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

doi:10.12140/j.issn.1000-7423.2023.06.010

Abstract

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

CLC Number:

R532.19

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.

Figures/Tables 4

References 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