我国不同利什曼病流行区利什曼原虫分离株核糖体内转录间隔区1的多态性分析
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摘要
目的分析我国不同利什曼病流行区的利什曼原虫分离株核糖体内转录间隔区1 (ITS-1)序列的多态性。方法实验室培养10个分离自我国不同利什曼病流行区的利什曼原虫分离株,包括四川/甘肃山丘型内脏利什曼病流行区的SC6和Cy分离株,新疆喀什人源型内脏利什曼病流行区的801和KS-2分离株,新疆伽师荒漠型内脏利什曼病流行区的JIASHI-1、 JIASHI-5、 XJ771分离株以及新疆克拉玛依皮肤利什曼病流行区的KXG-Xu、KXG-Liu和KXG-927分离株。收集利什曼原虫前鞭毛体,提取DNA, PCR扩增各利什曼原虫分离株的ITS-1并送测序,用GENEDOC软件对ITS-1序列进行比对分析,从GenBank下载利什曼原虫其他分离株的ITS-1序列,构建系统进化树,分析我国利什曼原虫分离株的亲缘关系。结果我国10个利什曼原虫分离株均扩增出单一的条带。序列分析结果显示, 801和KS-2分离株的ITS-1片段为316 bp,与MHOM/IN/80/DD8参照株比较其碱基突变率为0; JIASHI-1、 JIASHI-5和XJ771分离株为313 bp,碱基突变率为1.6%; KXG-Xu、 KXG-Liu和KXG-927分离株为312 bp,碱基突变率为1.6%; Cy和SC6分离株为318 bp,碱基突变率为0.6%。系统进化树显示,这10个利什曼原虫分离株ITS-1序列聚集成2个群和3个亚群,其中JIASHI-1、 JIASHI-5和XJ771分离株聚集为A亚群, KXG-Xu、 KXG-Liu和KXG-927分离株聚集为B亚群, A和B亚群聚集于群Ⅰ,为婴儿利什曼原虫; Cy、SC6、 801和KS2分离株聚集为C亚群,属群Ⅱ,为杜氏利什曼原虫。结论我国不同利什曼病流行区的利什曼原虫分离株的ITS-1序列存在多态性。
Objective The polymorphism of the internal transcribed spacer 1(ITS-1) sequence of Leishmania isolates from different endemic areas in China was analyzed to understand the genetic variation and relationship a-mong different Leishmania isolates. Methods Total 10 Chinese Leishmania isolates, including SC6 and Cy isolates from endemic areas of mountainous visceral leishmaniasis in Sichuan/Gansu Province( Leishmania donovani); 801 and KS-2 isolates from endemic areas of anthroponotic visceral leishmaniasis in Kashi, Xinjiang( L. donovani); JIASHI-1,JIASHI-5 and XJ771 isolates from endemic areas of desert-type visceral leishmaniasis in Jiashi, Xinjiang(L. infantum); KXG-Xu, KXG-Liu and KXG-927 isolates from endemic areas of cutaneous leishmaniasis in Karamay, Xinjiang(L. infantum), were maintained in the laboratory culture. The genomic DNAs were extracted from the cultured promastigotes and the ITS-1 was PCR amplified using gene-specific primers. The PCR products of amplified ITS-1 were sequenced and the obtained sequences from different isolates were aligned with reference ITS-1 sequences of Leishmania deposited in GenBank using GENEDOC software. The phylogenetic tree was constructed based on their genetic variation and relationship. Results A single band of ITS-1 with size of about 310 bp was amplified from all 10 Chinese Leishmania isolates. The sequence analysis identified that ITS-1 amplified from anthroponotic L. donovani801 and KS-2 isolates contained 316 bp with sequence identical to ITS-1 sequence from L. donovani reference strain MHOM/IN/80/DD8 without any mutation. The ITS-1 amplified from desert-type L. infantum JIASHI-1, JIASHI-5 and XJ771 isolates were 313 bp with 1.6% sequence variation. The ITS-1 amplified from cutaneous L. infantum KXGXu, KXG-Liu and KXG-927 isolates were 312 bp with 1.6% sequence variation. The ITS-1 amplified from mountainous L. donovani Cy and SC6 isolates were 318 bp with 0.6% sequence variation. Phylogenetic analysis of the ITS-1 sequences showed that the 10 Leishmania isolates were branched into three subgroups and two clusters according to the different species and endemic areas. The L. infantum isolates from Jiashi, Xinjiang(JIASHI-1, JIASHI-5 and XJ771) were closely related and clustered as subgroup A. The L. infantum isolates from Karamay, Xinjiang(KXG-Xu, KXG-Liu and KXG-927) are clustered as subgroup B. Subgroup A and B are closely related to L.infantum reference as cluster Ⅰ. The L. donovani isolates Cy, SC6, 801 and KS2 are closely related as subgroup C, fallen into cluster Ⅱ of L. donovani. Conclusion ITS-1 sequences of Leishmania isolates from different leishma niasis endemic areas in China showed different polymorphism related to different Leishmania species and endemic areas.
