山羊腔阔盘吸虫的形态学观察和分子鉴定
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摘要
为了从形态学和分子水平对山羊腔阔盘吸虫(Eurytrema coelomaticum)的种类鉴定提供依据,对采集自广东佛山发病山羊胰脏的阔盘吸虫在形态学观察的基础上,抽提虫体基因组DNA,通过PCR方法对虫体核糖体18SrRNA基因和线粒体cox1基因进行扩增,将产物与pMD18-T载体连接,转化到大肠埃希菌DH5α感受态细胞进行克隆、测序和序列分析。结果获得虫体18SrRNA基因的大小为1 922bp,线粒体cox 1基因大小为444bp。序列分析显示,获得的18SrRNA基因与GenBank收录的腔阔盘吸虫(E.coelomaticum,登录号为DQ401035)同源性高达99%;线粒体cox1基因与GenBank收录的胰阔盘吸虫(E.pancreaticum,登录号为KC535544)的同源性仅为90%。研究结果从分子水平证明采集自广东佛山山羊体的阔盘吸虫为腔阔盘吸虫(E.coelomaticum),对山羊阔盘吸虫病的防控有指导意义。
In order to provide the basis for the identification of Eurytrema coelomaticumin goats from morphological and molecular level,genomic DNA of the parasites,which were collected from the pancreas of a goat with clinical symptoms in Foshan,Guangdong province,was extracted based on the morphological observation.Then PCR was used to amplify the 18 SrRNA gene and cox1 gene of this parasite.The amplified products were then ligated with PMD18-T vector and transformed into Escherichia coli DH5ɑfor cloning and sequencing.The 18 SrRNA gene sequence was 1 922 bp,and the cox 1 gene sequence was 444 bp.Based on BLAST analysis,the 18 SrRNA gene sequence had 99%similarity to E.coelomaticumin GenBank(accession no DQ401035),and the cox 1 gene sequence had 90% similarity to E.pancreaticum(accession no KC535544).The results proved that the parasites collected from the pancreas of a goat were molecularly identified as E.coelomaticum,which is of significance for the prevention and control of E.coelomaticumin goats.
In order to provide the basis for the identification of Eurytrema coelomaticumin goats from morphological and molecular level,genomic DNA of the parasites,which were collected from the pancreas of a goat with clinical symptoms in Foshan,Guangdong province,was extracted based on the morphological observation.Then PCR was used to amplify the 18 SrRNA gene and cox1 gene of this parasite.The amplified products were then ligated with PMD18-T vector and transformed into Escherichia coli DH5ɑfor cloning and sequencing.The 18 SrRNA gene sequence was 1 922 bp,and the cox 1 gene sequence was 444 bp.Based on BLAST analysis,the 18 SrRNA gene sequence had 99%similarity to E.coelomaticumin GenBank(accession no DQ401035),and the cox 1 gene sequence had 90% similarity to E.pancreaticum(accession no KC535544).The results proved that the parasites collected from the pancreas of a goat were molecularly identified as E.coelomaticum,which is of significance for the prevention and control of E.coelomaticumin goats.
引文
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[16] Oral A B,Soyder A,MalatyalíE,et al.Genotyping of Echinococcus granulosus isolates by sequencing of mitochondrial cytochrome C oxidase subunit 1(cox1)gene in Aydin[J].Mikrobiyol Bul,2018,52(2):198-205.
[2]唐仲璋,唐崇惕.牛、羊二种阔盘吸虫及矛形双腔吸虫的流行学及生物学的研究[J].动物学报,1977,23(3):267-282.
[3] Hornok S,Corduneanu A,Kontschán J,et al.Analyses of separate and concatenated cox1and 18SrRNA gene sequences indicate that the bat piroplasm Babesia vesperuginis is phylogenetically close to Cytauxzoonfelis and the'prototheilerid'Babesia conradae[J].Acta Vet Hung,2018,66(1):107-115.
[4] Kirillova V,Prakas P,Calero-Bernal R,et al.Identification and genetic characterization of Sarcocystis arctica,and Sarcocystis lutrae,in red foxes(Vulpes vulpes)from Baltic States and Spain[J].Parasit Vectors,2018,11(1):173.
[5] Gjerde B,Giacomelli S,Bianchi A,et al.Morphological and molecular characterization of four Sarcocystis,spp.including Sarcocystis linearis,n.sp.from roe deer(Capreolus capreolus)in Italy[J].Parasitol Res,2017,116(4):1317-1338.
[6]郑亚东,骆学农,施程洪,等.腔阔盘吸虫18SrRNA及亲缘关系分析[J].中国寄生虫学与寄生虫病杂志,2006,24(5):345-248.
[7] Bowles J,Blair D,Mcmanus D P.Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing[J].Mol Biochem Parasitol,1992,54(2):165-173.
[8]郑旭.胰阔盘吸虫分子种系发生的研究[D].黑龙江大庆:黑龙江八一农垦大学,2014.
[9]郝桂英.山羊源枝睾阔盘吸虫的形态和分子鉴定[J].中国预防兽医学报,2017,39(10):852-854.
[10] Chang Q C,Liu G H,Gao J F,et al.Sequencing and characterization of the complete mitochondrial genome from the pancreatic fluke Eurytrema pancreaticum(trematoda:Dicrocoeliidae)[J].Gene,2016,576(1):160-165.
[11]贾晓旭,唐修君,樊艳凤,等.华东地区地方鸡品种mtDNA控制区遗传多样性[J].生物多样性,2017,25(5):540-548.
[12]李瑶瑶,刘云国,刘凌霄,等.基于线粒体基因ND1、COX1和Cytb的双壳贝类分子系统进化研究[J].福建农林大学学报:自然科学版,2018(2):188-197.
[13]胡双双,吴炳耀,何江,等.江苏口岸褐家鼠线粒体DNA细胞色素C氧化酶亚基Ⅰ基因的遗传多态性研究[J].中国媒介生物学及控制杂志,2017,28(1):31-34.
[14] Rodrigues M S,Morelli K A,Jansen A M.Cytochrome c oxidase subunit 1gene as a DNA barcode for discriminating Trypanosoma cruzi DTUs and closely related species[J].Parasit Vectors,2017,10(1):488.
[15]王雨璇,刘钰婷,徐琨尧,等.结膜吸吮线虫形态学鉴定及基于cox1基因的系统发育分析[J].基因组学与应用生物学,2018,37(2):704-709.
[16] Oral A B,Soyder A,MalatyalíE,et al.Genotyping of Echinococcus granulosus isolates by sequencing of mitochondrial cytochrome C oxidase subunit 1(cox1)gene in Aydin[J].Mikrobiyol Bul,2018,52(2):198-205.