Transcriptional profiling and functional characterization of the Hc-NHR-49 gene in ivermectin resistance of Haemonchus contortus

Abstract Background The barber’s pole worm (BPW), Haemonchus contortus , poses a significant threat to sheep health and livestock husbandry. Control has historically relied on synthetic anthelmintics such as ivermectin; however, widespread resistance to this drug has emerged. The functional role of nuclear hormone receptors (NHRs) in H. contortus remains poorly understood. Methods The Hc-NHR-49 gene was polymerase chain reaction (PCR)-amplified and subjected to bioinformatic analysis. Expression profiles of the gene in resistant and sensitive strains across different developmental stages and under ivermectin stress were examined using Quantitative reverse transcription PCR (RT‑qPCR). Polyclonal antibodies were generated in mice via recombinant prokaryotic expression and validated by Western blot. The spatial expression pattern of Hc‑NHR‑49 was further determined by immunohistochemistry. Finally, RNA interference followed by larval head swing assays was performed to assess its functional role in ivermectin response. Results In this study, we cloned and characterized the nuclear hormone receptor gene Hc-NHR-49 from H. contortus . The full-length complementary DNA (cDNA) is 1272 bp, encoding a 423-amino-acid protein. Bioinformatics analysis revealed that Hc-NHR-49 is highly conserved across species, suggesting functional similarity. Recombinant Hc-NHR-49 was expressed and purified. Polyclonal antibodies raised in mice specifically recognized native Hc-NHR-49 in somatic extracts, as confirmed by Western blot. Immunohistochemical localization showed that Hc-NHR-49 is widely distributed, with particularly high expression in the intestine, uterus, ovaries, and testes. Transcript levels were detected throughout all developmental stages in both ivermectin-susceptible and -resistant strains. To investigate its association with ivermectin resistance, worms were exposed to the half maximal effective concentration (EC 50 ) of ivermectin, which elicited a plastic expression response of Hc-NHR-49 . RNA interference (RNAi)-mediated knockdown of Hc-NHR-49 increased ivermectin susceptibility in resistant parasites. Conclusions Collectively, our results suggest that Hc-NHR-49 is implicated in ivermectin resistance in H. contortus . These findings contribute to a deeper understanding of resistance mechanisms and could inform the future development of alternative control measures.