山西省绵羊捻转血矛线虫的感染现状及遗传变异分析

doi:10.12140/j.issn.1000-7423.2023.06.010

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (6): 733-738.doi: 10.12140/j.issn.1000-7423.2023.06.010

山西省绵羊捻转血矛线虫的感染现状及遗传变异分析

马志雅¹(), 谢世臣¹, 贺渊惠¹, 高文伟¹, 刘卿¹, 朱兴全¹^,², 郑文斌¹^,^*()

1 山西农业大学动物医学学院，太谷 030801
2 云南农业大学动物医学院，云南省高校兽医公共卫生重点实验室，昆明 650201

收稿日期:2022-04-26 修回日期:2023-09-22 出版日期:2023-12-30 发布日期:2023-12-19
通讯作者: * 郑文斌（1991-），男，博士，副教授，从事重要动物寄生虫和人兽共患寄生虫的感染与致病机制研究。E-mail：wenbinzheng1@126.com
作者简介:马志雅（1999-），女，硕士研究生，从事兽医寄生虫病学研究。E-mail：mzy16635048046@126.com
基金资助:
山西省“1331工程”(20211331-13);山西农业大学高层次人才科研专项(2021XG001);云南省专家工作站项目(202005AF150041)

Infection status and genetic variation analysis of Haemonchus contortus in sheep in Shanxi Province

MA Zhiya¹(), XIE Shichen¹, HE Yuanhui¹, GAO Wenwei¹, LIU Qing¹, ZHU Xingquan¹^,², ZHENG Wenbin¹^,^*()

1 College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China
2 Key Laboratory of Veterinary Public Health of Higher Education of Yunnan Province, College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China

Received:2022-04-26 Revised:2023-09-22 Online:2023-12-30 Published:2023-12-19
Contact: * E-mail: wenbinzheng1@126.com
Supported by:
Fund for Shanxi “1331 Project”(20211331-13);Special Research Fund of Shanxi Agricultural University for High-level Talents(2021XG001);Yunnan Expert Workstation Project(202005AF150041)

摘要/Abstract

摘要：

目的了解山西省绵羊捻转血矛线虫的感染现状及遗传变异情况。方法采集山西省晋北的山阴县、晋中的祁县和晋南的稷山县绵羊粪样，并提取粪样基因组DNA。利用捻转血矛线虫种特异性引物，扩增样品的核糖体内转录间隔区-2（ITS-2）部分序列，经凝胶电泳观察和DNA测序分析鉴定其种属，评估不同地区绵羊捻转血矛线虫的感染现状。取PCR阳性样品，采用扩增捻转血矛线虫ITS全长序列的保守引物，PCR扩增ITS全长序列并测序，在DNAMAN 9.0中与GenBank中已公开的捻转血矛线虫ITS-2和ITS全长序列进行比对，查找碱基变异位点。使用DNAstar 7.1软件计算捻转血矛线虫ITS序列的碱基含量，并对GenBank中已报道的不同地区、不同宿主来源的18条捻转血矛线虫ITS全长序列及本研究获得的2条ITS全长序列进行序列相似性分析，用最大似然法构建基于ITS全长序列的系统进化树，分析捻转血矛线虫的遗传变异情况。结果共采集401份绵羊粪样，其中69份粪样扩增出ITS-2片段（占17.2%）；序列分析结果显示，获得的ITS-2部分序列与捻转血矛线虫（GenBank登录号OQ674251）的序列相似性为100%，故鉴定为捻转血矛线虫。其中山阴县、稷山县和祁县的绵羊捻转血矛线虫阳性率分别为47.4%（64/135）、3.0%（5/169）和0（0/97）。69份ITS-2阳性的粪样中，4份样品成功扩增出ITS全长序列，其存在两种基因型，且这两种基因型之间仅在ITS-1序列中226 bp处存在1个碱基差异。基于捻转血矛线虫ITS全长序列的序列相似性分析发现，两种基因型之间的变异率仅为0.1%；18条不同地区和不同宿主来源的捻转血矛线虫的序列间变异率为0~2.6%。系统进化树分析结果显示，本研究中鉴定的序列（GenBank登录号OP518297和OP518298）与来自美国（GenBank登录号EU086378）、老挝（GenBank登录号AB908961）和中国其他地区（GenBank登录号HQ844231）的序列处于同一分支上；捻转血矛线虫的群体遗传结构无明显的地理和宿主相关性。结论山西省调查地区的绵羊捻转血矛线虫感染率较高，捻转血矛线虫的遗传变异率较低，与其他地区、不同宿主来源的捻转血矛线虫之间无明显差异。

