Evaluation of the value of K26 sequence applied in identification of Leishmania isolates in China

doi:10.12140/j.issn.1000-7423.2020.02.008

Abstract

Abstract:

Objective To evaluate the application of Leishmania K26 sequence in identification of Leishmania isolates in China.Methods Sixteen Leishmania isolates collected from different domestic endemic areas and 6 international reference strains were cultured for collection of promastigotes, from which DNA was extracted for amplification of K26 sequence by PCR. The K26 sequence was confirmed by sequencing. The sequence alignment was made using the ClustalX2 software. A phylogenetic tree was constructed based on the K26 sequences of highly-homologous Leishmania isolates downloaded from the GenBank database, to analyze the genetic relationship among the Leishmania isolates from different endemic areas in China.Results PCR amplification showed a specific band from all the 16 isolates and the DD8 international reference strain, while no specific band was found from five non-L. donovani complex strains. Sequence analysis indicates that all 3 isolates 801, KS-2 and KS-6 from the endemic areas of anthroponotic leishmaniasis generated a 920 bp fragment in PCR; 4 isolates XJ771, JIASHI-1, JIASHI-2 and JIASHI-5 from endemic areas of natural-sourced leishmaniasis all generated a 491 bp fragment in amplification reaction; 4 strains Cy, SC6, Hebei-Lv and Shanxi-Yang from endemic areas of zoonosis (specifically canine) leishmaniasis generated a 404 bp in amplification; 5 strains KXG-Xu, KXG-Liu, KXG-65, KXG-918 and KXG-927 from endemic areas of cutaneous leishmaniasis all generated a 449 bp fragment in amplification. Among the endemic areas, the K26 sequences of intra-area isolates showed 100% homology, while the sequences of enter-area isolates displayed a lower homology (40.2%-91.4%). Phylogenetic tree analysis showed that the 16 isolates of Leishmania in China clustered into 2 groups and 3 subgroups, among which nine isolates including KXG-Xu, KXG-Liu, KXG-65, KXG-918, KXG-927, XJ771, JIASHI-1, JIASHI-2, and JIASHI-5 clustered into subgroup A, while five isolates Cy, SC6, Hebei-Lv, Shanxi-Yang and L. infantum clustered into subgroup B, and both subgroups clustered into groupⅠ. The 801, KS-2 and KS-6 isolates clustered with L. donovani as subgroup C, which further clustered with the DD8 international reference isolate into group Ⅱ.Conclusion The K26 sequences of Leishmania can be used for identification of Leishmania isolates in China.

Key words: Leishmaniasis, Leishmania, Isolate, K26, Phylogenetic tree, China

CLC Number:

R382.22

Jian-xiu LIU, Chun-hua GAO, Yue-tao YANG, Bin ZHENG, Jun-yun WANG. Evaluation of the value of K26 sequence applied in identification of Leishmania isolates in China[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(2): 181-187.

Figures/Tables 5

Fig. 1

Table 1

The amplified K26 sequence length and GenBank accession numbers of Leishmania isolates in China and international reference isolates

流行区 Endemic area	分离株 Isolate	世界卫生组织编码 WHO code	来源 Location	宿主 Host	序列长度/bp Sequence length/bp	GenBank登录号 Accession no.
人源型 Anthroponotic type	801	MHOM/CN/1980/801	新疆喀什Kashi, Xinjiang	人Human	920	MN702441
	KS-2	MHOM/CN/1996/KS-2	新疆喀什Kashi, Xinjiang	人Human	920	MN702442
	KS-6	MHOM/CN/1996/KS-6	新疆喀什Kashi, Xinjiang	人Human	920	MN688111
自然疫源型 natural-sourced leishmaniasis	JIASHI-1	MHOM/CN/2008/JIASHI-1	新疆伽师Jiashi, Xinjiang	人Human	491	MN688113
	JIASHI-2	MHOM/CN/2008/JIASHI-2	新疆伽师Jiashi, Xinjiang	人Human	491	MN688114
	JIASHI-5	MHOM/CN/2008/JIASHI-5	新疆伽师Jiashi, Xinjiang	人Human	491	MN688115
	XJ771	IWUI/CN/1977/XJ771	新疆巴楚Bachu, Xinjiang	白蛉Sandfly	491	MN688112
人犬共患型 zoonosis (specifically canine) leishmaniasis	SC6	MHOM/CN/1986/SC6	四川九寨沟Jiuzhaigou, Sichuan	人Human	404	MN688116
	Cy	MCAN/CN/2008/Cy	甘肃武都Wudu, Gansu	犬Canine	404	MN688117
	Shanxi-Yang	MHOM/CN/2018/Shanxi-Yang	山西阳泉Yangquan, Shanxi	人Human	404	MN688118
	Hebei-Lv	MHOM/CN/2018/ Hebei-Lv	河北邯郸Handan, Hebei	人Human	404	MN688119
皮肤利什曼病 Cutaneous leishmaniasis	KXG-Xu	MHOM/CN/1994/KXG-Xu	新疆克拉玛依 Karamay, Xinjiang	人Human	449	MN688120
	KXG-927	IMJW/CN/1992/KXG-927	新疆克拉玛依 Karamay, Xinjiang	白蛉Sandfly	449	MN688121
	KXG-918	IMJW/CN/1991/KXG-918	新疆克拉玛依 Karamay, Xinjiang	白蛉Sandfly	449	MN688122
	KXG-65	IMJW/CN/1987/KXG-65	新疆克拉玛依 Karamay, Xinjiang	白蛉Sandfly	449	MN688123
	KXG-Liu	MHOM/CN/1994/KXG-Liu	新疆克拉玛依 Karamay, Xinjiang	人Human	449	MN688124
国际参照株 International reference strains	DD8	MHOM/IN/1980/DD8	印度India	人Human	641	MN702440
	5AS	MHOM/SU/73/5ASKH	土库曼Turkmenistan	人Human	-
	2903	MHOM/BR/75/M2903	巴西Brazil	人Human	-
	3720	MROH/SU/80/CLONE3720	乌兹别克Uzbek	大沙鼠Big gerbils	-
	L100	MHOM/ET/72/L100	埃塞俄比亚Ethiopia	人Human	-
	K27	MHOM/SU/74/K27	阿塞拜疆Azerbaijan	人Human	-

