Polymorphism analysis of candidate genes for ivermectin resistance in Haemonchus contortus

doi:10.12140/j.issn.1000-7423.2022.04.018

Abstract

Abstract:

The whole genomic DNA of susceptible strains (Hc-S) and resistance strains (Hc-R) of Haemonchus contortus was extracted and the target fragments of candidate genes were amplified by synthesised primers and sequenced directly by PCR. Then DNA sequencing results were analysed using Lasergene7.0 software. EditSeq program was used to intercept DNA fragments containing the coding sequence (CDS) region where specific single nucleotide polymorphism (SNP) sites were located, and MegAlign program was used for sequence alignment and single nucleotide and amino acid polymorphism analysis. The results showed no difference in all nucleotide sites of the HCOI00378500 gene in 25 Hc-S samples, while among 35 Hc-R samples, 8 samples showed a total of 32 SNP sites with 17 amino acid mutations, of which SNPs with the highest mutation rate accounted for 8.6%. The CDS region of the HCOI00506600 gene sequence was mutated at the 75th base of all samples in 40 Hc-R (T > C), and 7 samples of the 21 Hc-S had mutations (T > C), in which the amino acid was changed from isoleucine (Ile, I) to threonine (Thr, T). Among the 38 susceptible strains successfully sequenced, there are 4 SNPs at sites 38, 101, 125 and 134 of the HCOI01200700 gene; among the 13 successfully sequenced resistant strains, there are 6 SNPs at sites 32, 48, 71, 86, 101 and 131. The 38th, 125th and 134th positions of the HCOI01200700 gene showed G/A polymorphism (52.6%, 20/38), T/C polymorphism (57.9%, 22/38) and C/A polymorphism (57.9%, 22/38), respectively. There was at least one 38G, 125T or 134C in the Hc-S, while 38A, 125C or 134A in the Hc-R. Two candidate positive molecules were analysed and identified, including a candidate functional gene HCOI00506600 and a candidate marker HCOI01200700 for the diagnosis of IVM resistance.

Key words: Ivermectin, Haemonchus contortus, Anthelmintic resistance, Candidate gene, Single nucleotide polymorphism

CLC Number:

R383.19

LUO Xiao-ping, LI Jun-yan, GAO Wa, GEN Wan-heng, WANG Rui, HU Wei, QIAO Lei, SHAO Guo-yu, YANG Xiao-ye. Polymorphism analysis of candidate genes for ivermectin resistance in Haemonchus contortus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(4): 536-539.