Objective The polymorphism of the internal transcribed spacer 1(ITS-1) sequence of Leishmania isolates from different endemic areas in China was analyzed to understand the genetic variation and relationship a-mong different Leishmania isolates. Methods Total 10 Chinese Leishmania isolates, including SC6 and Cy isolates from endemic areas of mountainous visceral leishmaniasis in Sichuan/Gansu Province( Leishmania donovani); 801 and KS-2 isolates from endemic areas of anthroponotic visceral leishmaniasis in Kashi, Xinjiang( L. donovani); JIASHI-1,JIASHI-5 and XJ771 isolates from endemic areas of desert-type visceral leishmaniasis in Jiashi, Xinjiang(L. infantum); KXG-Xu, KXG-Liu and KXG-927 isolates from endemic areas of cutaneous leishmaniasis in Karamay, Xinjiang(L. infantum), were maintained in the laboratory culture. The genomic DNAs were extracted from the cultured promastigotes and the ITS-1 was PCR amplified using gene-specific primers. The PCR products of amplified ITS-1 were sequenced and the obtained sequences from different isolates were aligned with reference ITS-1 sequences of Leishmania deposited in GenBank using GENEDOC software. The phylogenetic tree was constructed based on their genetic variation and relationship. Results A single band of ITS-1 with size of about 310 bp was amplified from all 10 Chinese Leishmania isolates. The sequence analysis identified that ITS-1 amplified from anthroponotic L. donovani801 and KS-2 isolates contained 316 bp with sequence identical to ITS-1 sequence from L. donovani reference strain MHOM/IN/80/DD8 without any mutation. The ITS-1 amplified from desert-type L. infantum JIASHI-1, JIASHI-5 and XJ771 isolates were 313 bp with 1.6% sequence variation. The ITS-1 amplified from cutaneous L. infantum KXGXu, KXG-Liu and KXG-927 isolates were 312 bp with 1.6% sequence variation. The ITS-1 amplified from mountainous L. donovani Cy and SC6 isolates were 318 bp with 0.6% sequence variation. Phylogenetic analysis of the ITS-1 sequences showed that the 10 Leishmania isolates were branched into three subgroups and two clusters according to the different species and endemic areas. The L. infantum isolates from Jiashi, Xinjiang(JIASHI-1, JIASHI-5 and XJ771) were closely related and clustered as subgroup A. The L. infantum isolates from Karamay, Xinjiang(KXG-Xu, KXG-Liu and KXG-927) are clustered as subgroup B. Subgroup A and B are closely related to L.infantum reference as cluster Ⅰ. The L. donovani isolates Cy, SC6, 801 and KS2 are closely related as subgroup C, fallen into cluster Ⅱ of L. donovani. Conclusion ITS-1 sequences of Leishmania isolates from different leishma niasis endemic areas in China showed different polymorphism related to different Leishmania species and endemic areas.
引文
[1]高春花,管立人,汪俊云,等.利什曼原虫的传播机制及传媒蛉种的研究进展[J].国际医学寄生虫病杂志,2007,34(3):113-118.
[2]Desjeux P.Leishmaniasis:current situation and new perspectives[J].Comp Immunol Microbiol Infect Dis,2004,27(5):305-318.
[3]Alvar J,Vélez ID,Bern C,et al.Leishmaniasis worldwide and global estimates of its incidence[J].PLoS One,2012,7(5):e35671.
[4]Guan LR,Shen WX.Recent advances in visceral leishmaniasis in China[J].Southeast Asian J Trop Med Public Health,1991,22(3):291-298.