关键词: 捻转血矛线虫, 内转录间隔区, PCR, 系统进化树, 绵羊, 山西

Abstract:

Objective To understand the prevalence and genetic variation of Haemonchus contortus in sheep in Shanxi Province. Methods Fecal samples were collected from sheep in Shanyin County in Northern Shanxi, Qi County in Central Shanxi and Jishan County in Southern Shanxi for extraction of genomic DNA, of which the partial sequence of H. contortus second internal transcribed spacer 2 (ITS-2) was amplified using H. contortus specific primers by PCR and sequenced to identify the parasite species by gel electrophoresis and evaluate the H. contortus prevalence in sheep in different regions of Shanxi Province. The full-length sequence of ITS of H. contortus was amplified by PCR using conserved primers from ITS-2 positive samples and was sequenced. The ITS-2 sequences and ITS full-length sequences of H. contortus obtained in this study were aligned with the corresponding sequences available in GenBank to examine the base mutation site by DNAMAN 9.0. The base content of H. contortus ITS sequence was calculated using DNAstar 7.1 software. Moreover, the sequence homolohy was analyzed on 18 ITS full-length sequences of H. contortus from different regions and hosts, which were available in GenBank (n = 18), and the 2 ITS full-length sequences obtained in this study. Based on the ITS full-length sequence, phylogenetic tree was constructed using maximum likelihood method to analyze the genetic variation in H. contortus. Results A total of 401 sheep fecal samples were collected, of which 69 samples were successfully amplified with the target ITS-2 fragment (17.2%). The ITS-2 sequences obtained in this study showed 100% sequence identity with that of H. contortus (accession number OQ674251) and were identified as H. contortus. The positive rate of H. contortus was 47.4% (64/135), 3.0% (5/169) and 0 (0/97) in sheep in Shanyin County, Jishan County and Qi County of Shanxi Province, respectively. In addition, the complete ITS sequences of H. contortus were successfully amplified from 4 H. contortus-positive fecal samples, showing two genotypes with a single nucleotide variation at the 226 bp position of ITS-1 locus. Based on the sequence identity analysis of the complete ITS sequences of H. contortus, it was found that the variation rate between the two genotypes identified in this study was only 0.1%, and the variation rate of the 18 sequences from different regions of the world and hosts were 0-2.6%. The analysis of ITS sequences and phylogenetic tree showed that the sequences identified in this study, OP518297 and OP518298, were on the same branch with sequences from the United States (accession number EU086378), Laos (accession number AB908961) and other regions of China (accession number HQ844231), and there was no significant difference among ITS sequences of H. contortus from different regions of the world and hosts. Conclusion The H. contortus infection rate in sheep in the surveyed regions of Shanxi Province was comparatively high, while the genetic variation rate of the parasite detected was low, which showed no significant difference from those of H. contortus found in different regions and hosts.

Key words: Haemonchus contortus, Internal transcribed spacer, PCR, Phylogenetic tree, Sheep, Shanxi

中图分类号:

R532.19

马志雅, 谢世臣, 贺渊惠, 高文伟, 刘卿, 朱兴全, 郑文斌. 山西省绵羊捻转血矛线虫的感染现状及遗传变异分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(6): 733-738.

MA Zhiya, XIE Shichen, HE Yuanhui, GAO Wenwei, LIU Qing, ZHU Xingquan, ZHENG Wenbin. Infection status and genetic variation analysis of Haemonchus contortus in sheep in Shanxi Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(6): 733-738.

