Polymorphism analysis of internal transcribed spacer 1 of Leishmania isolates from different endemic areas in China

doi:10.12140/j.issn.1000-7423.2019.02.014

Abstract

Abstract:

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 among 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. donovani 801 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 KXG-Xu, 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 Ⅱof L. donovani. Conclusion ITS-1 sequences of Leishmania isolates from different leishmaniasis endemic areas in China showed different polymorphism related to different Leishmania species and endemic areas.

Key words: Leishmaniasis, Leishmania, Isolate, Internal transcribed spacer 1, Genotyping, China

CLC Number:

R382.22

Fang-zhou CHENG, Chun-hua GAO, Yue-tao YANG, Jun-yun WANG. Polymorphism analysis of internal transcribed spacer 1 of Leishmania isolates from different endemic areas in China[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(2): 196-201.

Figures/Tables 5

Table 1

Fig. 1

Table 2

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种名Species	世界卫生组织编码WHO code	分离来源国Source country	登录号Accession no.
杜氏利什曼原虫 L. donovani	MHOM/IN/80/DD8	印度 India	AJ000292
	MHOM/KE/75/H9	肯尼亚Kenya	EU326228
	MHOM/ET/08/DM309-C11	埃塞俄比亚Ethiopia	FN182209
	MHOM/ET/08/DM309-C13	埃塞俄比亚Ethiopia	FN182206
婴儿利什曼原虫 L. infantum	MHOM/IR/04/IPI-UN10	伊朗Iran	GQ444144
	MHOM/UZ/2007/MUA	乌兹别克斯坦Uzbekistan	FM164416
	MHOM/UZ/2007/OBA	乌兹别克斯坦Uzbekistan	FM164419
	MHOM/BR/74/PP75	巴西 Brazil	EU326227
埃塞俄比亚利什曼原虫L. aethiopica	MHOM/ET/94/1470	埃塞俄比亚Ethiopia	FN677344
热带利什曼原虫 L. tropica	MHOM/AF/88/KK27	阿富汗Afghanistan	GQ913688
	MHOM/SU/60/OD	苏联 Soviet Union	EU326226

分离株Isolate	世界卫生组织编码 WHO code	虫种 Species	来源 Location	宿主 Host	序列长度/bp Sequence length/bp	GenBank登录号 GenBank accession no.
KXG-Xu	MHOM/CN/1994/KXG-Xu	婴儿利什曼原虫 L. infantum	新疆克拉玛依 Karamay, Xinjiang	患者 Human	312	MH450079^a
KXG-Liu	MHOM/CN/1994/KXG-Liu	婴儿利什曼原虫 L. infantum	新疆克拉玛依 Karamay, Xinjiang	患者 Human	312	MH450080^a
KXG-927	IMJW/CN/1992/KXG-927	婴儿利什曼原虫 L. infantum	新疆克拉玛依 Karamay, Xinjiang	白蛉 Sandfly	312	MH450082^a
JIASHI-1	MHOM/CN/2008/JIASHI-1	婴儿利什曼原虫 L. infantum	新疆伽师 Jiashi, Xinjiang	患者 Human	313	GQ367486^b
JIASHI-5	MHOM/CN/2008/JIASHI-5	婴儿利什曼原虫 L. infantum	新疆伽师 Jiashi, Xinjiang	患者 Human	313	MH450075^a
XJ771	IWUI/CN/1977/XJ771	婴儿利什曼原虫 L. infantum	新疆伽师 Jiashi, Xinjiang	白蛉 Sandfly	313	MH450076^a
SC6	MHOM/CN/1986/SC6	杜氏利什曼原虫 L. donovani	四川九寨沟 Jiuzhaigou, Sichun	患者 Huaman	318	MH450071^a
Cy	MCAN/CN/2008/Cy	杜氏利什曼原虫 L. donovani	甘肃武都 Wudu, Gansu	犬 Canine	318	MH450070^a
801	MHOM/CN/1980/801	杜氏利什曼原虫 L. donovani	新疆喀什 Kashi, Xinjiang	患者 Human	316	GQ367489^b
KS-2	MHOM/CN/1996/KS-2	杜氏利什曼原虫 L. donovani	新疆喀什 Kashi, Xinjiang	患者 Human	316	MH450077^a