新疆小亚璃眼蜱的鉴定及其携带牛环形泰勒虫病原DNA检测
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摘要
通过对璃眼蜱形态学及璃眼蜱属特异性基因(ITS-2)和其携带病原基因(Tams1)PCR扩增及其PCR产物的克隆、测序、同源性比较等,鉴定和检测采自新疆吐鲁番地区疑似牛体表璃眼蜱(n=50)及其携带病原状况。结果显示,采集的蜱虫经形态学鉴定均为小亚璃眼蜱,PCR扩增的璃眼蜱种属特异性ITS-2基因和环形泰勒虫Tams1基因序列大小分别为491 bp和534 bp。本试验获得的ITS-2序列与Gen Bank中已记载的小亚璃眼蜱ITS-2序列一致,与形态学鉴定结果相符,故这些璃眼蜱属于小亚璃眼蜱种(Hyalomma anatolicum)。在检测的50只璃眼蜱中,携带牛环形泰勒虫的有3只,阳性率为6%(3/50),并发现所扩增的携带病原目的基因序列与已登录记载的牛环形泰勒虫基因序列相似性为99%。结果表明,在传统形态学分类的基础上,借助分子生物学方法对璃眼蜱种属鉴定及无典型硬蜱种的鉴定具有简易、准确度高等特点,而且在璃眼蜱种属确定的基础上分离其携带的病原更有实际意义。
This study identified and detected cattle body surface Hyalomma( n = 50) of suspected case and its carrying pathogenic condition in Turpan,Xinjiang,through morphology identification and PCR amplification of the specific gene( ITS-2) and carried pathogenic gene( Tams1) of Hyalomma koch,and the cloning,sequencing and homology analysis of PCR products. The result showed that acquired ticks were Hyalomma anatolicum by morphological identification,gene sequence of Hyalomma koch ITS-2 gene and Theileria annulata Tams1 was 491 bp and 534 bp,respectively. Acquired ITS-2 sequence was closer to the recorded Hyalomma anatolicum in Gen Bank,consistent with morphological identification results. So these Hyalomma belong to Hyalomma anatolicum. In the 50 detected ticks,there were three cattles carrying Theileria annulata,6%( 3 /50) positive rate,and amplified carrying pathogenic purpose fragment was similarity99% with the recorded Theileria annulata gene. The results indicated that identification methods of molecular biology could easily and accurately identify Hyalomma species and untypical tick species.
This study identified and detected cattle body surface Hyalomma( n = 50) of suspected case and its carrying pathogenic condition in Turpan,Xinjiang,through morphology identification and PCR amplification of the specific gene( ITS-2) and carried pathogenic gene( Tams1) of Hyalomma koch,and the cloning,sequencing and homology analysis of PCR products. The result showed that acquired ticks were Hyalomma anatolicum by morphological identification,gene sequence of Hyalomma koch ITS-2 gene and Theileria annulata Tams1 was 491 bp and 534 bp,respectively. Acquired ITS-2 sequence was closer to the recorded Hyalomma anatolicum in Gen Bank,consistent with morphological identification results. So these Hyalomma belong to Hyalomma anatolicum. In the 50 detected ticks,there were three cattles carrying Theileria annulata,6%( 3 /50) positive rate,and amplified carrying pathogenic purpose fragment was similarity99% with the recorded Theileria annulata gene. The results indicated that identification methods of molecular biology could easily and accurately identify Hyalomma species and untypical tick species.
引文
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[11]DOliveira C,Van Der Welde M,Habela M A,et al.Detection of Theileria annulata in blood samples of carrier cattle by PCR[J].J Clin Microbiol,1995,33(10):2665-2669.
[12]Azizi H,Shiran B,Farzaneh D A,et al.Detection of Theileria annulata by PCR and its comparison with smear method in native carrier cows[J].Biotechnology,2008,7(3):574-577.
[13]Dumanl N,Aktas M,Cetinkaya B,et al.Prevalence and distribution of tropical Theileriosis in eastern Turkey[J].Vet Parasitol,2005,127(1):9-15.
[14]简予健,黄家雨,马素贞,等.牛环形泰勒虫巢式PCR诊断方法的建立[J].新疆农业大学学报,2008,31(6):59-62.
[15]王振宝,刘启生,哈森,等.牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用[J].动物医学进展,2012,33(5):59-63.
[16]王振宝,巴音查汗.小亚璃眼蜱的鉴定及其生物学特性研究[J].新疆农业大学学报,2009,32(2):63-66.
[17]罗建勋,殷宏,关贵全,等.小亚璃眼蜱和亚洲璃眼蜱对环形泰勒虫传播能力的研究[J].中国兽医科技,2003,33(2):22-23.
[2]阿布力克木·阿不都热西提,李冰,蒋卫,等.吐鲁番地区首次发现小亚璃眼蜱的报告[J].地方病通报,2006(6):10.
[3]宋世荣,李良荣.我国北方各省区牛环形泰勒焦虫病调查报告[J].贵州畜牧兽医,2000,24(3):9-10.
[4]吕文祥,吕文顺,张其才,等.我国牛蜱传性血液原虫的虫种调查和分布特征的研究[J].中国兽医科技,1995,25(8):13-16.
[5]康发亮.蜱分类的方法及我国蜱分类的研究进展[J].安徽农业科学,2011,39(33):495-497.
[6]范雄林,朱朝军.蜱分类学研究进展[J].中国媒介生物学及控制杂志,1998,9(5):355-385.
[7]李晓娟,杨毅梅.核糖体DNA内转录间隔区序列标记在寄生虫学中的应用[J].大理学院学报,2008,4(7):79-81.
[8]Schittger L,Katzer F,Biermann R,et al.Characterization of a polymorphic Theileria annulata surface protein(TaSP)closely related to PIM of Theileria parva:implications for use in diagnostic tests and subunit vaccines[J].Mol Biochem Parasit,2002,120(2):247-256.
[9]Pipano E,Cahana M.Fluorescent antibody test for the serodiagnosis of Theileria annulata[J].J Parastitol,1969,55:765.
[10]Pipano E,Cahana M.Feller B,et al.A serological method for assessing the response to Theileria annulata immunization[J].Refuah Vet,1969,26:145-148.
[11]DOliveira C,Van Der Welde M,Habela M A,et al.Detection of Theileria annulata in blood samples of carrier cattle by PCR[J].J Clin Microbiol,1995,33(10):2665-2669.
[12]Azizi H,Shiran B,Farzaneh D A,et al.Detection of Theileria annulata by PCR and its comparison with smear method in native carrier cows[J].Biotechnology,2008,7(3):574-577.
[13]Dumanl N,Aktas M,Cetinkaya B,et al.Prevalence and distribution of tropical Theileriosis in eastern Turkey[J].Vet Parasitol,2005,127(1):9-15.
[14]简予健,黄家雨,马素贞,等.牛环形泰勒虫巢式PCR诊断方法的建立[J].新疆农业大学学报,2008,31(6):59-62.
[15]王振宝,刘启生,哈森,等.牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用[J].动物医学进展,2012,33(5):59-63.
[16]王振宝,巴音查汗.小亚璃眼蜱的鉴定及其生物学特性研究[J].新疆农业大学学报,2009,32(2):63-66.
[17]罗建勋,殷宏,关贵全,等.小亚璃眼蜱和亚洲璃眼蜱对环形泰勒虫传播能力的研究[J].中国兽医科技,2003,33(2):22-23.