人体混合感染巨片吸虫和巨片吸虫与肝片吸虫的中间型
Faschiola gigantica and Intermediate Form between Faschiola gigantica and Faschola hepatica from a Mixed Infected Body
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摘要: 目的 通过线粒体nad1和细胞核ITS1序列, 对从云南省玉溪地区一女性片形吸虫病患者体内取出的2条片形吸虫成虫进行虫种鉴定, 获知寄生于人体的片形吸虫种类.方法 片形吸虫标本保存于75%乙醇溶液, 提取全基因组DNA, PCR扩增线粒体基因NADH脱氢酶亚基1 (nad1) 和细胞核r DNA的第一内转录间隔区 (ITS1) 序列, 正反引物双向测序.所测序列与Gen Bank中Fasciola属相关同源序列组成DNA数据集, 进行ITS1单倍型分析和构建nad1的NJ系统发育树.结果测得片形吸虫标本的ITS1和nad1序列长度分别是422 bp和542bp.2条片形吸虫标本的nad1序列完全一致, 属于F.gigantica (Fg型) .但是ITS1单倍型不一致, 标本YXGD01在该基因中属于F.gigantica (Fg型) , 标本YXGD02属于F.hepatica (Fh型) .基于nad1 DNA数据集构建的NJ系统发育树, 明显分为F.gigantica和F.hepatica 2个类群.结论 基于线粒体nad1和细胞核ITS1序列, 从云南省玉溪地区一片形吸虫病患者体内取出的2条片形吸虫属于不同类型, 标本YXGD01鉴定为F.gigantica, 标本YXGD02可能是F.gigantica与F.hepatica杂交生成的中间型个体, 但需要更多的实验证据.Abstract: Objective To identify two Fasciola spp., Sample YXGD01 and YXGD02, collected from one female fasciolosis patient living in Yuxin, Yunnan Province in China using DNA marker nad1 and ITS1, and to investigate causative agents of Zoonoses fasciolosis in humans. Me thods Two Fasciola fluckes collected from one female patient were preserved in 75% ethyl alcohol. The mitochondrial NADH dehydrogenase subunit 1 (nad1) gene and first internal transcribed spacer (ITS1) of nuclear ribosomal DNA (r DNA) of individual trematodes were amplified by polymerase chain reaction (PCR) . Then the PCR product were sequenced in forward and reverse direction. The sequences analyzed by comparing with related sequences of Fasciola available in Genbank database.Using the DNA database, to identify Fasciola spp. by haplotype analysis for ITS1 and construct phylogenetic tree by NJ method for nad1. Re s ults 422 bp ITS1 sequences and 542 bp nad1 sequences were obtained respectively for thetwo Fasciola fluckes sequenced in our study. Using haplotype analysis method, both Fasciola fluckes with the identical nad1 haplotype similar toF. gigiantica (Fg) haplogoup, showed Fg type to sample YXGD01 and Fh type to sample YXGD02 in ITS1 respectively. The NJ phylogenetic tree inferred from nad1 displays distinct F. gigantica goup and F. hepatica goup. Conclusion This study identified the two Fasciola spp. from one fasciolosis patient living in Yuxi, Yunnan Province usingmitochondria nad1 and nuclear ITS1. The sample YXGD01 should belong toF.gigantica in molecular characteristics. The confusion result between ITS1 and nad1 haplotype of sample YXGD02 infers that it may belong to one special type of intermediate form Fasciola between F. hepatica and F. gigantica, but more experimental proof is needed.
