Genetic Diversity Of Isotype-1 ?-tubulin Gene Associated With Benzimidazole Resistance In Haemonchus Contortus Populations In China

Haemonchus contortus is one of the major pathogens affecting small rumiant livestock around the world,particularly in tropical and subtropical regions.The adults mainly live in the abomasum and small intestine,and suck blood from the abomasum of ruminant hosts leading to anaemia,oedema,diarrhoea and even death.Due to the lack of commercial vaccine against haemonchosis,application of anthelmintic is still the primary measure for the treatment of H.contortus infection.As a consequence of excessive use of drugs,resistance against all anthemintinc has developed to be a worldwide problem and even some regions have occurred multiple drug resistance.In order to develop effective strategies to delay the emergence of drug resistance,it is imperative to understand the rise and spread of drug resistance mutations.Benzimidazole is one of the three most commonly used anthelmintics,and BZ resistance in nematodes is the best understood at the molecular level.It has been proved that three resistance-associated non-synonymous single nucleotide polymorphisms(SNPs)(F167Y,E198 A and F200Y)in the isotype-1 ?-tubulin gene have been associated with benzimidazole resistance in H.contortus.A large number of studies on BZ resistance in H.contortus have been reported abroad and recently some studies have focused on the rise and spread of BZ resistance mutations,however research in this aera is relatively lacking in China.Hence,in the present study,we explored the isotype-1 ?-tubulin gene in H.contortus from eight populations in China to learn about the presence and distribution of BZ resistance.The genetic diversity and origin of resistance mutation were also analyzed.(1)Genetic diversity of isotype-1 ?-tubulin gene(1)Genetic diversity of isotype-1 ?-tubulin geneIn total of 192 male H.contortus from eight populations in Hubei,Hebei,Yunnan,Shaanxi,Guangxi,Inner mongolia,Liaoning and Heilongjiang were used to amplify the isotype-1 ?-tubulin gene and PCR coupled direct sequencing method was used to detect three SNPs.Among Chinese H.contortus populations,F167 Y was not detected but different frequencies of E198 A and F200 Y were observed.Six distinct genotypes were identified.In addition,the resistant allele frequencies of E198 A and F200 Y were 0-70%and 0-31%,respectively.The genetic diversity and phylogenetic analysis were performed based on the gene pool of eight populations.The results showed that high haplotype diversities(0.455-0.939)and high nucleotide diversities(0.018-0.039)were present in each population.Low pairwise FST ranging from 0.00907 to 0.30732 between the eight populations were obtained,indicating a low level of genetic differentiation among populations due to high gene flow.AMOVA analysis showed that 93% of the genetic variance was partitioned within populations and the different geographical origins and host species of H.contortus populations had little effect(<2%)on the genetic structure.The phylogenetic relationship was analyzed by three different methods.The results revealed two haplotypes possessing resistant SNP E198 A in two distinct groups in the network,with each containing at least one susceptible haplotype,indicating two independent origins of E198 A.The seven haplotypes possessing resistant SNP F200 Y were found in three distinct groups in the network,and these haplotypes clustered with at least one susceptible haplotype,suggesting at least three different origins of this SNP.(2)Development of tetra primer ARMS-PCR methodBased on the results of the above study,a tetra primer ARMS-PCR was developed to detect the E198 A in the isotype-1 ?-tubulin gene and the accuracy of the results was compared with that of direct sequencing.The results showed that optimization of PCR reaction system,especially the proportion of inner and outer primers,could achieve better amplification effect.Three different profiles which indicats three genotypes could be seen clearly and intuitively through the gel.433 bp and 200 bp indicated susceptible homozygous(SS).433 bp and 284 bp indicated resistant homozygous(RR).433 bp,284bp and 200 bp indicated heterozygous(RS).Finaly,it shows that the method can be successfully applied to the detection of E198 A,which has high accuracy and is easy to perform.In conclusion,this is the first detection of all three BZ resistance-associated SNPs in the isotype-1 ?-tubulin gene of H.contortus populations in China.Meanwhile,the genetic diversity and origin of resistance mutation were also analyzed.These findings fill the gaps in this area in China,providing a theoretical basis for subsequent research and control strategy.