Characteristics of genetic differentiation of Echinococcus multilocularis and E. shiquicus in Qinghai region

doi:10.12140/j.issn.1000-7423.2024.03.005

Abstract

Abstract:

Objective To analyze the genetic differentiation characteristics of Echinococcus multilocularis and E. shiquicus in Qinghai region to provide theoretical support for the prevention and control of echinococcosis in Qinghai Province. Methods Small mammals were captured in the main natural endemic areas of Echinococcus spp. in Yushu, Guoluo, and Huangnan Tibetan Autonomous Prefecture and were dissected to collect cysts. The genomic DNA from cysts tissue was extracted and the cytochrome oxidase 1 (cox1) gene was amplified using PCR and sequenced. DnaSP v6, Iqtree, BEAST v2.7.4 and other software were used for haplotype analysis, nucleotide polymorphism analysis, construction of a phylogenetic tree, and estimation of the divergence time of the Echinococcus genus. Results A total of 55 hydatid cysts were obtained from 2 864 small mammals. All 55 cyst samples were amplified for cox1 bands with a length of approximately 800 bp, of which 37 were E. multilocularis, and 18 were E. shiquicus, the prevalence of E. multilocularis in Neodon fuscus was 1.96% (37/1884). The prevalence of E. shiquicus in Ochotona curzoniae was 1.84% (18/980). In the 37 cox1 sequences of E. multilocularis, there were 5 haplotypes in the 37 cox1 sequences of E. multilocularis with EmH3 being the predominant one (33/37), the haplotype diversity index was 0.207, the nucleotide diversity index was 0.033 55, and there were 156 variable sites. In the 18 cox1 sequences of E. shiquicus, There were 8 haplotypes, with the EsH2 haplotype being the predominant one (8/18), the haplotype diversity index was 0.778, the nucleotide diversity index was 0.060 52, and there were 14 variable sites. Thirteen haplotypes of E. multilocularis and E. shiquensis were uploaded to GenBank. The accession numbers of haplotypes EmH1-EmH5 are OR821706, OR821707, OR830343, OR830344, OR826123, respectively. The accession numbers of haplotypes ESH1-ESH8 are OR835156, OR835157, OR830376, OR830378, OR831110, OR875250, OR835161, OR841080. The phylogenetic tree shows that the 5 haplotypes of E. multilocularis were clustered together with the Asian strain of E. multilocularis, and the 8 haplotypes of E. shiquicus were clustered with E. shiquicus in the GenBank. The divergence time based on the cox1 gene showed that the common ancestor of E. granulosus, E. multilocularis, E. shiquicus, E. oligarthrus and E. vogeli existed approximately 5.67 million years ago (Mya) (95% CI: 4.72-6.66 Mya), and the average divergence time for E. granulosus, E. shiquicus and E. multilocularis was approximately 2.02 Mya (95% CI: 1.51-2.49 Mya). Conclusion E. multilocularis and E. shiquicus in Qinghai region have high genetic diversity, with EmH3 haplotype dominating E. multilocularis and EsH2 haplotype dominating E. shiquicus.

Key words: Echinococcus multilocularis, Echinococcus shiquicus, Haplotype, cox1 gene, Phylogenetic analysis, Divergence time

CLC Number:

R383.33

FU Yong, ZHANG Haining, CHEN Wangkai, SHI Zhenghe, ZHANG Xueyong, GUO Zhihong, DUO Hong, SHEN Xiuying, MENG Ru, LI Zhi. Characteristics of genetic differentiation of Echinococcus multilocularis and E. shiquicus in Qinghai region[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(3): 309-315.

