羊流产衣原体的分离培养及间接免疫荧光法鉴定
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- 英文论文题名：Isolation and Identification of ovine Chlamydia abortus with indirect immunofluorescence
- 论文作者：赵荣 ; 李兆才 ; 曹小安 ; 周继章
- 英文论文作者：ZHAO Rong ; LI Zhao-cai ; CAO Xiao-an ; ZHOU Ji-zhang ; Key Laboratory of Animal Virology of the Ministry of Agriculture/Lanzhou Veterinary Research Institute ; Chinese Acedemy of Agricultural Sciences
- 年：2016
- 作者机构：中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室;
- 论文关键词：流产衣原体 ; PCR-RFLP ; 间接免疫荧光法 ; 鉴定
- 英文论文关键词：Chlamydia abortus ; PCR-RFLP ; Indirect immunofluorescence ; identification
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S852.67
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：8
- 文件大小：799k
- 原文格式：D
- 会议级别：全国
摘要
目的基于流产衣原体MOMP单克隆抗体的间接免疫荧光法检测流产衣原体包涵体抗原。方法将收集到的疑似或确定为衣原体感染的羊流产胎儿的肝、脾、肺、肾、胃内容物等的混合物接种7日龄鸡胚卵黄囊进行培养,收集4~9日龄死亡鸡胚的卵黄囊膜作为样本,作PCR-RFLP检测;将PCR阳性样本接种于Hela229细胞,培养48 h,以鼠抗流产衣原体MOMP单克隆抗体为一抗,FITC标记的羊抗鼠Ig G抗体为二抗,用间接免疫荧光法检测流产衣原体包涵体,同时用Giemsa染色对流产衣原体包涵体进行观察。结果间接免疫荧光染色结果,在Hela229细胞浆内可见呈绿色荧光的衣原体包涵体,Giemsa染色可见蓝紫色包涵体。结论基于流产衣原体MOMP单克隆抗体的间接免疫荧光法检测流产衣原体包涵体的敏感性较高。
Objective To detect whole cell inclusions of C.abortus use of the indirect immunofluorescence(IIF) method with anti-Chlamydia abortus MOMP monoclonal antibody.Methods The liver,spleen,lung,kidney,stomach contents will be collected from the suspected or identified Chlamydia abortus infected fetal,meanwhile these mixed suspension from the infected fetal was inoculated into the SPF egg yolk sac membrane on day 7.The yolk sac membrane of the dead chick embryo was collected during the day 4 to 9,as a sample for detection of PCR-RFLP.The PCR positive samples were inoculated on Hela229 cells,for 48 h.Then,whole cell inclusions of C.abortus were detected by IIF with mouse anti-C.abortus MOMP monoclonal antibody as the first antibody,FITC labeled Goat anti-mouse Ig G as the second antibody.At the same time,traditional Giemsa staining was also used to identify whole cell inclusions of C.abortus.Results Indirect immunofluorescence staining results showed that the chlamydial inclusion of green fluorescence could be seen in the cytoplasm of Hela229 cells,and Giemsa staining showed purple inclusion.Conclusion An indirect immunofluorescence assay based on anti-C.abortus MOMP monoclonal antibody was used to identify whole cell inclusions of C.abortus,showing high sensitivity.
Objective To detect whole cell inclusions of C.abortus use of the indirect immunofluorescence(IIF) method with anti-Chlamydia abortus MOMP monoclonal antibody.Methods The liver,spleen,lung,kidney,stomach contents will be collected from the suspected or identified Chlamydia abortus infected fetal,meanwhile these mixed suspension from the infected fetal was inoculated into the SPF egg yolk sac membrane on day 7.The yolk sac membrane of the dead chick embryo was collected during the day 4 to 9,as a sample for detection of PCR-RFLP.The PCR positive samples were inoculated on Hela229 cells,for 48 h.Then,whole cell inclusions of C.abortus were detected by IIF with mouse anti-C.abortus MOMP monoclonal antibody as the first antibody,FITC labeled Goat anti-mouse Ig G as the second antibody.At the same time,traditional Giemsa staining was also used to identify whole cell inclusions of C.abortus.Results Indirect immunofluorescence staining results showed that the chlamydial inclusion of green fluorescence could be seen in the cytoplasm of Hela229 cells,and Giemsa staining showed purple inclusion.Conclusion An indirect immunofluorescence assay based on anti-C.abortus MOMP monoclonal antibody was used to identify whole cell inclusions of C.abortus,showing high sensitivity.
引文
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[8]谢昌元,王光华,李明录,等.青海省羊衣原体病综合防制措施[J].青海畜牧兽医杂志,2013,43(3):39-40.XIE Chang-yuan,WANG Guang-hua,LI Ming-lu,et al.Comprehensive preventive and control measures of the ovine Chlamydiosis in Qinghai Province[J].Qinghai Journal of Animal and Veterinary Sciences,2013,43(3):39-40.
[9]林治涌,梁督军,张志珍,等.陕西省羊衣原体病的诊治[J].中国兽医杂志,1994,20(6):24-25.LIN Chang-yuan,LIANG Du-jun,ZHANG Zhi-zhen,et al.Diagnosis and treatment of the ovine Chlamydiosis in Shaanxi Province[J].Chinese Journal of Veterinary Medicine,1994,20(6):24-25.
