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