[5]Lu HG,Zhong L,Guan LR,et al.Separation of Chinese Leishmania isolates into five genotypes by kinetoplast and chromosomal DNA heterogeneity[J].Am J Trop Med Hyg,1994,50(6):763-770.
[6]Zhou XN,Lv S,Yang GJ,et al.Spatial epidemiology in zoonotic parasitic diseases:insights gained at the 1st International Symposium on Geospatial Health in Lijiang,China,2007[J].Parasit Vectors,2009,2:10.
[7]管立人,瞿靖琦,柴君杰.中国利什曼病的现状和对开展防治工作的若干建议[J].地方病通报,2000,15(3):49-52.
[8]Wang JY,Gao CH,Yang YT,et al.An outbreak of the desert sub-type of zoonotic visceral leishmaniasis in Jiashi,Xinjiang Uygur Autonomous Region,People’s Republic of China[J].Parasitol Int,2010,59(3):331-337.
[9]Wang JY,Cui G,Chen HT,et al.Current epidemiological profile and features of visceral leishmaniasis in people’s republic of China[J].Parasit Vectors,2012,5:31.
[10]《中华传染病杂志》编辑委员会.中国利什曼原虫感染诊断和治疗专家共识[J].中华传染病杂志,2017,35(9):513-518.
[11]Guan LR,Yang YQ,Qu JQ,et al.Discovery and study of cutaneous leishmaniasis in Karamay of Xinjiang,West China[J].Infect Dis Poverty,2013,2:20.
[12]管立人.我国内脏利什曼病的现状和对防治工作的展望[J].中国寄生虫学与寄生虫病杂志,2009,27(5):394-397.
[13]Tashakori M,Mahnaz T,Kuhls K,et al.Leishmania major:genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer[J].Acta Trop,2006,98(1):52-58.
[14]Subba Raju BV,Gurumurthy S,Kuhls K,et al.Genetic typing reveals monomorphism between antimony sensitive and resistant Leishmania donovani isolates from visceral leishmaniasis or post kala-azar dermal leishmaniasis cases in India[J].Parasitol Res,2012,111(4):1559-1568.
[15]Ghatee MA,Sharifi I,Kuhls K,et al.Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran[J].Exp Parasitol,2014,144:44-51.
[16]Karamian M,Kuhls K,Hemmati M,et al.Phylogenetic structure of Leishmania tropica in the new endemic focus Birjand in East Iran in comparison to other Iranian endemic regions[J].Acta Trop,2016,158:68-76.
[17]田玉,陈建平,胡孝素.山丘疫区杜氏利什曼原虫核糖体基因内转录间隔区的克隆及序列分析[J].中国寄生虫学与寄生虫病杂志,2004,22(5):294-296.
[18]el Tai NO,Osman OF,el Fari M,et al.Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing[J].Trans R Soc Trop Med Hyg,2000,94(5):575-579.
[19]Kuhls K,Mauricio IL,Pratlong F,et al.Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex[J].Microbes Infect,2005,7(11/12):1224-1234.
[20]Tamura K,Stecher G,Peterson D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Mol Biol Evol,2013,30(12):2725-2729.
[21]Schonian G,Cupolillo E,Mauricio I.Molecular evolution and phylogeny of Leishmania[M].Drug Resistance in Leishmania Parasites,2013:19-57.
[22]Rioux JA,Lanotte G,Serres E,et al.Taxonomy of Leishmania.Use of isoenzymes.Suggestions for a new classification[J].Ann Parasitol Hum Comp,1990,65(3):111-125.
[23]Cupolillo E,Grimaldi Júnior G,Momen H,et al.Intergenic region typing(IRT):a rapid molecular approach to the characterization and evolution of Leishmania[J].Mol Biochem Parasitol,1995,73(1/2):145-155.
[24]Quispe Tintaya KW,Ying X,Dedet JP,et al.Antigen genes for molecular epidemiology of leishmaniasis:polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein63 in the Leishmania donovani complex[J].J Infect Dis,2004,189(6):1035-1043.
[25]丁丹,高春花,杨玥涛,等.我国不同流行区利什曼原虫分离株动基体小环DNA序列分析[J].中国人兽共患病学报,2013,29(11):1055-1059.
[26]汪俊云.利什曼原虫DNA重复序列在虫种鉴定上的价值分析[J].中国人兽共患病学报,2005,21(4):304-308.
