Genetic diversity and differentiation time of human isolates of Echinococcus granulosus and E. multilocularis from Qinghai

doi:10.12140/j.issn.1000-7423.2022.05.007

Abstract

Abstract:

Objective To analyzed the genetic diversity, genetic differences between populations and differentiation time of Echinococcus granulosus and E. multilocularis of Qinghai isolates, in order to provide scientific basis for species tracing and prevention and control of Echinococcus in Qinghai Province, China. Methods For genetic analysis, 50 liver lesion samples were collected from hospitalized echinococcosis patients in the Affiliated Hospital of Qinghai University to extract genomic DNA and amplify mitochondrial dehydrogenase 1 gene (nad1). Sequence multiple alignment was performed using Clustal X v2.0 software. Geographic informatics mapping of patients’ residence was constructed using ArcGIS software. Sequence haplotype analysis was made with DnaSP v6 software. Modeltest 3.7 software and PAUP*4.0B10 software were used to calculate the minimum optimal nucleic acid evolution model. The Bayesian’s phylogenetic evolution tree was constructed with MrBayes-3.2.7 software. The differentiation time of each node in the phylogenetic tree was estimated with the Bayesian method using BEAST v2.6.3 software. Results We successfully identified 48 Echinococcus lesion samples specimen and obtained the full length of complete nad1 gene of 894 bp. Among them, 13 samples were identified as the G1 genotype of E. granulosus, and 35 samples as E. multilocularis. All the sequences showed > 99% similarity to those in GenBank. Four haplotypes were identified as H1-H4 in the two species respectively; H3 was the dominant haplotype in E. granulosus samples(10/13), which is present in Xining, Guoluo, Yushu, Haidong, Haibei and Huangnan. H2 haplotype was found dominant in E. multilocular samples (51.4%,18/35), which is present in Xining, Guoluo, Yushu, and Haidong. The phylogenetic tree showed that E. granulosus and G1 genotype clustered into one branch, and E. multilocularis and Asian strain clustered into one branch. The results of differentiation time showed that the nearest common ancestor of E. granulosus, E. multilocularis, E. vogeli and E. oligarthrus was about 5.5 Mya (95% confidence interval 4.5-6.5 Mya), and the differentiation time of E. granulosus and E. multilocularis was about 2.5 Mya (95% confidence interval 2.3-4.1 Mya). Conclusion Both human E. granulosus and E. multilocularis in Qinghai Province show high genetic diversity. E. granulosus was found of G1 genotype, with H3 as the dominant haplotype, while in E. multilocularis samles H2 is the dominant. The two speies are widely distributed throughout Qinghai Province. The two species of Echinococcus exhit closer genetic relationship and differentiation timing.

Key words: Echinococcus granulosus, Echinococcus multilocularis, nad1 gene, Haplotype, Differentiation time

CLC Number:

R383.33

WU De-fang, FU Yong, REN Bin, ZHANG Yao-gang, XU Xiao-lei, PANG Ming-quan, FAN Hai-ning. Genetic diversity and differentiation time of human isolates of Echinococcus granulosus and E. multilocularis from Qinghai[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(5): 610-615.