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Key words:
- Faschiola gigantica /
- Faschiola hepatica /
- Intermediate form /
- Mixed infection /
- ITS1 /
- nad1 /
- Yunnan province
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[1] [1]URQUHART G M, ARMOUR J, DUNCAN J L, et al.Veterinary parasitology[M].2nd ed.Oxford:Blackwell Science, 1996:307. [2] [2]SPITHILL T W, DALTON J P.Progress in development of liver fluke vaccines[J].Parasitol Today, 1998, 14 (6) :224-228. [3] [3]MAS-COMA S, VALERO M A, BARGUES M D.Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control[J].Adv Parasitol, 2009, 69 (1) :141-146. [4] [4]HASSEB A N, SHAZLY A M, ARAFA M A, et al.A review on fascioliasis in Egypt[J].J Egypt Soc Parasitol, 2002, 32 (1) :317-354. [5] [5]ISHII Y, NAKAMURA-UCHIYAMA F, NAWA Y.A praziquantel-ineffective fascioliasis case successfully treated with triclabendazole[J].Parasitology International, 2002, 51 (2) :205-209. [6] [6]MAS-COMA M S, ESTEBAN J G, BARGUES M D.Epidemiology of human fascioliasis:a review and proposed new classification[J].Bull World Health Organ, 1999, 77 (4) :340-346. [7] [7]AI L, CHEN MX, ALASAAD S, et al.Genetic characterization, species differentiation and detection of Fasciola spp.by molecular approaches[J].Parasit Vectors, 2011, 4 (1) :101. [8]许隆祺, 余森海, 徐淑慧.中国人体寄生虫分布与危害[M].北京:人民卫生出版社, 2000:152-153. [9]李雍龙.人体寄生虫学[M].第7版.北京:人民卫生出版社, 2008:99-101. [10]陈木新, 艾琳, 许学年, 等.云南省大理州大片形吸虫群体感染26例分析[J].中国地方病杂志, 2012, 31 (6) :595-598. [11]张国丽, 苏慧勇, 周俊, 等.云南省宾川县28例大片形吸虫病患者的临床分析[J].中华实验和临床感染病杂志, 2014, 8 (5) :667-670. [12] [12]SARWAR, M M.Fasciola indica Varma, a synonym of Fasciola gigantica Cobbold[J].Biologia, 1957, 3 (2) :168-175. [13] [13]YAMAGUTI S.Systema Helminthum.Vol.Ⅰ, The Digenetic Trematodes of Vertebrates, part I[M].New York:Interscience Publishers Inc, 1958:839-841. [14] [14]ITAGAKI T, SUTSUMI K.Triploid form of Fasciola in Japan:genetic relationships between Fasciola hepatica and Fasciola gigantica determined by ITS-2 sequence of nuclear r DNA[J].International Journal for Parasitology, 1998, 28 (5) :777-781. [15]杜礼义, 张印泉, 廖祖荫, 等.湘、鄂边区牛、羊片形吸虫感染调查[J].中国人兽共患病学报, 1994, 10 (2) :55-56. [16]尹怀志, 叶冰莹, 汪溥钦.肝片吸虫和巨片吸虫染色体和同工酶的研究[J].福建师范大学学报 (自然科学版) , 1990, 6 (1) :85-90. [17] [17]HENEBERG P.Phylogenetic data suggest the reclassification of Fasciola jacksoni (Digenea:Fasciolidae) as Fascioloides jacksoni comb.nov[J].Parasitology Research, 2013, 112 (4) :1679-1689. [18] [18]REDDY P V, SUBRAMANYAMS.Chromosome studies in the liver fluke, Fasciola gigantica Cobbold, 1856, from Andra Pradesh[J].Current Science, 1973, 42 (8) :288-291. [19] [19]PERIAGO M V, VALERO M A, SAYED M, et al.First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta, Egypt[J].Infection Genetics and Evolution, 2008, 8 (1) :51-58. [20] [20]ALI H, AI L, SONG H Q, et al.Genetic characterisation of Fasciola samples from different host species and geographical localities revealed the existence of F.hepatica and F.gigantica in Niger[J].Parasitol Res, 2008, 102 (5) :1021-1024. [21] [21]HUANG W Y, HE B, WANG C R, et al.Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence[J].Vet Parasitol, 2004, 120 (1-2) :75-83. [22] [22]ITAGAKI T, KIKAWA M, SAKAGUCHI K, et al.Genetic characterization of parthenogenic Fasciola sp.in Japan on the basis of the sequences of ribosomal and mitochondrial DNA[J].Parasitology, 2005, 131 (5) :679-685. [23] [23]ADLARD R D, BARKER S C, BLAIR D, et al.Comparison of the second internal transcribed spacer (ribosomal DNA) from populations and species of Fasciolidae (Digenea) [J].Int J Parasitol, 1993, 23 (3) :423-425. [24] [24]HASHIMOTO K, WATANOBE T, LIU C X, et al.Mitochondrial DNA and nuclear DNA indicate that the Japanese Fasciola species is F.gigantica[J].Parasitology Research, 1997, 83 (3) :220-225. [25] [25]AGATSUMA T, ARAKAWA Y, IWAGAMI M, et al.Molecular evidence of natural hybridization between Fasciola hepatica and F.gigantica[J].Parasitol Int, 2000, 49 (3) :231-238. [26] [26]CHOE S E, NGUYEN T T, KANG T G, et al.Genetic analysis of Fasciola isolates from cattle in Korea based on second internal transcribed spacer (ITS-2) sequence of nuclear ribosomal DNA[J].Parasitology Research, 2011, 109 (3) :833-839. [27] [27]ITAGAKI T, KIKAWA M, TERASAKI K, et al.Molecular characterization of parthenogenic Fasciola sp.in Korea on the basis of DNA sequences of ribosomal ITS1 and mitochondrial NDI gene[J].J Vet Med Sci, 2005, 67 (11) :1115-1118. [28] [28]ALASAAD S, HUANG C Q, LI Q Y, et al.Characterization of Fasciola samples from different host species and geographical localities in Spain by sequences of internal transcribed spacers of r DNA[J].Parasitol Res, 2007, 101 (5) :1245-1250. [29] [29]AMOR N, HALAJIAN A, FARJALLAH S, et al.Molecular characterization of Fasciola spp.from the endemic area of northern Iran based on nuclear ribosomal DNA sequences[J].Experimental Parasitology, 2011, 128 (3) :196-204. [30] [30]LIU G H, GASSER R B, YOUNG N D, et al.Complete mitochondrial genomes of the‘intermediate form’of Fasciola and Fasciola gigantica, and their comparison with F.hepatica[J].Parasites&Vectors, 2014, 7 (1) :150. [31] [31]ITAGAKI T, SAKAGUCHI K, TERASAKI K, et al.Occurrence of spermic diploid and aspermic triploid forms of Fasciola in Vietnam and their molecular characterization based on nuclear and mitochondrial DNA[J].Parasitol Int, 2009, 58 (1) :81-85. [32] [32]LE T H, DE N V, AGATSUMA T, et al.Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam[J].International Journal for Parasitology, 2008, 38 (6) :725. [33]张莉莉, 王会珍, 刘钢.云南肝片吸虫病首例报告[J].中国病原生物学杂志, 2005, 18 (5) :344-345. [34]艾琳, 陈木新, 吕山, 等.云南宾川地区牛羊片形吸虫感染调查及分子鉴定[J].热带医学杂志, 2013, 13 (6) :791-794. [35] [35]HALL T A.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT[J].Nucl Acids Symp, 1999, 41 (1) :95-98. [36] [36]KUMAR S, STECHER G, TAMURA K.MEGA7:Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[J].Molecular Biology and Evolution, 2016, 33 (7) :1870-1874. [37] [37]ITAGAKI T, ICHINOMIYA M, FUKUDA K, et al.Hybridization experiments indicate incomplete reproductive isolating mechanism between Fasciola hepatica and Fasciola gigantica[J].Parasitology, 2011, 138 (10) :1278-1284. [38] [38]BRENNAN G P, FAIRWEATHER I, TRUDGETT A, et al.Understanding triclabendazole resistance[J].Exp Mol Pathol, 2007, 82 (2) :104-109.
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