[27]张春莹,宋兴勃,严可宁,等.中国不同流行疫区利什曼原虫分离株的微卫星多态性分析[J].现代预防医学,2014,41(10):1852-1855.
[28]Schonian G,Nasereddin A,Dinse N,et al.PCR diagnosis and characterization of Leishmania in local and imported clinical samples[J].Diagn Microbiol Infect Dis,2003,47(1):349-358.
[29]ZemanováE,Jirk M,Mauricio IL,et al.Genetic polymorphism within the leishmania donovani complex:correlation with geographic origin[J].Am J Trop Med Hyg,2004,70(6):613-617.
[30]Zelazny AM,Fedorko DP,Li L,et al.Evaluation of 7SLRNA gene sequences for the identification of Leishmania spp.[J].Am J Trop Med Hyg,2005,72(4):415-420.
[31]Berzunza-Cruz M,Cabrera N,Crippa-Rossi M,et al.Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana[J].Parasitol Res,2002,88(10):918-925.
[32]管立人,许永湘,左新平,等.新疆克拉玛依皮肤利什曼病传播媒介的研究[J].地方病通报,1996,11(1):38-41.
[33]Yang BB,Guo XG,Hu XS,et al.Species discrimination and phylogenetic inference of 17 Chinese Leishmania isolates based on internal transcribed spacer 1(ITS1)sequences[J].Parasitol Res,2010,107(5):1049-1065.
[34]Shang LM,Peng WP,Jin HT,et al.The prevalence of canine Leishmania infantum infection in Sichuan Province,southwestern China detected by real time PCR[J].Parasit Vectors,2011,4(1):173-177.
[35]张春莹,黄玉霞,袁余,等.热休克蛋白70(hsp70)基因对利什曼原虫中国分离株的系统发育分析[J].中国人兽共患病学报,2014,30(2):163-168.
[2]Desjeux P.Leishmaniasis:current situation and new perspectives[J].Comp Immunol Microbiol Infect Dis,2004,27(5):305-318.
[3]Alvar J,Vélez ID,Bern C,et al.Leishmaniasis worldwide and global estimates of its incidence[J].PLoS One,2012,7(5):e35671.
[4]Guan LR,Shen WX.Recent advances in visceral leishmaniasis in China[J].Southeast Asian J Trop Med Public Health,1991,22(3):291-298.
[5]Lu HG,Zhong L,Guan LR,et al.Separation of Chinese Leishmania isolates into five genotypes by kinetoplast and chromosomal DNA heterogeneity[J].Am J Trop Med Hyg,1994,50(6):763-770.
[6]Zhou XN,Lv S,Yang GJ,et al.Spatial epidemiology in zoonotic parasitic diseases:insights gained at the 1st International Symposium on Geospatial Health in Lijiang,China,2007[J].Parasit Vectors,2009,2:10.
[7]管立人,瞿靖琦,柴君杰.中国利什曼病的现状和对开展防治工作的若干建议[J].地方病通报,2000,15(3):49-52.
[8]Wang JY,Gao CH,Yang YT,et al.An outbreak of the desert sub-type of zoonotic visceral leishmaniasis in Jiashi,Xinjiang Uygur Autonomous Region,People’s Republic of China[J].Parasitol Int,2010,59(3):331-337.
[9]Wang JY,Cui G,Chen HT,et al.Current epidemiological profile and features of visceral leishmaniasis in people’s republic of China[J].Parasit Vectors,2012,5:31.
[10]《中华传染病杂志》编辑委员会.中国利什曼原虫感染诊断和治疗专家共识[J].中华传染病杂志,2017,35(9):513-518.
[11]Guan LR,Yang YQ,Qu JQ,et al.Discovery and study of cutaneous leishmaniasis in Karamay of Xinjiang,West China[J].Infect Dis Poverty,2013,2:20.
[12]管立人.我国内脏利什曼病的现状和对防治工作的展望[J].中国寄生虫学与寄生虫病杂志,2009,27(5):394-397.
[13]Tashakori M,Mahnaz T,Kuhls K,et al.Leishmania major:genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer[J].Acta Trop,2006,98(1):52-58.