Figures/Tables 4

References 21

[1]	Laing R, Gillan V, Devaney E. Ivermectin-old drug, new tricks?[J]. Trends Parasitol, 2017, 33(6): 463-472.
[2]	Yates DM, Portillo V, Wolstenholme AJ. The avermectin receptors of Haemonchus contortus and Caenorhabditis elegans[J]. Int J Parasitol, 2003, 33(11):1183-1193.
[3]	Wolstenholme AJ, Rogers AT. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics[J]. Parasitology, 2005, 131 Suppl: S85-S95.
[4]	Portillo V, Jagannathan S, Wolstenholme AJ. Distribution of glutamate-gated chloride channel subunits in the parasitic nematode Haemonchus contortus[J]. J Comp Neurol, 2003, 462(2):213-222.
[5]	Kotze AC, Prichard RK. Anthelmintic resistance in Haemonchus contortus: history, mechanisms and diagnosis[J]. Adv Parasitol, 2016, 93: 397-428.
[6]	Blackhall WJ, Liu HY, Xu M, et al. Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus[J]. Mol Biochem Parasitol, 1998, 95(2): 193-201.
[7]	Njue AI, Hayashi J, Kinne L, et al. Mutations in the extracellular domains of glutamate-gated chloride channel alpha3 and beta subunits from ivermectin-resistant Cooperia oncophora affect agonist sensitivity[J]. J Neurochem, 2004, 89(5): 1137-1147.
[8]	Kotze AC, Hunt PW, Skuce P, et al. Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions[J]. Int J Parasitol Drugs Drug Resist, 2014, 4(3): 164-184.
[9]	Lespine A, Ménez C, Bourguinat C, et al. P-glycoproteins and other multidrug resistance transporters in the pharmacology of anthelmintics: prospects for reversing transport-dependent anthelmintic resistance[J]. Int J Parasitol Drugs Drug Resist, 2011, 2: 58-75.
[10]	Redman E, Sargison N, Whitelaw F, et al. Introgression of ivermectin resistance genes into a susceptible Haemonchus contortus strain by multiple backcrossing[J]. PLoS Pathog, 2012, 8(2): e1002534.
[11]	Luo XP, Shi XN, Yuan CX, et al. Genome-wide SNP analysis using 2b-RAD sequencing identifies the candidate genes putatively associated with resistance to ivermectin in Haemonchus contortus[J]. Parasit Vectors, 2017, 10(1): 31.
[12]	Barrere V, Falzon LC, Shakya KP, et al. Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: comparison of detection methods for drug resistance[J]. Vet Parasitol, 2013, 198(1/2): 159-165.
[13]	Yang X, Lei WQ, Di WD, et al. Investigation of benzimidazole resistance-associated SNPs in the isotype-1 β-tubulin gene in Haemonchus contortus populations in Yili, Xinjiang[J]. Chin J Vet Med, 2018, 54(9): 8-11, 122. (in Chinese)
	( 杨新, 雷卫强, 邸文达, 等. 新疆伊犁地区捻转血矛线虫种群Ⅰ型β微管蛋白基因苯并咪唑抗药性相关单核苷酸多态性调查[J]. 中国兽医杂志, 2018, 54(9): 8-11, 122.)
[14]	Luo XP, Wang PL, Li JY, et al. Characteristics of albendazole resistance of different ivermectin-resistant Haemonchus contortus isolates in China[J]. Chin J Vet Sci, 2021, 41(7): 1301-1309, 1347. (in Chinese)
	( 罗晓平, 王鹏龙, 李军燕, 等. 不同耐伊维菌素捻转血矛线虫国内分离株耐阿苯达唑特性[J]. 中国兽医学报, 2021, 41(7): 1301-1309, 1347.)
[15]	Nogimori T, Ogami K, Oishi Y, et al. ABCE1 acts as a positive regulator of exogenous RNA decay[J]. Viruses, 2020, 12(2): 174.
[16]	Tian Y, Han X, Tian DL. The biological regulation of ABCE1[J]. IUBMB Life, 2012, 64(10): 795-800.
[17]	Iida A, Saito S, Sekine A, et al. Catalog of 605 single-nucleotide polymorphisms (SNPs) among 13 genes encoding human ATP-binding cassette transporters: ABCA4, ABCA7, ABCA8, ABCD1, ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8[J]. J Hum Genet, 2002, 47(6): 285-310.
[18]	Riou M, Guégnard F, Sizaret PY, et al. Drug resistance is affected by colocalization of P-glycoproteins in raft-like structures unexpected in eggshells of the nematode Haemonchus contortus[J]. Biochem Cell Biol, 2010, 88(3): 459-467.
[19]	Riou M, Koch C, Kerboeuf D. Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation[J]. Parasitol Res, 2005, 95(4): 266-272.
[20]	Chen KY, Featherstone DE. Discs-large (DLG) is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila[J]. BMC Biol, 2005, 3: 1.
[21]	Montgomery JM, Zamorano PL, Garner CC. MAGUKs in synapse assembly and function: an emerging view[J]. Cell Mol Life Sci, 2004, 61(7/8): 911-929.

基因	引物序列（5′→3′）	产物长度/bp	退火温度/℃
HCOI00378500	F：AGAAGCATGAGCATTTGCAG- TACA R：TCTCCGAGCGGTTGTTCTGA	809	56
HCOI00506600	F：CCACTCTGCGTCTATGACCGT R：AGCGCATAAGGTAGGGAGGA	762	60
HCOI01200700	F：CATGATGGGCGTCTCGGAGT R：TCGATCATGCCGCATCCTCA	692	56

位点	氨基酸变化情况敏感/耐药	敏感株样品数（百分比/%）	耐药株样品数（百分比/%）
7	K/E	25（100）	1（2.9）
10	F/I	25（100）	1（2.9）
12	V/F	25（100）	1（2.9）
13	E/D	25（100）	1（2.9）
23	A/S	25（100）	1（2.9）
26	V/E	25（100）	2（5.7）
39	A/V	25（100）	1（2.9）
40	R/T	25（100）	2（5.7）
41	K/N	25（100）	1（2.9）
43	Q/L	25（100）	3（8.6）
47	D/H	25（100）	1（2.9）
65	E/Q	25（100）	1（2.9）
69	V/G	25（100）	3（2.9）
69	V/A	25（100）	1（2.9）
70	G/C	25（100）	2（5.7）
70	G/D	25（100）	1（2.9）
72	S/F	25（100）	1（2.9）

虫株	突变位点碱基（百分比/%）
虫株	32(C/T)	38(G/A)	48(C/T)	71(A/C)	86(T/A)	101(A/G)	125(C/T)	131(T/A)	134(C/A)
敏感虫株（38）	C(100)	G(52.6)	C(100)	A(100)	T(100)	G(36.8)	T(57.9)	T(100)	C(57.9)
耐药虫株（13）	T(23.1)	A(100)	T(15.4)	C(7.7)	A(23.1)	G(38.5)	C(100)	A(23.1)	A(100)