考株亲缘关系97%以上。4.Balb/c小鼠致病性试验和奶山羊回归试验证实分离菌株具有很强的致病性。5.陕西富平、陇县和云南泸西3个地区奶山羊干酪性淋巴结炎血清抗体阳性率为59.92%。其中,富平、陇县和泸西奶山羊干酪性淋巴结炎血清抗体阳性率分别为:71.50%、67.50%和25.47%;半放牧半舍饲养殖、集约化养殖和合作社养殖下奶山羊干酪性淋巴结炎血清抗体阳性率分别为:25.47%、67.66%和75%。6.经两样本百分数检验发现:成年奶山羊比其他生理阶段的山羊对奶山羊干酪性淋巴结炎更易感;与其他地区奶山羊相比,云南泸西奶山羊对山羊干酪性淋巴结炎最不易感。在不同养殖模式下,合作社养殖奶山羊对山羊干酪性淋巴结炎最易感。结论:1.成功分离3株山羊干酪性淋巴结炎病原菌;2.明确陕西富平、陇县和云南泸西奶山羊CLA现行流行情况。
[Abstract]:Large-scale breeding of dairy goats has become the main development direction of dairy goat industry in China. Due to the competitive advantage of goat milk prices, there are more and more areas where milk goats are raised, such as Henan, Heilongjiang, Fujian, Guizhou and other provinces. In Shaanxi and Yunnan, the main provinces of dairy goat farming in China, large-scale farming has been widely accepted by farmers. However, dairy goat infectious disease is still one of the major factors restricting the healthy development of dairy goat industry, especially caseous lymphadenitis (CLA)., which is difficult to cure. Sick dairy goats not only have weight loss, poor production performance, reproductive dysfunction and other disease symptoms. Especially, the milk of lactating sheep contains a certain number of pathogens, which causes a great danger to the health of consumers who like to eat raw goat milk. Cheese lymphadenitis of dairy goats can be seen in many farms in China, but more accurate epidemiological survey data are still blank, which is one of the main problems to be solved in this study. In this study, milk goat farms in Fu, Longxian and Luxi, Yunnan Province were selected to collect abscess from goat abscess suspected to be caseous lymphadenitis. Bacteria isolation culture, microscopic observation and biochemical test were used. The pathogenic bacteria were isolated and identified by synergistic hemolysis test and animal pathogenicity test. Indirect ELISA was used to detect the CLA antibody in the collected milk goat serum. The results are as follows: 1. Three strains were isolated from sheep samples collected from sheep in Fuping, Longxian and Luxi, Yunnan. The isolated strains grew barren on the ordinary Agar plate and formed a small colony with gray white round and dry while that of wheat Kang Kai did not grow on the Agar plate. On the blood Agar plate, there were grayish white, opaque, concentric circle, irregular edge, narrow 尾 hemolytic circle colony. After passage, the hemolysis phenomenon disappeared. The surface of the liquid culture medium of LB formed white bacterial membrane, and the bottom of the tube was granular and flocculent. Microscopical examination showed Gram-positive staining, no capsule, no spores, no movement, mostly stick or club shape. The biochemical characteristics of bacteria are basically consistent with those of Corynebacterium pseudotuberculosis described in Berger's Manual of bacterial Identification. On the sheep blood plate, the isolated strain showed characteristic synergistic hemolysis with Erythrococcus equisetii. 3. Molecular biology analysis showed that the relationship between the three strains and the reference strain of Corynebacterium pseudotuberculosis in Gen Bank was more than 97%. 4. The pathogenicity test of Balb / c mice and the regression test of dairy goat confirmed that the isolated strains had strong pathogenicity. 5. The positive rate of serum antibodies against caseous lymphadenitis of dairy goats in three areas of Fuping, Longxian and Luxi of Yunnan was 59.92. The positive rates of antibodies against caseous lymphadenitis in Fuping, Longxian and Luxi dairy goats were 67.50% and 25.47%, respectively. The positive rates of serum antibodies against caseous lymphadenitis of dairy goats in intensive farming and cooperative farming were 67.66% and 75.6%, respectively. It was found that adult dairy goats were more susceptible to cheesy lymphadenitis than those in other physiological stages, and Luxi dairy goats were the least susceptible to caseous lymphadenitis compared with other dairy goats. In different breeding modes, dairy goats were most susceptible to caseous lymphadenitis in cooperative farming. Conclusion 1. Three strains of caseous lymphadenitis were isolated successfully. To determine the current prevalence of CLA in Fuping, Longxian and Luxi dairy goats in Yunnan.
【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】：2017
【分类号】：S858.27

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