The Study On Polymorphism Of P-glycoprotein Gene In Ivermectin Resistance Strains Of Haemonchus Contortus

The anthelmintic resistance of sheep gastrointestinal nematodes has been a serious problem in the world, among which resistance of Haemonchus contortus is the worst one. But the study in this area was less in China and it was also necessary to the control of parasitic diseases in sheep husbandry. Three aspects of P-glycoprotein gene of Haemonchus contortus with drug-resistant were studied in this paper. The results were as follows:(1) With Haemonchus contortus of Ivermectin-resistant、Albendazole-resistant and sensitive strains from sheep as samples, partial sequence of P-glycoprotein gene had been amplification, followed by cloning and sequencing. The result showed that the homology of P-glycoprotein gene was higher in sensitive strains, and it could be up to99.1percent. But the homology of P-glycoprotein gene between the resistant strains and the sensitive strains was lower, which was between80.2percent and93.0percent. The nucleotide had multiple mutations in the resistant strains. According to the analysis on two open reading frames, and compared with the sensitive strains, the sixth amino acid of the ivermectin-resistant or the albendazole-resistant strains had changed into valine (Val, V) from glutamic acid (Glu, E).(2) With the Ivermectin-resistant strains of Haemonchus contortus in sheep as samples, the primers were designed according to partial sequence of the open reading frames (ORF) in P-glycoprotein gene. Then the polymorphism of this fragment was studied by PCR-SSCP, followed by sequencing to the gene of the different genotypes respectively. The result indicated that there were four mutated nucleotide bases in the ORF of the two genotypes, but the amino acid sequences were identical. Compared with sensitive strains, the sixth amino acid of the nematodes had mutated from Glu to Val, which showed that it was usual in Ivermectin-resistant strains of Haemonchus contortus.(3) With the Ivermectin-resistant and sensitive strains of Haemonchus contortus from sheep as samples, PCR-RFLP was used to analyzed PCR products of P-glycoprotein. The result showed that the PCR products of sensitive group were resistive to the restriction enzyme-Kpn Ⅰ, while Ivermectin-resistant group were vulnerable to the enzyme. The statistics data showed,10nematodes of ivermectin-susceptible strain were all homozygote (BB). In20nematodes of ivermectin-resistance strain, there were6ivermectin-resistant homozygotes (AA,30%),13heterozygotes (AB,65%) and only1ivermectin-susceptible homozygote (BB,5%). The heterozygote (AB) was the most dominant genotype, followed by ivermectin-resistant homozygotes (AA), and ivermectin-susceptible homozygote (BB) was the least. The resistance allele A was the most dominant gene, and take a percentage of62.5%, while allele B was only37.5%.