Figures/Tables 4

References 39

[1]	Casulli A, Barth TFE, Tamarozzi F. Echinococcus multilocularis[J]. Trends Parasitol, 2019, 35(9): 738-739. doi: S1471-4922(19)30109-6 pmid: 31182385
[2]	Gong QL, Ge GY, Wang Q, et al. Meta-analysis of the prevalence of Echinococcus in dogs in China from 2010 to 2019[J]. PLoS Negl Trop Dis, 2021, 15(4): e0009268.
[3]	Torgerson PR, Keller K, Magnotta M, et al. The global burden of alveolar echinococcosis[J]. PLoS Negl Trop Dis, 2010, 4(6): e722.
[4]	Deplazes P, Rinaldi L, Alvarez Rojas CA, et al. Global distribution of alveolar and cystic echinococcosis[J]. Adv Parasitol, 2017, 95: 315-493.
[5]	Craig PS, Giraudoux P, Wang ZH, et al. Echinococcosis transmission on the Tibetan Plateau[J]. Adv Parasitol, 2019, 104: 165-246.
[6]	Mathy A, Hanosset R, Adant S, et al. The carriage of larval Echinococcus multilocularis and other cestodes by the musk rat (Ondatra zibethicus) along the Ourthe River and its tributaries (Belgium)[J]. J Wildl Dis, 2009, 45(2): 279-287.
[7]	Beiromvand M, Akhlaghi L, Fattahi Massom SH, et al. Molecular identification of Echinococcus multilocularis infection in small mammals from Northeast, Iran[J]. PLoS Negl Trop Dis, 2013, 7(7): e2313.
[8]	Kui Y, Xue CZ, Wang X, et al. Progress of echinococcosis control in China, 2022[J]. Chin J Parasitol Parasit Dis, 2024, 42(1): 8-16. (in Chinese)
	(蒉嫣, 薛垂召, 王旭, 等. 2022年全国棘球蚴病防治工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 8-16.) doi: 10.12140/j.issn.1000-7423.2024.01.002
[9]	Kui Y, Xue CZ, Wang X, et al. Progress of echinococcosis control in China, 2021[J]. Chin J Parasitol Parasit Dis, 2023, 41(2): 142-148. (in Chinese)
	(蒉嫣, 薛垂召, 王旭, 等. 2021年全国棘球蚴病防治进展[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(2): 142-148.) doi: 10.12140/j.issn.1000-7423.2023.02.003
[10]	Oksanen A, Siles-Lucas M, Karamon J, et al. The geographical distribution and prevalence of Echinococcus multilocularis in animals in the European Union and adjacent countries: a systematic review and meta-analysis[J]. Parasit Vectors, 2016, 9(1): 519.
[11]	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.
[12]	Dán Á, Rónai Z, Széll Z, et al. Prevalence and genetic characterization of Echinococcus spp. in cattle, sheep, and swine in Hungary[J]. Parasitol Res, 2018, 117(9): 3019-3022. doi: 10.1007/s00436-018-5977-5 pmid: 29934692
[13]	Xiao N, Qiu JM, Nakao M, et al. Echinococcus shiquicus, a new species from the Qinghai-Tibet Plateau Region of China: discovery and epidemiological implications[J]. Parasitol Int, 2006, 55(Suppl): S233-S236.
[14]	Yan HB, Li L, Li WH, et al. Echinococcus shiquicus in Qinghai-Tibet Plateau: population structure and confirmation of additional endemic areas[J]. Parasitology, 2021, 148(7): 879-886.
[15]	Zhu GQ, Yan HB, Li L, et al. First report on the phylogenetic relationship, genetic variation of Echinococcus shiquicus isolates in Tibet Autonomous Region, China[J]. Parasit Vectors, 2020, 13(1): 590.
[16]	Wu YT, Li L, Xu FL, et al. Establishment of a secondary infection laboratory model of Echinococcus shiquicus metacestode using BALB/c mice and Mongolian jirds (Meriones unguiculatus)[J]. Parasitology, 2023, 150(9): 813-820.
[17]	Zhang YG, Ma YY, Cao DP, et al. Bioinformatic analyses on sequences of the complete mitochondrial genomes of Echinococcus genus[J]. Chin J Zoonoses, 2019, 35(3): 271-277. (in Chinese)
	(张耀刚, 马艳艳, 曹得萍, 等. 棘球属绦虫线粒体基因组全序列生物信息学分析[J]. 中国人兽共患病学报, 2019, 35(3): 271-277.)
[18]	Han XM, Jian YN, Zhang XY, et al. Genetic characterization of Echinococcus isolates from various intermediate hosts in the Qinghai-Tibetan Plateau Area, China[J]. Parasitology, 2019, 146(10): 1305-1312.