Figures/Tables 5

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

References 30

[1]	Wu WP,, Wang H,, Wang Q, et al. A nationwide sampling survey on echinococcosis in China during 2012—2016[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 1-14. (in Chinese)
	( 伍卫平,, 王虎,, 王谦, 等. 2012—2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14.)
[2]	Nakao M,, McManus DP,, Schantz PM, et al. A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes[J]. Parasitology, 2007, 134(Pt 5): 713-722. pmid: 17156584
[3]	Bowles J,, Blair D,, McManus DP. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing[J]. Mol Biochem Parasitol, 1992, 54(2): 165-173. doi: 10.1016/0166-6851(92)90109-W
[4]	Nakao M,, Xiao N,, Okamoto M, et al. Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis[J]. Parasitol Int, 2009, 58(4): 384-389. doi: 10.1016/j.parint.2009.07.010
[5]	Zhu WJ. Studies on the molecular phylogeny of two types Echinococcus in the Qinghai Province[D]. Xining: Qinghai University, 2018: 4-5. (in Chinese)
	( 朱文君. 青海省两型棘球属绦虫分子种系发生的研究[D]. 西宁: 青海大学, 2018: 4-5.)
[6]	Wei YH,, Liu H,, Li WJ, et al. Analysis of nad1 gene polymorphisms of Echinococcus granulosus isolates from humans in Ali region of Tibet[J]. Chin J Parasitol Parasit Dis, 2020, 38(1): 17-21, 29. (in Chinese)
	( 魏玉环,, 刘华,, 李武军, 等. 西藏阿里地区细粒棘球蚴人体分离株nad1基因多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 17-21, 29.)
[7]	Yu JF,, Gui Z,, Wu L, et al. Cloning and sequence analysis of the partial nad1 gene within mitochondrial DNA of Echinococcus granulosus[J]. Chin J Zoonoses, 2020, 36(7): 534-538. (in Chinese)
	( 于晶峰,, 桂峥,, 武琳, 等. 细粒棘球蚴线粒体nad1基因的克隆与序列分析[J]. 中国人兽共患病学报, 2020, 36(7): 534-538.)
[8]	Wu YT,, Li L,, Zhu GQ, et al. Mitochondrial genome data confirm that yaks can serve as the intermediate host of Echinococcus canadensis (G10) on the Tibetan Plateau[J]. Parasit Vectors, 2018, 11(1): 166. doi: 10.1186/s13071-018-2684-0
[9]	Larkin MA,, Blackshields G,, Brown NP, et al. Clustal W and clustal X version 2.0[J]. Bioinformatics, 2007, 23(21): 2947-2948. doi: 10.1093/bioinformatics/btm404 pmid: 17846036
[10]	Rozas J,, Ferrer-Mata A,, Sánchez-Delbarrio JC, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets[J]. Mol Biol Evol, 2017, 34(12): 3299-3302. doi: 10.1093/molbev/msx248 pmid: 29029172
[11]	Ronquist F,, Teslenko M,, van der Mark P, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[J]. Syst Biol, 2012, 61(3): 539-542. doi: 10.1093/sysbio/sys029 pmid: 22357727
[12]	Drummond AJ,, Ho SY,, Phillips MJ, et al. Relaxed phylogenetics and dating with confidence[J]. PLoS Biol, 2006, 4(5): e88. doi: 10.1371/journal.pbio.0040088
[13]	Knapp J,, Nakao M,, Yanagida T, et al. Phylogenetic relationships within Echinococcus and Taenia tapeworms (Cestoda ∶ Taeniidae): an inference from nuclear protein-coding genes[J]. Mol Phylogenet Evol, 2011, 61(3): 628-638. doi: 10.1016/j.ympev.2011.07.022
[14]	Alvi MA,, Ohiolei JA,, Saqib M, et al. Echinococcus granulosus (sensu stricto) (G1, G3) and E. ortleppi (G5) in Pakistan: phylogeny, genetic diversity and population structural analysis based on mitochondrial DNA[J]. Parasit Vectors, 2020, 13(1): 347. doi: 10.1186/s13071-020-04199-8
[15]	Sharma M,, Sehgal R,, Fomda BA, et al. Molecular characterization of Echinococcus granulosus cysts in north Indian patients: identification of G1, G3, G5 and G6 genotypes[J]. PLoS Negl Trop Dis, 2013, 7(6): e2262. doi: 10.1371/journal.pntd.0002262