[10]罗意,周碧君,张华,等.贵州省7个地区主要山羊流产疫病的流行病学调查[J].畜牧与兽医,2013,45(6):62-65.LUO Yi,ZHOU Bi-jun,ZHANG Hua,et al.Epidemiological investigation on the abortion of the goats in 7regions of Guizhou Province[J].Animal Husbandry&Veterinary Medicine,2013,45(6):62-65.
[11]李云龙.云南省楚雄州猪衣原体病血清流行病学调查报告[J].当代畜牧,2014,11:29-30.LI Yun-long.Serum epidemiological investigation of Chlamydiosis in swine in Chuxiong Prefecture,Yunnan Province[J].Contemporary Animal Husbandry,2014,11:29-30.
[12]KALENDER(H.),et al.Identification of Chlamydophila abortus infection in aborting ewes and goats in Eastern Turkey.[J]Revue Méd.Vét.,2013,164(6):295-301.
[13]CREALAN J.,Mc CULLOUGH L.:Evaluation of strain specifc primer sequnces from an abortifacient strain of ovine Chlamydophila abortus(Chlamydia psittaci)for the detection of EAE by PCR[J],FEMS Microbiol.Lett.,2000,190,103-108.
[14]Borel et al.Mixed infections with Clamydia and porcine epidemic diarrhea virus-a new in vitro model of chlamydial persistence[J].BMC Microbiology,2010,10:201-209.
[15]?NG?R H.,?ET?NKAYA B.,A?IK M.N.,KARAHAN M.,BULUT H.:Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-Polymerase Chain Reaction[J].J.Vet.Med.,2004,50,43-45.
[2]吴移谋.衣原体[M].1版.北京:人民卫生出版社,2012:60+201.WU Yi-mou,Chlamydia[M].1st ed.Beijing:People's Medical Publishing House,2012:60+201.(in Chinese)
[3]Fukushi H,Nojiri K,Hirai K.Monoclonal antibody typing of Chlamydia psittaci strains derived from avian and mammalian species[J].Journal of Clinical Microbiology,1987,25(10):1978-1981.
[4]Thomson NR,Yeats C,Bell K,et al.The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation[]J.Genome Research,2005,15(5):629-640.
[5]闫少侠.猪流产嗜性衣原体单抗制备及阻断ELISA检测方法的建立[D].武汉:华中农业大学,2013.YAN Shao-xia.Production of Monoclonal Antibodies and Establishment of Blocking ELISA for Detection of Chlamydophila Abortus in Swine[D].Wuhan:Huazhong Agricultural University,2013.(in Chinese)
[6]韩宏军.猪衣原体病的诊治[J].中国畜牧兽医文摘,2014,30(8):148.HAN Hong-jun.Diagnosis and treatment of Chlamydiosis in swine[J].China animal husbandry and veterinary Digest,2014,30(8):148.
[7]陈廷和,王守智,李景水,等.宁夏羊衣原体病的血清学诊断及预防试验[J].宁夏农林科技,1994,6:30-32.CHEN Ting-he,WANG Shou-zhi.LI Jing-shui,et al.Serological diagnosis and preventive test of Chlamydiosis Ningxia[J].Ningxia Journal of Agriculture and Forestry Science and Technology.1994,6:30-32.
[8]谢昌元,王光华,李明录,等.青海省羊衣原体病综合防制措施[J].青海畜牧兽医杂志,2013,43(3):39-40.XIE Chang-yuan,WANG Guang-hua,LI Ming-lu,et al.Comprehensive preventive and control measures of the ovine Chlamydiosis in Qinghai Province[J].Qinghai Journal of Animal and Veterinary Sciences,2013,43(3):39-40.
[9]林治涌,梁督军,张志珍,等.陕西省羊衣原体病的诊治[J].中国兽医杂志,1994,20(6):24-25.LIN Chang-yuan,LIANG Du-jun,ZHANG Zhi-zhen,et al.Diagnosis and treatment of the ovine Chlamydiosis in Shaanxi Province[J].Chinese Journal of Veterinary Medicine,1994,20(6):24-25.
[10]罗意,周碧君,张华,等.贵州省7个地区主要山羊流产疫病的流行病学调查[J].畜牧与兽医,2013,45(6):62-65.LUO Yi,ZHOU Bi-jun,ZHANG Hua,et al.Epidemiological investigation on the abortion of the goats in 7regions of Guizhou Province[J].Animal Husbandry&Veterinary Medicine,2013,45(6):62-65.
[11]李云龙.云南省楚雄州猪衣原体病血清流行病学调查报告[J].当代畜牧,2014,11:29-30.LI Yun-long.Serum epidemiological investigation of Chlamydiosis in swine in Chuxiong Prefecture,Yunnan Province[J].Contemporary Animal Husbandry,2014,11:29-30.
[12]KALENDER(H.),et al.Identification of Chlamydophila abortus infection in aborting ewes and goats in Eastern Turkey.[J]Revue Méd.Vét.,2013,164(6):295-301.
[13]CREALAN J.,Mc CULLOUGH L.:Evaluation of strain specifc primer sequnces from an abortifacient strain of ovine Chlamydophila abortus(Chlamydia psittaci)for the detection of EAE by PCR[J],FEMS Microbiol.Lett.,2000,190,103-108.
[14]Borel et al.Mixed infections with Clamydia and porcine epidemic diarrhea virus-a new in vitro model of chlamydial persistence[J].BMC Microbiology,2010,10:201-209.
[15]?NG?R H.,?ET?NKAYA B.,A?IK M.N.,KARAHAN M.,BULUT H.:Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-Polymerase Chain Reaction[J].J.Vet.Med.,2004,50,43-45.
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