鸡毒支原体(MG)疫苗株和野毒株的PCR鉴定

Development of PCR assay for differential detection of MG attenuated strains from field strains

针对由于鸡毒支原体(Mycoplasma gallisepticum,MG)不同毒株(针对TS-11、6/85、F株疫苗株和S6、R株野毒株)的特定序列,设计出特异性检测引物,对临床免疫MG疫苗株TS-11的鸡群以及父母代(A^H场)鸡群和祖代(I场)鸡群采集毛蛋气囊拭子,A场父母代种鸡群和I场祖代鸡群同时采集咽喉拭子进行MG的PCR鉴别诊断,对临床送检免疫6/85的客户鸡群(J场)采集病料,F株疫苗株进行MG的鉴别诊断,同时采集SPF鸡群咽喉拭子作为阴性对照群,验证该鉴别引物的特异性。结果显示:鉴别引物具有非常好的特异性,SPF鸡群MG的检测均为阴性,不存在假阳性的弊端;临床免疫MG疫苗株6/85的鸡群(J场)存在MG野毒株S6的感染,在免疫后1个月左右采集病料中仍能检出MG 6/85疫苗株。对免疫TS-11的父母代种鸡群(A^H场)和祖代种鸡群(I场)死亡的毛蛋气囊拭子,用PCR检测MG阳性率,结果显示:祖代鸡群(I场)后代死亡的毛蛋气囊评估结果为优,毛蛋气囊拭子MG的PCR鉴别引物检测均为阴性;而父母代鸡群后代死亡的毛蛋气囊从50周龄开始出现浑浊,MG的PCR鉴别引物可同时检出TS-11疫苗毒和野毒株,部分鸡群同时存在R株和S6株两种野毒感染;采集A场父母代种鸡群的咽喉拭子和后代啄壳死亡毛蛋气囊拭子,PCR检测结果显示种鸡群和后代可同时检出野毒株S6和疫苗株TS-11。这表明种鸡群MG的感染,可通过垂直传播感染其后代。本研究设计的MG鉴别引物,可将不同MG疫苗株(TS-11、F株、6/85)与野毒株(S6株、R株)区分,为临床MG的鉴别诊断和种鸡群的MG感染状态评估提供积极的指导意义。

In this study,specific detection primers(for TS-11,6/85,and F vaccine strains,and S6 and R wild strains)were designed for specific sequences of different Mycoplasma gallisepticum(MG)strains.For the chickens clinically immunized MG vaccine strain TS-11 and parent chickens(Farms A to H)and grandparent chickens(Farm I),air sacs swabs were collected from dead chicks hatched from eggs.From a parent stock group and grandparent chickens of Farm I,throat swabs were collected at the same time for MG PCR differential diagnosis.Disease materials from client chickens(Farm J)immunized with 6/85 of clinical delivery were also gathered.The MG differential diagnosis of F strain of vaccine was carried out,and the throat swabs of SPF chickens were collected as negative control to verify the specificity of the differential primers.The results showed that the primers had a high specificity.SPF chicken flocks was tested negative for MG,with no false positives.Infection with wild MG strain S6 was found in the chickens(Farm J)immunized with MG vaccine strain 6/85,and the MG 6/85 vaccine strain could still be detected in the collected samples about one month after immunization.The positive rates of MG primers were detected by PCR from the parents of TS-11 immunized chickens(Farms A to H)and the offspring of grandparent chickens(Farm I).Evaluation of air sacs from the dead chicks hatched from eggs of grandparent chicken flocks(Farm I)were excellent,and the PCR primers of the air sacs swabs MG were negative.The air sacs from the dead chicks hatched from eggs of the parent flocks began to present turbid at 50 weeks of age.The TS-11 vaccine virus and wild virus strains were detected simultaneously by PCR primers of MG.Two wild virus strains,R strain and S6 strain,were observed in some chicken flocks at the same time.The throat swabs from the parents and the air sacs swabs from the dead chicks hatched from eggs of the parent flocks from Farm A were collected for MG differential primers PCR detection.The results showed that the wild strain S6 and the vaccine strain TS-11 were detected simultaneously in the chickens and their offspring.This indicated that MG infection in chicken flocks might be transmitted vertically to their offspring.Therefore,the MG identification primers in this study might distinguish different MG vaccine strains(TS-11,F strains,6/85)from wild strains(S6 strains,R strains),which provided useful guidance for differential diagnosis and evaluation of MG infection status in chicken population.