Table 1

Fig. 2

Fig. 3

Table 2

References 19

[1]	Chen JP, Yang BB, Xu JN , et al. Epidemic status of kala-azar in China[ C]//Zoological Society Symposium in Sichuan Province. Chengdu, 2011: 138. (in Chinese)
	( 陈建平, 杨斌斌, 徐佳楠 , 等. 我国黑热病流行现状[C]// 四川省动物学会第九次会员代表大会暨第十届学术研讨会论文集. 成都, 2011: 138.)
[2]	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.
[3]	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.
[4]	Guan LR, Qu JQ, Chai JJ . Leishmaniasis in China-present status of prevalence and some suggestions on its control[J]. Endem Dis Bull China, 2000,15(3):49-52. (in Chinese)
	( 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.)
[5]	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.
[6]	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(1):20.
[7]	Alce TM, Gokool S ,McGhie D , et al. Expression of hydrophilic surface proteins in infective stages of Leishmania donovani[J]. Mol Biochem Parasitol, 1999,102(1):191-196.
[8]	Haralambous C, Antoniou M, Pratlong F , et al. Development of a molecular assay specific for the Leishmania donovani complex that discriminates L. donovani/Leishmania infantum zymodemes: a useful tool for typing MON-1[J]. Diagn Microbiol Infect Dis, 2008,60(1):33-42.
[9]	Kimura M . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[J]. J Mol Evol, 1980,16(2):111-120.
[10]	Saitou N, Nei M . The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Mol Biol Evol, 1987,4(4):406-425.
[11]	Felsenstein J . Confidence limits on phylogenies: an approach using the bootstrap[J]. Evolution, 1985,39(4):783-791.
[12]	Schönian G, Cupolillo E, Mauricio I . Molecular evolution and phylogeny of Leishmania[M] //Drug Resistance in Leishmania Parasites. Vienna: Springer Vienna, 2012: 15-44.
[13]	Guan LR, Yang YQ, Zhai JQ , et al. Study of cutaneous leishmaniasis in Karamay, Xinjiang[J]. Chin J Parasitol Parasit Dis, 1997,15(3):181-185. (in Chinese)
	( 管立人, 杨元清, 翟靖琦 . 新疆克拉玛依地区皮肤利什曼病的研究[J]. 中国寄生虫学与寄生虫病杂志, 1997,15(3):181-185.)
[14]	Cheng FZ, Gao CH, Yang YT , et al. Polymorphism analysis of internal transcribed spacer 1 of Leishmania isolates from different endemic areas in China[J]. Chin J Parasitol Parasit Dis, 2019,37(2):196-201. (in Chinese)
	( 程芳洲, 高春花, 杨玥涛 , 等. 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[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(2):196-201.)
[15]	Bu LY, Hu XS, Jing BQ, Yi TL . Sequence analysis of SSU rDNA variable regions of Leishmania isolates from hillly foci and plain foci of China[J]. Chin J Parasitol Parasit Dis, 2000,18(6):321-324. (in Chinese)
	( 卜玲毅, 胡孝素, 敬保迁 , 等. 我国内脏利什曼病山丘疫区与平原疫区利什曼原虫SSUrDNA多变区序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2000,18(6):321-324.)
[16]	Bu LY, Hu XS, Jing BQ , et al. Sequence analysis of SSU rDNA variable region of different Leishmania isolates of China[J]. Pract Parasit Dis, 2001,9(1):1-3. (in Chinese)
	( 卜玲毅, 胡孝素, 敬保迁 , 等. 利什曼原虫中国分离株SSU rDNA多变区序列分析[J]. 实用寄生虫病杂志, 2001,9(1):1-3.)
[17]	Yuan DM, Qin HX, Zhang JG , et al. Phylogenetic analysis of HSP70 and cyt b gene sequences for Chinese Leishmania isolates and ultrastructural characteristics of Chinese Leishmania sp[J]. Parasitol Res, 2017,116(2):693-702.
[18]	Cao DP, Guo XG, Chen DL , et al. Species delimitation and phylogenetic relationships of Chinese Leishmania isolates reexamined using kinetoplast cytochrome oxidase Ⅱ gene sequences[J]. Parasitol Res, 2011,109(1):163-173.
[19]	Guan W, Cao DP, Sun K , et al. Phylogenic analysis of Chinese Leishmania isolates based on small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA)[J]. Acta parasitologica, 2012,57(2):101-113.