鸡滑液囊支原体JS1株的分离鉴定及禽支原体、大肠杆菌、沙门菌多重PCR检测方法的建立

发布时间：2017-12-28 02:01

本文关键词：鸡滑液囊支原体JS1株的分离鉴定及禽支原体、大肠杆菌、沙门菌多重PCR检测方法的建立　出处：《山东农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：鸡滑液囊支原体感染是由滑液支原体(Mycoplasma synoviae,MS)引起的,鸡和火鸡的一种急慢性传染病,其临床以病鸡关节或足垫肿大、滑液囊和腱鞘发炎为主要特征,也可引起呼吸道疾病及气囊炎,严重的引起全身性感染,也可导致新城疫病毒、传染性支气管炎病毒、大肠杆菌等病原微生物的混合或继发感染。该病在世界范围内流行,多个国家的流行病学监测表明MS在商品代鸡群中有很高的流行率。近几年滑液支原体在中国的发病率有增多趋势,很多地区季节性发病相当严重。MS感染鸡群可导致跛行、产蛋量下降、生长发育迟缓及酮体降级等危害,严重影响鸡的生产性能,给世界商品养鸡产业造成巨大的经济损失。本研究从江苏某鸡场收集病鸡,对其进行支原体分离,获得一株疑似支原体的分离物,通过菌落观察、血清学实验、支原体16S r RNA通用引物测序鉴定及与实验室标准株MS WVU1853差异性鉴定,确定该分离物为滑液支原体,且不同于本实验室的MS标准株(MS WVU1853),因此将其命名为JS1株。本研究对滑液支原体SPF鸡攻毒模型进行了初步探索,将分离株MS JS1对21日龄SPF鸡进行攻毒,分别采用关节注射、肌肉注射、足垫注射和滴鼻四组不同攻毒方式,每组10只SPF鸡,攻毒剂量为每只1×109 CCU。结果表明攻毒方式不同,其发病率也不一致,其中关节攻毒组发病率最高,达9/10,其后依次是足垫攻毒组发病率为8/10,滴鼻攻毒组发病率为4/10,肌肉攻毒组发病率为0。除肌肉攻毒组无发病症状外,其余三种攻毒方式均能引起不同程度的特征性病变,即鸡关节囊肿、足垫肿大、胸部皮下囊肿、跛足以及鸡冠苍白等,对发病鸡部分剖检也能从关节、足垫或肺组织中分离到滑液支原体。针对滑液支原体(Mycoplasma synoviae,MS)的pdh A基因,鸡毒支原体(Mycopl asma gallisepticum,MG)的gap A基因,大肠杆菌(Escherichia coli,EC)的pho A基因,沙门菌(Salmonella,SA)的inv A基因分别设计2-3对特异性引物,通过筛选引物组合、优化体系条件,最后确定了一组最优引物组合和反应体系,建立了针对这四种禽常见致病菌的多重PCR检测方法。特异性实验证明该方法能特异性扩增出MS、MG、EC和SA的目的片段,其他非相关的禽致病菌则无扩增产物。敏感性检测发现MS和MG的检测灵敏度均达到1×102 CCU/反应,EC的检测灵敏度1×102 CFU/反应,SA的检测灵敏度1×103 CFU/反应。临床感染组织样本检测结果显示,多重PCR检测结果与分离鉴定结果一致,检测率可达83.8%。本实验对MS分离株JS1进行了分离鉴定,初步建立了滑液支原体对SPF鸡的攻毒模型,并成功建立能同时检测禽常见致病菌MS、MG、EC和SA的多重PCR检测方法,为滑液支原体感染流行病学调查研究以及进一步开展滑液支原体疫苗的研制奠定了基础。
[Abstract]:Mycoplasma synoviae infection by Mycoplasma synoviae (Mycoplasma synoviae, MS) caused by the chicken and Turkey for acute and chronic infectious diseases, the clinical disease in chicken joint or footpad swelling, synovial capsule and tendon sheath inflammation as the main feature, can also cause respiratory disease and airsacculitis, caused by severe systemic infection also, can lead to Newcastle disease virus, infectious bronchitis virus, Escherichia coli and other pathogenic microorganisms or mixed infection. The epidemic is prevalent throughout the world, and epidemiological surveillance in many countries shows that MS has a high prevalence rate in commercial chicken flocks. In recent years, the incidence of Mycoplasma synovium in China has been increasing, and the seasonal incidence of the disease is very serious in many areas. MS infected chickens can cause lameness, egg production, growth retardation and ketone body degradation, which seriously affect the production performance of chicken and cause huge economic losses to the world's chicken industry. This study collected the sick chicken from a chicken farm in Jiangsu, for the isolation of Mycoplasma isolates, obtained a strain of mycoplasma, through observation, serological test, Mycoplasma colony 16S R RNA universal primers and sequencing with standard laboratory strains of MS WVU1853 were identified, confirmed the isolate for Mycoplasma synoviae and is different from the laboratory. The MS standard strain (MS WVU1853), it will be named JS1 strain. This study conducted a preliminary exploration on Mycoplasma synoviae SPF chicken infection model, will isolate MS JS1 on the 21 day old SPF chickens were challenged by intraarticular injection, intramuscular injection, foot pad injection and intranasal inoculation of four different groups, 10 rats in each group of SPF chickens challenged dose for each 1 * 109 CCU. The results showed that the incidence of attack was different. The incidence of joint attack group was the highest, reaching 9/10, followed by foot pad attack group, the incidence rate was 8/10, the incidence of intranasal attack group was 4/10, and the incidence of muscle attack group was 0. In addition to the muscle inoculation group without symptoms, the other three kinds of virus attack methods can induce the characteristic lesions in different degrees, namely chicken articular cyst, foot pad swelling, chest subcutaneous cyst, lame and comb pale, on the incidence of chickens can be separated from the autopsy joint, foot pad or lung tissue to Mycoplasma synoviae. For Mycoplasma synoviae (Mycoplasma synoviae MS) PDH A gene of Mycoplasma gallisepticum (Mycopl ASMA, gallisepticum, MG) gap A gene in Escherichia coli (Escherichia, coli, EC) Pho A gene, Salmonella (Salmonella, SA) inv A gene specific primers were designed for 2-3, through the screening of primer combinations and the optimization of system conditions, finally identified a set of optimal primers and reaction system was established for the four common multiple PCR avian pathogen detection method. Specific experiments showed that the method could specifically amplify the target fragments of MS, MG, EC and SA, and other unrelated avian pathogenic bacteria had no amplification products. Sensitivity test showed that the detection sensitivity of MS and MG reached 1 * 102 CCU/ reaction. The sensitivity of EC was 1 * 102 CFU/, and the sensitivity of SA was 1 * 103 CFU/ reaction. The results of detection of tissue samples from clinical infection showed that the results of multiple PCR detection were in accordance with the results of isolation and identification, and the detection rate could reach 83.8%. JS1 of MS isolates were isolated and identified in the experiment, established the model of poison Mycoplasma synoviae to SPF chicken, and multiple PCR detection method for simultaneous detection of avian pathogenic bacteria MS, MG, EC and SA is established successfully, which laid the foundation for the epidemiological investigation of Mycoplasma synoviae infection and the development of the further development of Mycoplasma synoviae vaccine.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

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