| Haemonchus contortus is the most prevalent gastrointestinal nematode(GIN)of small ruminants,causing significant economic impacts on animal Husbandry.At present,the prevention and control of this parasite still mainly depends on broad-spectrum anthelmintics,of which ivermectin(IVM)is one of the most widely used anthelmintics,which plays an important role for the control of H.contortus infection.However,as the uncontrolled use has led to increasing its resistance,and the complex mechanisms underlying resistance are unresolved.In the present study,the H.contortus IVM resistant and susceptible strains were tested by three different resistance detection method,and to explore the IVM-resistant related genes and metabolites,transcriptomics and metabolomics were used to compare the differences of genes and metabolites between resistant and susceptible strains,and predict the potential drug-resistant mechanism,which provided a theoretical basis and data resources for the study of IVM drug-resistant mechanism.In addition,although the best understood benzimidazole resistance at the molecular level,its resistance is the most serious around the world.A previous study revealed that the mutation frequency of E198 A in isotype I β-tubulin gene is higher than that of F200 Y in H.contortus from various geographical populations in China.Therefore,to provide technical support for the real-time monitoring of benzimidazole resistance,a LAMP method for rapid detection of E198 A in β-tubulin locus was established in this study.The research contents of this thesis are as follows:1.Detection of anthelmintic resistance of IVM resistant strains of H.contortusIn this thesis,the H.contortus resistant and susceptible strains were tested by three different resistance detection method.The larval development assay results showed that the IC50 values were 0.218 ng/ml(95% CI: 0.208 to 0.227)and 1.291 ng/ml(95% CI: 1.209 to 1.377)for resistant and susceptible strains,respectively and the RR was calculated as5.9.The result of control test revealed that the percentage reduction in worm burdens was32.5%.In addition,the result of faecal egg count reduction test showed that compared with the before administration and un-treatment group,the FECR was-55% and 1% in the IVM administration group at day 14 post treatment(Table 2).The results of these three anthelmintic resistance tests confirmed that the Xinjiang strain was IVM resistant strain.2.Comparative analysis on transcriptomics of IVM resistant and sensitive strains of H.contortusSubsequently,the cDNA libraries of female and male worms of IVM resistant and susceptible strains of H.contortus were constructed,and a total of 17857 mRNAs and 441 miRNAs were identified,of which 16336 and 160 were known genes and miRNAs respectively,and 1521 and 281 were newly identified,respectively.In the resistant males,338 and 30 mRNA and miRNA were significantly up-regulated,205 and 21 were significantly down regulated;In the resistant females,104 and 4 mRNA and miRNA were significantly up-regulated,254 and 5 were significantly down regulated.In addition,among the differentially expressed genes(DEGs)identified some candidate genes with functions involved in receptor activities,transport,detoxification,structural constituent of cuticle,lipid binding,transport,metabolism and signalling pathway.In the differential miRNA analysis,miRNAs that may be related to IVM resistance were identified,namely mi R-1,mir-2,mi R-133 and mi R-5912 b.The results of GO analysis showed that the DEGs of male and female worms had similar enrichment trend,the up-regulated genes in the male and female were significantly enriched in intrinsic component of membrane,integral component of membrane,peptidase activity,molecular transducer activity,transport activity,oxidoreductase activity,oxidationreduction process,signaling receptor activity,proteolysis,lipid metabolic process,carbohydrate binding;The down-regulated genes in the male and female were significantly enriched in cellular component,structural molecular activity,structural constituent of cuticle,collagen trimer,proteolysis,ion transport,ligand-gated channel activity.Go enrichment of differential miRNA target gene of the male were significantly enriched in phosphorus metabolic process,cellular protein metabolic process,cellular modified amino acid biosynthetic process,cell part,neuron part,ion binding,protein binding,purine NTP-dependent helicase activity.Go enrichment of differential miRNA target gene of the female were significantly enriched in cellular protein metabolic process,small molecule metabolic process,phosphate-containing compound metabolic process,anion binding,ATP binding,purine nucleotide binding.KEGG results showed that the enrichment trend of DEGs between male and female was similar,and significantly enriched in lysosome,autophagy,apoptosis,sphingolipid signalling pathway,nicotine addiction,NOD-like receptor signaling pathway,glycosylphosphatidylinositol(GPI)-anchor biosynthesis,ABC transporters,Cytochrome P450.The analysis of miRNA target gene pathway of male and female also showed certain similarities,mainly including m TOR signal pathway,MAPK signal pathway,c AMP signal pathway,calcium signal pathway,lysine degradation,pyruvate metabolism,purine metabolism and TCA cycle.The functional classification analysis of mRNA and miRNA target genes showed that DEGs and miRNA target genes of male and female were mainly involved in the metabolic process,of which amino acid metabolism is the main one.Finally,eleven DEGs and six miRNAs were randomly selected and verified by RT-PCR,it was found that the results of RNA-seq were consistent with those of RT-PCR,which confirmed the credibility of sequencing data.3.Comparative analysis on metabolomics of IVM resistant and susceptible strains of H.contortusMetabonomic analysis of H.contortus IVM resistant and susceptible strains showed that a total of 42 categories and 705 metabolites were identified.A total of 86 differential metabolites were identified between resistant and susceptible strains,among them 17 upregulated and 69 down regulated,mainly including amino acids and their derivatives(28),organic acids and their derivatives(14),nucleotides and their derivatives(10),hormones(5),carnitines(5),etc.Based on KEGG pathway analysis,it was found that 86 differential metabolites were mainly enriched in purine metabolism,amino acid biosynthesis,glycine,serine and threonine metabolism,cysteine and methionine metabolism,tryptophan metabolism and riboflavin metabolism.Based on the above results,it is preliminarily speculated that amino acid metabolism and purine metabolism may be closely related to IVM drug resistance.4.Establishment of LAMP method for detecting H.contortus isotype I β-tubulin E198AIn this study,a LAMP method for the detection of benzimidazole resistance was established.The results show that the optimized LAMP reaction reagents and conditions could achieve the best detection efficiency,and accurately distinguish resistant genotypes from sensitive genotypes,and the experimental results were visualized by adding hydroxynaphthol blue dye(HNB).In addition,179 adult samples of H.contortus collected from eight different regions in China were detected,and detection rate of resistant genotypes was 8.3% ～ 87%,and the total detection rate was 37%.It shows that the established LAMP method can be applied to the detection of E198 A of isotype I β-tubulin gene and suitable for the rapid detection of field sample.In conclusion,this study compared the differences of gene transcription and metabolism between H.contortus IVM-resistant and sensitive strains by high-throughput sequencing,and analyzed the changes of gene transcription and metabolism between IVM-resistant and sensitive strains.Finally,a new hypothesis was put forward through joint analysis that amino acid metabolism and purine metabolism may be related to IVM resistance.This study not only provides valuable analytical data,but also lays a foundation for a better understanding of the mechanism of IVM resistance.In addition,a LAMP detection method for benzimidazole resistance was established to provide technical support for the real-time monitoring of benzimidazole resistance. |
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