图/表 4

参考文献 23

[1]	Sanders J, Xie Y, Gazzola D, et al. A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep[J]. Int J Parasitol Drugs Drug Resist, 2020, 14: 230-236. doi: 10.1016/j.ijpddr.2020.11.004
[2]	Ehsan M, Hu RS, Liang QL, et al. Advances in the development of anti-Haemonchus contortus vaccines: challenges, opportunities, and perspectives[J]. Vaccines (Basel), 2020, 8(3): 555.
[3]	Wang CQ, Li FF, Zhang ZZ, et al. Recent research progress in China on Haemonchus contortus[J]. Front Microbiol, 2017, 8: 1509. doi: 10.3389/fmicb.2017.01509
[4]	Luo XP, Gao W, Geng WH, et al. Investigation of gastrointestinal nematode infections in sheep in the northern suburb of Yuci district of Shanxi Province and description of Nematodirella longispiculata[J]. Anim Husb Feed Sci, 2020, 41(2): 96-100. (in Chinese)
	(罗晓平, 高娃, 耿万恒, 等. 山西榆次区北郊绵羊胃肠道线虫感染调查及长刺似细颈线虫的描述[J]. 畜牧与饲料科学, 2020, 41(2): 96-100.)
[5]	Ahmed M, Singh MN, Bera AK, et al. Molecular basis for identification of species/isolates of gastrointestinal nematode parasites[J]. Asian Pac J Trop Med, 2011, 4(8): 589-593. doi: 10.1016/S1995-7645(11)60152-1 pmid: 21914532
[6]	Gasser RB, Bott NJ, Chilton NB, et al. Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock: bionomic and biotechnological implications[J]. Biotechnol Adv, 2008, 26(4): 325-334. doi: 10.1016/j.biotechadv.2008.03.003 pmid: 18502076
[7]	Xu FF, Su XY, Long SR, et al. Application of molecular markers in the research of genetic diversity in medical helminths[J]. Chin Trop Med, 2023, 23(1): 83-89. (in Chinese)
	(徐方方, 苏晓艺, 龙绍蓉, 等. 分子标记在医学蠕虫遗传多态性研究中的应用[J]. 中国热带医学, 2023, 23(1): 83-89.) doi: 10.13604/j.cnki.46-1064/r.2023.01.15
[8]	Bott NJ, Campbell BE, Beveridge I, et al. A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep[J]. Int J Parasitol, 2009, 39(11): 1277-1287. doi: 10.1016/j.ijpara.2009.03.002 pmid: 19328802
[9]	Zarlenga DS, Barry Chute M, Gasbarre LC, et al. A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle[J]. Vet Parasitol, 2001, 97(3): 199-209. pmid: 11390072
[10]	Zhu X, Chilton NB, Jacobs DE, et al. Characterisation of Ascaris from human and pig hosts by nuclear ribosomal DNA sequences[J]. Int J Parasitol, 1999, 29(3): 469-478. pmid: 10333331
[11]	Mohammedsalih KM, Khalafalla A, Bashar A, et al. Epidemiology of strongyle nematode infections and first report of benzimidazole resistance in Haemonchus contortus in goats in South Darfur State, Sudan[J]. BMC Vet Res, 2019, 15(1): 184. doi: 10.1186/s12917-019-1937-2 pmid: 31164118
[12]	Rose H, Caminade C, Bolajoko MB, et al. Climate-driven changes to the spatio-temporal distribution of the parasitic nematode, Haemonchus contortus, in sheep in Europe[J]. Glob Chang Biol, 2016, 22(3): 1271-1285. doi: 10.1111/gcb.2016.22.issue-3
