鸡滑液囊支原体贵州株的分离鉴定及VlhA基因序列分析

Isolation and Identification of Mycoplasma synoviae Guizhou Strain and Sequence Analysis of VlhA Gene

【目的】获得鸡滑液囊支原体(Mycoplasma synoviae,MS)贵州流行株,以进一步开展其病原学及免疫学相关研究。【方法】从贵州省织金县某肉鸡场共采集107份疑似MS感染病鸡的咽拭子、肿胀关节和脚垫、肿胀组织渗出物进行PCR检测,对PCR筛选的阳性组织样本进行MS分离培养,并进行瑞氏染色、生化试验及VlhA基因序列分析。【结果】PCR检测筛选到20份阳性样本,从关节和爪垫阳性样本中分离到1株疑似MS,分离株在平板上生长出“煎蛋状”菌落;瑞氏染色可见蓝色细小的球形菌;生化试验中,分离株分解葡萄糖和麦芽糖,但不分解精氨酸、尿素等,与MS标准株生化结果一致;分离培养物经PCR扩增得到821 bp的目的片段;分离株VlhA基因核苷酸相似性分析表明该分离株为MS,与GenBank登录的国内其他地区流行株相似性有地域差异。与安徽、重庆、福建、广东、广西、湖北、湖南、江苏和云南等地区流行株相似性在90.3%~99.7%之间;该分离株与湖南MS流行株相似性最高,达到99.7%;但与国外参考流行株的相似性低;VlhA基因遗传进化树分析显示,该分离株与国内参考株亲缘关系较近,与国外参考株亲缘关系较远。【结论】本研究成功从引起关节和足垫肿胀的鸡中分离鉴定了1株MS贵州流行株,该分离株存在一定变异,为贵州省MS的分子生物特征及本病的诊断和防控研究提供了基础依据。

【Objective】The aim of this study was to obtain Mycoplasma synoviae(MS)strains in Guizhou,to further develop its etiology and immunology related research.【Method】A total of 107 samples of pharyngeal swabs,swollen joints and claw pads and swollen tissue exudate suspected to be infect with MS were collected from a broiler farm in Zhijin county,Guizhou province and detected by PCR.The samples tested positive by PCR were used to isolate and culture MS,Wright staining,biochemical test and sequence analysis of the VlhA gene were performed.【Result】20 positive samples were tested by PCR,only 1 strain of MS was isolated from the positive samples of joint and claw pad,and the colony formed“egg-shaped”on solid medium.The isolated strain was small blue spherical stained by Wright’s in the microscope.In biochemical tests,the isolates could decompose glucose and maltose,but not arginine and urea,which was consistent with the biochemical results of MS standard strains.The target fragment of 821 bp was amplified by PCR.Nucleotide similarity analysis of the VlhA gene of the isolated strain indicated that the strain was MS,and there were regional differences in similarity with other endemic strains in GenBank.The similarity with the epidemic strains in Anhui,Chongqing,Fujian,Guangdong,Guangxi,Hubei,Hunan,Jiangsu and Yunnan was 90.3%to 99.7%.The most similar strain was MS epidemic strain in Hunan,which reached 99.7%.But the similarity with foreign reference strains was low.The phylogenetic analysis of VlhA gene showed that this strain was closely related to the domestic reference strain,but far from the foreign.【Conclusion】In this study,a Guizhou endemic MS strain was successfully isolated and identified from chickens causing swelling of joints and foot pads with some variation.This study provided a basis for the molecular and biological characteristics of MS in Guizhou and the diagnosis and control of this disease.