Study On Polymorphisms Of P-gp-9 And GluCl-gbr-2b Genes Of Ivermectin Resistant Isolates In Haemonchus Contortus

The resistance of sheep gastrointestinal nematodes to anthelmintic drugs has become a common problem all over the world,among which the resistance of Haemonchus contortus to ivermectin is the most serious.In view of the need for the prevention and control of parasitic diseases in the production practice of sheep breeding industry,in this paper,the P-gp-9 gene and GluCl-gbr-2b gene of native isolates of Haemonchus contortus were studied with international standard strains as control,in order to understand the mechanism of ivermectin resistance.The results were as follows:The sequences of GluCl-gbr-2b gene fragments of Haemonchus contortus were compared and analyzed by MEGA5.1 biological software,and it was found that 20 bases of GluCl-gbr-2b gene fragments of international standard drug-resistant strain Hc-R and local drug-resistant isolates WSHc-001,WSHc-003 and WSHc-138 were common mutation points with international standard sensitive strain as control.However,when the isolated sensitive strain YCHc-022 was used as control,the GluCl-gbr-2b gene fragments of Hc-R,WSHc-001,WSHc-003 and WSHc-138 only had a common mutation at position 422,indicating that the resistance of Haemonchus contortus to ivermectin may be associated with a mutation at a base site on the GluCl-gbr-2b gene fragment,and its nucleotide polymorphism may be used as a potential molecule markers for the detection of drug resistance.By comparing and analyzing the ORF sequence of P-gp-9 gene,it was found that the ORF sequence of international standard sensitive strain Hc-S was identical with that of isolated sensitive strain YCHc-022,and the ORF sequence of international standard drug resistant strain Hc-R was also consistent with that of WSHc-001,WSHc-003 and WSHc-138.Moreover,compared with the sensitive strain,the ORF nucleotide sequence of the P-gp-9 gene of the resistant strain mutated from CCA to CTA,and amino acid from proline to leucine at the 89th base.At the same time,the polymorphism of this fragment was analyzed by PCR-SSCP method,and the results showed that drug-resistant and sensitive strains were obviously divided into two genotypes.Therefore,this method could be considered for the field detection of ivermectin-resistant strains of Haemonchus contortus.