[13]	Domke AV, Chartier C, Gjerde B, et al. Prevalence of gastrointestinal helminths, lungworms and liver fluke in sheep and goats in Norway[J]. Vet Parasitol, 2013, 194(1): 40-48. doi: 10.1016/j.vetpar.2012.12.023 pmid: 23298563
[14]	Zhao XL, Eyeldege, Hai Y, et al. Study on infections and drug resistance of Haemonchus contortus of sheep[J]. J Domest Anim Ecol, 2019, 40(11): 70-72. (in Chinese)
	(赵学亮, 额叶勒德格, 海鹰, 等. 绵羊捻转血矛线虫流行病学调查及耐药性检测[J]. 家畜生态学报, 2019, 40(11): 70-72.)
[15]	Guan C, Liu X, Ren Y, et al. Investigation on infection species and infection rate of digestive nematodes of sheep in Zhangjiakou[J]. J Hebei N Univ Nat Sci Ed, 2018, 34(9): 37-40, 44. (in Chinese)
	关琛, 刘峡, 任一, 等. 张家口地区羊消化道线虫感染虫种及感染率的调查[J]. 河北北方学院学报(自然科学版), 2018, 34(9):37- 40, 44.)
[16]	Yi JX, Yi LD, Lin L. Investigation onparasitic nematode infection in sheep in some areas of Yueyang City[J]. Hunan J Anim Sci Vet Med, 2021(5): 9-10. (in Chinese)
	(易劲翔, 易立冬, 林莉. 岳阳市部分地区羊寄生线虫感染的调查[J]. 湖南畜牧兽医, 2021(5): 9-10.)
[17]	Eyeldeg, Cao ZF, Hai Y, et al. Studies on infections and drug resistance of gastrointestinal nematodes in sheep in Uxin Banner[J]. Chin Vet Sci, 2018, 48(6): 735-742. (in Chinese)
	(额叶勒德格, 曹增富, 海鹰, 等. 乌审旗地区绵羊消化道线虫感染情况及耐药性的调查[J]. 中国兽医科学, 2018, 48(6): 735-742.)
[18]	Luo XP, Li JY, Gao W, et al. Polymorphism analysis of candidate genes for ivermectin resistance in Haemonchus contortus[J]. Chin J Parasitol Parasit Dis, 2022, 40(4): 536-539, 544. (in Chinese)
	(罗晓平, 李军燕, 高娃, 等. 捻转血矛线虫耐伊维菌素候选基因的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 536-539, 544.)
[19]	Chen XD, Wang TY, Shi YQ, et al. Analysis of the expressed IncRNA related to albendazole resistance of Haemonchus contortus[J]. Chin J Parasitol Parasit Dis, 2022, 40(4): 540-544. (in Chinese)
	(陈昕迪, 王腾宇, 石雅琴, 等. 捻转血矛线虫阿苯达唑耐药相关长链非编码RNA的表达分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 540-544.)
[20]	Shu L, Liu XZ, Lv FL. Research progress on the effect of ivermectin against parasitic diseases and parasitic infections[J]. J Pathog Biol, 2022, 17(8): 972-977. (in Chinese)
	(舒磊, 刘星卓, 吕芳丽. 伊维菌素抗寄生虫病和寄生虫感染的作用研究进展[J]. 中国病原生物学杂志, 2022, 17(8): 972-977.)
[21]	Li FF, Zhang T, Zhou CX, et al. Progresses of haemonchosis[J]. Chin J Anim Infect Dis, 2019, 27(3): 107-111. (in Chinese)
	(李芳芳, 张挺, 周彩显, 等. 捻转血矛线虫病的研究进展[J]. 中国动物传染病学报, 2019, 27(3): 107-111.)
[22]	Yan RF, Song XK, Xu LX, et al. Phylogenetic analysis of Haemonchus contortus based on ITS sequence[J]. Chin J Anim Vet Sci, 2012, 43(7): 1117-1122. (in Chinese)
	(严若峰, 宋小凯, 徐立新, 等. 基于ITS序列的捻转血矛线虫系统进化分析[J]. 畜牧兽医学报, 2012, 43(7): 1117-1122.)
[23]	Hussain T, Periasamy K, Nadeem A, et al. Sympatric species distribution, genetic diversity and population structure of Haemonchus isolates from domestic ruminants in Pakistan[J]. Vet Parasitol, 2014, 206(3/4): 188-199. doi: 10.1016/j.vetpar.2014.10.026

