【作者】 张磊；

【导师】 朱瑞良；

【作者基本信息】 山东农业大学 ， 兽医， 2010， 硕士

【摘要】 鸡毒支原体(Mycoplasma Gallisepticum,MG)是鸡的慢性呼吸道病(Chronic Respiratory Disease,CRD)的病原体,其特征为呼吸啰音、咳嗽、流浆液性鼻液,气囊炎,严重时张口呼吸。通常与病毒、细菌协同感染,导致呼吸道症状加剧,使死亡率增高。MG感染在国内禽群中相当普遍,感染后鸡死亡率一般很低,主要表现为产蛋率、孵化率降低,出栏期延长和饲料利用率降低,对该病的预防和控制程序也额外增加了成本,同时造成禽产品中药物残留,给养禽业造成严重的经济损失。本研究通过对山东济南某鸡场(舍)的患呼吸道疾病的鸡进行支原体的分离,通过菌落观察及细菌L型恢复试验,分离纯化了一个支原体分离物。对所分离的支原体分离物进行了菌落形态观察、菌落红细胞吸附试验、生理生化及血清学鉴定,结果该分离物被鉴定为鸡毒支原体,命名为JN株。JN株在不含葡萄糖的液体培养基中,加入四氮唑能使培养基颜色变橙红色,在固体培养基表面所形成的菌落呈典型的油煎蛋状外观,大小约0.1～0.4mm,抗MG血清对JN株的代谢有抑制作用。通过JN株在不同血清培养基的生长情况,可以看出该菌株易于在猪血清培养基中生长,可以代替价格较贵且来源有限的马血清。从MG对鸡气管环的致病性试验结果可看出JN菌株对气管环纤毛的损伤较大,能够引起鸡胚气管环纤毛运动快速停止,致病力较F疫苗株强。同时,SDS-PAGE电泳图谱显示JN株、F株之间的蛋白条带有一定差异,可以说明JN株的致病性与结构蛋白之间存在一定的相关性。针对鸡毒支原体(MG)、鸡滑液囊支原体(Mycoplasma synoviae,MS)和火鸡支原体(Mycoplasma meleagridis,MM)设计了3对引物,大小分别为MG 732bp,MS 208bp,MM 850bp,通过单一PCR反应,分别得到了相应的目的片段,并且建立的多重PCR反应优化后,可同时得到3条目的条带,与单一PCR扩增结果一致。实验表明,由此建立的多重PCR方法可用于MG、MS和MM的同时检测和鉴别诊断,且简便快捷,灵敏度高,特异性强。针对鸡传染性支气管炎病毒(IBV)、禽流感病毒(AIV)、鸡新城疫病毒(NDV)和鸡传染性喉气管炎病毒(ILTV)等4种主要呼吸道传染病病原设计了4对特异性引物,大小分别为:IBV 1720bp、AIV 550bp、NDV 300bp、ILTV 647bp,与MG、MS建立多重RT-PCR,反应优化后可同时扩增出了清晰的目的条带。实验表明,建立的多重RT-PCR可用于混合感染上述6种呼吸道传染病病原的鉴别诊断。通过药物浸泡法、温差法和熏蒸法分别处理支原体污染严重的种蛋,出雏8周后对支原体抗体阳性率进行检测,结果为:百毒杀处理组13%,红霉素处理组17%,温差法处理组9%,熏蒸法处理组18%,而未经处理的MG阳性种蛋支原体阳性检出率却高达86.4%,尽管孵化率有不同程度的下降,但都能够显著降低MG的垂直传播。通过对孵化率、MG抗体检出率和35日龄平均体重、110日龄平均体重等指标进行综合评估,建议在生产实践中应用温差处理法可有效地降低MG的感染。总之,本研究对分离株进行了分离鉴定并建立了分子生物学检测方法,为今后进一步开展基因工程疫苗研制以及鸡毒支原体感染的综合性防治研究奠定了基础。更多还原

【Abstract】 Mycoplasma gallisepticum (MG) is a primary pathogen causing chronic respiratory disease in chicken, which is characterized by coughing, nasal discharge, rattling and air-saccaulitis. Usually chickens suffered co-infection with virus and bacteriumd, and led to forced-breathing and increased mortality. MG infection is common in flocks in our country, but mortality is low, which mainly led to high unhealthy chicken rate and egg production of layer dropped, feed conversion efficiency reduced and the period of onset to market prolonged, the cost of prevention added. At the same time, it could cause drug residua in poultry products. MG infection may result in economic losses in the poultry industry.One isolates was got from the infected or suspicious infected chickens came from a pheasantry of Shandong Jinan. On the basis of typical clone shape with frig egg and bacteria recovery test, 1 of mycoplama isolates was purified and was identified as Mycoplasma gallisepticum by colony morphology observation, red adsorption test colony, physiological and biochemical and serological appraisal. The isolate was named as JN. JN strain in liquid medium with tetrazolium and without glucose make medium color orange-red, their colonies in agar-solidified medium surface are typical of Fried egg shape, about 0.1~0.4 mm, colony can absorb 1% of red cell of chicken, MG antiserum can make JN strain metabolic inhibition.Through growing of the JN strain in different serum medium, it was easier in the pig serum medium, replacing expensive and less sources of horse serum. From the chicken annulus trachealis’spathogenic test of MG, JN strain can cause annulus trachealis cilia damage more, can make them dead quickly, pathogenicity larger than strains F. At the same time, SDS-PAGE map showed JN strain structural protein was different from F strains structural protein. So, we can conclude the correlation between the pathogenicity and structural proteins.Three primers were designed for mycoplasma gallisepticum (MG), mycoplasma synoviae (MS) and mycoplasma meleagridis (MM) and the size were MG 732bp,MS 208bp,MM 850bp. Through a single polymerase chain reaction (PCR), the corresponding segment can be got, under the optimized conditions, the multiple PCR established can also get three segments, the same as single PCR. Experiment showed that the multiple PCR can be used to detection of MS, MG and MM, convenient, high sensitivity and specificity.Aiming at chicken infective bronchitis virus (IBV), avian influenza virus (AIV), Newcastle disease virus (NDV), avian infectious laryngotracheitis virus (ILTV), four primers were designed and the sizes are IBV 1720bp, AIV 550bp, NDV 300bp, ILTV 647bp. And multiple RT-PCR was establisheded with MG, MS. Under the optimized conditions, it can simultaneously got corresponding segment clearly. Experiments indicated that the multiple RT-PCR can be used to identify and diagnose mixed infecting six kinds of respiratory infectious disease.Drug soaking, temperature fluctuation and fuming were used to treat Breeding eggs. The results demonstrated that three methods could drop the hatchery rate to some degree, but the vertical transmission infection of mycoplasma gallisepticum in chickens was cut down dramatically. Comprehensive index from hatchery rate, positive rate antibody against MG and average body weight of 35-day-old chicken, suggested that the temperature fluctuation method was more available for elimination of vertical transmission of MG in chickens.The research isolated and identified JN strain, and the molecular biological detection method was established. And more, the study laid a foundation for the research of genetic engineering vaccine and the comprehensive prevention and cure of the infection of MG in the future.更多还原