[14]Subba Raju BV,Gurumurthy S,Kuhls K,et al.Genetic typing reveals monomorphism between antimony sensitive and resistant Leishmania donovani isolates from visceral leishmaniasis or post kala-azar dermal leishmaniasis cases in India[J].Parasitol Res,2012,111(4):1559-1568.
[15]Ghatee MA,Sharifi I,Kuhls K,et al.Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran[J].Exp Parasitol,2014,144:44-51.
[16]Karamian M,Kuhls K,Hemmati M,et al.Phylogenetic structure of Leishmania tropica in the new endemic focus Birjand in East Iran in comparison to other Iranian endemic regions[J].Acta Trop,2016,158:68-76.
[17]田玉,陈建平,胡孝素.山丘疫区杜氏利什曼原虫核糖体基因内转录间隔区的克隆及序列分析[J].中国寄生虫学与寄生虫病杂志,2004,22(5):294-296.
[18]el Tai NO,Osman OF,el Fari M,et al.Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing[J].Trans R Soc Trop Med Hyg,2000,94(5):575-579.
[19]Kuhls K,Mauricio IL,Pratlong F,et al.Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex[J].Microbes Infect,2005,7(11/12):1224-1234.
[20]Tamura K,Stecher G,Peterson D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Mol Biol Evol,2013,30(12):2725-2729.
[21]Schonian G,Cupolillo E,Mauricio I.Molecular evolution and phylogeny of Leishmania[M].Drug Resistance in Leishmania Parasites,2013:19-57.
[22]Rioux JA,Lanotte G,Serres E,et al.Taxonomy of Leishmania.Use of isoenzymes.Suggestions for a new classification[J].Ann Parasitol Hum Comp,1990,65(3):111-125.
[23]Cupolillo E,Grimaldi Júnior G,Momen H,et al.Intergenic region typing(IRT):a rapid molecular approach to the characterization and evolution of Leishmania[J].Mol Biochem Parasitol,1995,73(1/2):145-155.
[24]Quispe Tintaya KW,Ying X,Dedet JP,et al.Antigen genes for molecular epidemiology of leishmaniasis:polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein63 in the Leishmania donovani complex[J].J Infect Dis,2004,189(6):1035-1043.
[25]丁丹,高春花,杨玥涛,等.我国不同流行区利什曼原虫分离株动基体小环DNA序列分析[J].中国人兽共患病学报,2013,29(11):1055-1059.
[26]汪俊云.利什曼原虫DNA重复序列在虫种鉴定上的价值分析[J].中国人兽共患病学报,2005,21(4):304-308.
[27]张春莹,宋兴勃,严可宁,等.中国不同流行疫区利什曼原虫分离株的微卫星多态性分析[J].现代预防医学,2014,41(10):1852-1855.
[28]Schonian G,Nasereddin A,Dinse N,et al.PCR diagnosis and characterization of Leishmania in local and imported clinical samples[J].Diagn Microbiol Infect Dis,2003,47(1):349-358.
[29]ZemanováE,Jirk M,Mauricio IL,et al.Genetic polymorphism within the leishmania donovani complex:correlation with geographic origin[J].Am J Trop Med Hyg,2004,70(6):613-617.
[30]Zelazny AM,Fedorko DP,Li L,et al.Evaluation of 7SLRNA gene sequences for the identification of Leishmania spp.[J].Am J Trop Med Hyg,2005,72(4):415-420.
[31]Berzunza-Cruz M,Cabrera N,Crippa-Rossi M,et al.Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana[J].Parasitol Res,2002,88(10):918-925.
[32]管立人,许永湘,左新平,等.新疆克拉玛依皮肤利什曼病传播媒介的研究[J].地方病通报,1996,11(1):38-41.
[33]Yang BB,Guo XG,Hu XS,et al.Species discrimination and phylogenetic inference of 17 Chinese Leishmania isolates based on internal transcribed spacer 1(ITS1)sequences[J].Parasitol Res,2010,107(5):1049-1065.
[34]Shang LM,Peng WP,Jin HT,et al.The prevalence of canine Leishmania infantum infection in Sichuan Province,southwestern China detected by real time PCR[J].Parasit Vectors,2011,4(1):173-177.
[35]张春莹,黄玉霞,袁余,等.热休克蛋白70(hsp70)基因对利什曼原虫中国分离株的系统发育分析[J].中国人兽共患病学报,2014,30(2):163-168.
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