基因 Gene	引物名称 Primer name	引物序列(（5'→3'） Sequences (5'→3')	产物长度/bp Length/bp
ITS-2	HAE	CAAATGGCATTTGTCTTTTAG	265
ITS-2	NC2	TTAGTTTCTTTTCCTCCGCT	265
E-ITS	628-F	CATTTTCGTCTTGGGCGATAT	2 810
E-ITS	NC2	TTAGTTTCTTTTCCTCCGCT	2 810
ITS	NC5	GTAGGTGAACCTGCGGAAGGATCATT	885
ITS	NC2	TTAGTTTCTTTTCCTCCGCT	885

山西省绵羊捻转血矛线虫的感染现状及遗传变异分析

Infection status and genetic variation analysis of Haemonchus contortus in sheep in Shanxi Province

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 4

参考文献 23

相关文章 15

编辑推荐

Metrics

本文评价

[1]	原慧真, 李栋梁, 程书琪, 菅复春. 芽囊原虫体外培养特性的研究[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(5): 631-635.
[2]	徐银, 刘婷, 徐慧, 曾小军, 兰炜明, 龚志红, 戴坤教, 邱婷婷, 郝先, 谢曙英. PCR-CRISPR/Cas12a华支睾吸虫囊蚴检测方法的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 421-426.
[3]	蔡长煌, 张芝平, 卓鸣莺, 郭理平, 傅志辉, 刘亦若. 基于虫卵内转录间隔区2序列分析诊断麝猫后睾吸虫和华支睾吸虫感染[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 427-433.
[4]	黎玉琼, 于有利, 郜军荣, 刘芸芸, 李红兵, 牛晓昊. 宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 476-479.
[5]	兰炜明, 徐慧, 徐银, 邱婷婷, 谢曙英, 邓凤林, 胡绍良, 刘欢, 郭家钢, 曾小军. 荧光定量PCR用于日本血吸虫感染高危环境早期预警的研究[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 502-505.
[6]	李美, 肖宁, 夏志贵. 基于无性期18S rDNA特异性引物检测5种疟原虫qPCR的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 36-43.
[7]	刘继兵, 赵洪喜. 基于18S rRNA的胎毛滴虫感染PCR诊断方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 682-685.
[8]	罗晓平, 李军燕, 高娃, 耿万恒, 王瑞, 胡薇, 乔蕾, 邵国玉, 杨晓野. 捻转血矛线虫耐伊维菌素候选基因的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 536-539.
[9]	陈昕迪, 王腾宇, 石雅琴, 毛晓伟, 闫旭, 苏娅, 温海峰, 王文龙. 捻转血矛线虫阿苯达唑耐药相关长链非编码RNA的表达分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 540-544.
[10]	江莉, 张耀光, 刘红霞, 王真瑜, 朱民, 吴寰宇. 疟疾蚊媒监测多重PCR方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 159-167.
[11]	刘路瑶, 杨聪山, 章翔, 程蓉蓉, 洪宇航, 吴成龙, 孙萌, 陈木新, 艾琳, 徐前明. 孔雀源异刺线虫的形态学特征及分子鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(6): 816-820.
[12]	付梅花, 王旭, 韩帅, 喻文杰, 杨毅, 高明军, 刘剑峰, 官亚宜, 王莹, 李春阳, 施丹丹, 伍卫平. 四川省色达县野外棘球绦虫终末宿主粪便污染情况及特征[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(5): 687-695.
[13]	马琳, 张铮, 王安礼, 刘东立. 检测利什曼原虫的实时荧光定量PCR的建立及应用[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 548-552.
[14]	郑玉华, 白永飞, 帖萍, 闫昌福, 王婷, 王晶莹, 王三桃, 蔡剑. 2010—2019年山西省人群内脏利什曼病流行特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(3): 352-358.
[15]	吴冬妮, 孙凌聪, 张华勋, 万伦, 张娟, 曹慕民, 夏菁. 湖北省输入性卵形疟的实验室检测能力分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(3): 406-409.