[16]	Jabbar A,, Narankhajid M,, Nolan MJ, et al. A first insight into the genotypes of Echinococcus granulosus from humans in Mongolia[J]. Mol Cell Probes, 2011, 25(1): 49-54. doi: 10.1016/j.mcp.2010.11.001
[17]	Alvarez Rojas CA,, Ebi D,, Gauci CG, et al. Microdiversity of Echinococcus granulosus sensu stricto in Australia[J]. Parasitology, 2016, 143(8): 1026-1033. doi: 10.1017/S0031182016000445 pmid: 27041115
[18]	Zhong XQ,, Wang N,, Hu DD, et al. Sequence analysis of cytb gene in Echinococcus granulosus from Western China[J]. Korean J Parasitol, 2014, 52(2): 205-209. doi: 10.3347/kjp.2014.52.2.205
[19]	Liu Q,, Cao LL,, Zhang YG, et al. Genotypes of Echinococcus granulosus in animals from Yushu, northeastern China[J]. Vector Borne Zoonotic Dis, 2013, 13(2): 134-137. doi: 10.1089/vbz.2012.1050
[20]	Wang JH,, Wang N,, Hu DD, et al. Genetic diversity of Echinococcus granulosus in southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene[J]. Sci World J, 2014, 2014: 867839.
[21]	Liu H,, Xiao N,, Yang SJ, et al. Epidemiological characteristics of canine Echinococcus infection in Qinghai-Tibet Plateau of China[J]. Chin J Schisto Control, 2017, 29(2): 129-138. (in Chinese)
	( 刘辉,, 肖宁,, 杨诗杰, 等. 青藏高原地区犬棘球绦虫感染的流行病学特征[J]. 中国血吸虫病防治杂志, 2017, 29(2): 129-138.)
[22]	Torgerson PR,, Keller K,, Magnotta M, et al. The global burden of alveolar echinococcosis[J]. PLoS Negl Trop Dis, 2010, 4(6): e722. doi: 10.1371/journal.pntd.0000722
[23]	Deplazes P,, Rinaldi L,, Alvarez Rojas CA, et al. Global distribution of alveolar and cystic echinococcosis[J]. Adv Parasitol, 2017, 95: 315-493.
[24]	Deng T,, Wang XM,, Wang SQ, et al. Evolution of the Chinese Neogene mammalian faunas and its relationship to uplift of the Tibetan Plateau[J]. Adv Earth Sci, 2015, 30(4): 407-415. (in Chinese) doi: 10.1167/j.issn.1001-8166.2015.04.0407
	( 邓涛,, 王晓鸣,, 王世骐, 等. 中国新近纪哺乳动物群的演化与青藏高原隆升的关系[J]. 地球科学进展, 2015, 30(4): 407-415.) doi: 10.1167/j.issn.1001-8166.2015.04.0407
[25]	de Schepper S,, Gibbard PL,, Salzmann U, et al. A global synthesis of the marine and terrestrial evidence for glaciation during the Pliocene Epoch[J]. Earth Sci Rev, 2014, 135: 83-102. doi: 10.1016/j.earscirev.2014.04.003
[26]	Tomiya S,, Tseng ZJ. Whence the beardogs? Reappraisal of the Middle to Late Eocene ‘Miacis’ from Texas, USA, and the origin of Amphicyonidae (Mammalia, Carnivora)[J]. R Soc Open Sci, 2016, 3(10): 160518. doi: 10.1098/rsos.160518
[27]	Steinthorsdottir M,, Coxall HK,, de Boer AM, et al. The miocene: the future of the past[J]. Paleoceanogr Paleoclimatol, 2021, 36(4): 1-71.
[28]	Johnson WE,, Eizirik E,, Pecon-Slattery J, et al. The late Miocene radiation of modern Felidae: a genetic assessment[J]. Science, 2006, 311(5757): 73-77. doi: 10.1126/science.1122277 pmid: 16400146
[29]	An ZS,, Wang SM,, Wu XH, et al. Eolian evidence from the Chinese Loess Plateau: the onset of the Late Cenozoic Great Glaciation in the Northern Hemisphere and Qinghai-Xizang Plateau uplift forcing[J]. Sci China Ser D Earth Sci, 1999, 42(3): 258-271. doi: 10.1007/BF02878963
[30]	Sun J,, Liu T. Stratigraphic evidence for the uplift of the Tibetan Plateau between -1.1 and -0.9 Myr ago[J]. Quat Res, 2000, 54(3): 309-320. doi: 10.1006/qres.2000.2170

分型 Species identification	序列数 No. sequence	单倍型数量 No. haplotype	单倍型多样性（HD）Haplotype diversity(HD)	核苷酸多样性（π） Nucleotide diversity(π)	单倍型（频率） Haplotype (Frequency)
细粒棘球绦虫 E. granulosus	13	4	0.423	0.000 69	EgH1（1）,EgH2（1）, EgH3（10）,EgH4（1）
多房棘球绦虫 E. multilocularis	35	4	0.588	0.000 76	EmH1（2）,EmH2（18）,EmH3（14）,EmH4（1）