[19]	Wang LY. Study on the relation of distribution of intermediate host of Echinococcus multilocularis to the environmental factors in southern Qinghai Plateau[D]. Beijing: Chinese Center for Disease Control and Prevention, 2009: 6-13. (in Chinese)
	(王立英. 青南高原多房棘球绦虫中间宿主分布与环境因素关系的研究[D]. 北京: 中国疾病预防控制中心, 2009: 6-13.)
[20]	Jiang WB, Liu N, Zhang GT, et al. Specific detection of Echinococcus spp. from the Tibetan fox (Vulpes ferrilata) and the red fox (V. Vulpes) using copro-DNA PCR analysis[J]. Parasitol Res, 2012, 111(4): 1531-1539.
[21]	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
[22]	Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability[J]. Mol Biol Evol, 2013, 30(4): 772-780. doi: 10.1093/molbev/mst010 pmid: 23329690
[23]	Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[J]. Bioinformatics, 2009, 25(15): 1972-1973. doi: 10.1093/bioinformatics/btp348 pmid: 19505945
[24]	Nguyen LT, Schmidt HA, von Haeseler A, et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies[J]. Mol Biol Evol, 2015, 32(1): 268-274.
[25]	Kalyaanamoorthy S, Minh BQ, Wong TKF, et al. ModelFinder: fast model selection for accurate phylogenetic estimates[J]. Nat Methods, 2017, 14(6): 587-589. doi: 10.1038/nmeth.4285 pmid: 28481363
[26]	Xie JM, Chen YR, Cai GJ, et al. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees[J]. Nucleic Acids Res, 2023, 51(W1): W587-W592.
[27]	Bouckaert R, Heled J, Kühnert D, et al. BEAST 2: a software platform for Bayesian evolutionary analysis[J]. PLoS Comput Biol, 2014, 10(4): e1003537.
[28]	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.
[29]	Wang X, Liu JY, Zuo QQ, et al. Echinococcus multilocularis and Echinococcus shiquicus in a small mammal community on the eastern Tibetan Plateau: host species composition, molecular prevalence, and epidemiological implications[J]. Parasit Vectors, 2018, 11(1): 302.
[30]	Li TY, Chen XW, Zhen R, et al. Widespread co-endemicity of human cystic and alveolar echinococcosis on the eastern Tibetan Plateau, northwest Sichuan/southeast Qinghai, China[J]. Acta Trop, 2010, 113(3): 248-256. doi: 10.1016/j.actatropica.2009.11.006 pmid: 19941830
[31]	Ma JY, Wang H, Lin GH, et al. Molecular identification of Echinococcus species from eastern and southern Qinghai, China, based on the mitochondrial cox1 gene[J]. Parasitol Res, 2012, 111(1): 179-184.
[32]	Wu CC, Zhang WB, Ran B, et al. Genetic variation of mitochondrial genes among Echinococcus multilocularis isolates collected in Western China[J]. Parasit Vectors, 2017, 10(1): 265.
[33]	Li CY, Guan YY, Wu WP, et al. Progress of researches on infection with two species of Echinococcus causing human diseases in animal hosts and influencing factors[J]. Chin J Schisto Control, 2022, 34(2): 194-199. (in Chinese)
	(李春阳, 官亚宜, 伍卫平, 等. 两种致病人体棘球绦虫动物宿主感染及影响因素研究进展[J]. 中国血吸虫病防治杂志, 2022, 34(2): 194-199.)
[34]	Fu MH, Wang X, Han S, et al. Advances in research on echinococcoses epidemiology in China[J]. Acta Trop, 2021, 219: 105921.
[35]	Zhou NN, Wang MX, Cui L, et al. Genetic variation of Empoasca vitis (Göthe) (Hemiptera ∶ Cicadellidae) among different geographical populations based on mtDNA COⅠ complete sequence[J]. Acta Ecol Sin, 2014, 34(23): 6879-6889. (in Chinese)
	(周宁宁, 王梦馨, 崔林, 等. 基于COⅠ基因全长序列的假眼小绿叶蝉地理种群遗传分化研究[J]. 生态学报, 2014, 34(23): 6879-6889.)
[36]	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.
[37]	Clark PU, Dyke AS, Shakun JD, et al. The last glacial maximum[J]. Science, 2009, 325(5941): 710-714. doi: 10.1126/science.1172873 pmid: 19661421
[38]	Wu YB, Liu YG, Yi CL, et al. Impact of Tibetan glacier change on the Asian climate during the last glacial maximum[J]. Acta Sci Nat Univ Pekin, 2019, 55(1): 159-170. (in Chinese)
	(毋宇斌, 刘永岗, 易朝路, 等. 末次冰盛期青藏高原冰川变化对亚洲气候的影响[J]. 北京大学学报(自然科学版), 2019, 55(1): 159-170.)
[39]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2): e00075-e00018.