CircRNAs, a novel class of ncRNA family, are endogenous transcriptional products involved in various biological and physiological processes in plants and animals. However, almost no information is available for circRNAs of parasitic helminths. In the present study, the circRNAs repertoire was comprehensively explored in Haemonchus contortus, a blood-sucking parasitic nematode of ruminants. In total, 20073 circRNAs were identified and annotated from three key developmental stages/genders of H. contortus including the free-living infective third-stage larvae (L3, 18883), parasitic adult female (Af, 3491), and male worms (Am, 2550) via deep-sequencing technology and bioinformatic analysis. Among these identified circRNAs, 71% were derived from exonic regions of protein-coding genes. The number of circRNAs transcribed from the X chromosome (4704) was higher than that from Chromosome I-V (3143, 3273, 3041, 3030, 2882). The amount of highly expressed circRNAs in third-stage larvae was significantly more abundant than that in adult stage. 15948 and 16847 circRNAs were differentially expressed between Af and L3s and between Am and L3, respectively. Among them, 13409 circRNAs existed in both comparisons. Furthermore, 1119 circRNAs were differentially expressed between Af_and_Am. GO enrichment analysis indicated that source genes of circRNAs differentially expressed between Am and L3 as well as between Af and L3 were significantly enriched in many biological processes, primarily including signaling, signal transduction and cell communication terms. KEGG analysis revealed that parental genes of differentially expressed circRNAs were mainly related to metabolism (pyruvate metabolism, glycerophospholipid metabolism, and carbon metabolism), MAPK signaling pathway, and phosphatidylinositol signaling system. Moreover, many circRNAs contained one or more miRNA potential binding sites, suggesting that they could regulate gene expression at the post-transcriptional level. Furthermore, the correctness of head-to-tail back splicing site and alternative circularization events were verified by Sanger sequencing using both divergent and convergent primers. Finally, the reliability of RNA-Seq data and the resistance of circRNAs to RNase R digestion were confirmed by quantitative RT-PCR. Taken together, our findings provide a foundation for elucidating the regulatory mechanisms of circRNAs in H. contortus, which will advance the understanding of circRNAs in parasitic nematodes.

Background Ivermectin (IVM) is one of the most important and widely used anthelmintics in veterinary medicine. However, its efficacy is increasingly compromised by widespread resistance, and the exact mechanism of IVM resistance remains unclear for most parasitic nematodes, including Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants. Methods In this study, an H. contortus IVM-resistant strain from Zhaosu, Xinjiang, China, was isolated and assessed by the control test, faecal egg count reduction test (FECRT) and the larval development assay (LDA). Subsequently, comparative analyses on the transcriptomics of IVM-susceptible and IVM-resistant adult worms of this parasite were carried out using RNA sequencing (RNA-seq) and bioinformatics. Results In total, 543 (416 known, 127 novel) and 359 (309 known, 50 novel) differentially expressed genes (DEGs) were identified in male and female adult worms of the resistant strain compared with those of the susceptible strain, respectively. In addition to several previously known candidate genes which were supposed to be associated with IVM resistance and whose functions were involved in receptor activity, transport, and detoxification, we found some new potential target genes, including those related to lipid metabolism, structural constituent of cuticle, and important pathways such as antigen processing and presentation, lysosome, autophagy, apoptosis, and NOD1-like receptor signalling pathways. Finally, the results of quantitative real-time polymerase chain reaction confirmed that the transcriptional profiles of selected DEGs (male: 8 genes, female: 10 genes) were consistent with those obtained by the RNA-seq. Conclusions Our results indicate that IVM has multiple effects, including both neuromuscular and non-neuromuscular targets, and provide valuable information for further studies on the IVM resistance mechanism in H. contortus. Graphical Abstract

Parasitic nematodes are important pathogens that infect animals, causing significant economic losses globally. Current repeated treatments have led to widespread anthelmintic resistance in nematode populations, so vaccine development offers an alternative control approach. However, only one effective vaccine (named Barbervax) has been developed to protect animals against one of the most pathogenic nematodes of ruminants—Haemonchus contortus (the barber’s pole worm). This vaccine contains a dominant component, Concanavalin A (Con A) purified H11 antigen, which has been shown to induce high levels (>85%) of immune protection in sheep breeds, but in goat breeds, the immunoprotection test of this native protein is still lacking. Here, we evaluated the protective efficacy of low-dose Con A-purified proteins for controlling the H. contortus infection in goats. Four-month-old Boer goats were equally divided into two vaccinated groups of 5 μg and 10 μg native proteins, and one adjuvant control. Each goat was immunized subcutaneously thrice and then challenged with 7000 infective third-stage larvae (L3s). The fecal egg count (FEC), degree of anemia, antibody levels of serum and abomasum mucosa, as well as worm burdens, were detected in experimental goats. Our results showed that both 5 μg and 10 μg vaccinated groups induced the effective protection in goats, reduced mean FEC by 71.8% and 68.6%, and mean worm burdens by 69.8% and 61.6%, respectively, compared to the adjuvant control. In addition, we detected that the serum antibody responses to the Con A-purified proteins were dominated by the IgG subtype, but the mucosal antibody responses were not detected. These data demonstrate Con A-purified proteins induced effective immunoprotection in goats, and underline their significance for controlling this widespread parasite.

Resistance to anthelmintics such as ivermectin (IVM) is currently a major problem in the treatment of Haemonchus contortus, an important parasitic nematode of small ruminants. Although many advances have been made in understanding the IVM resistance mechanism, its exact mechanism remains unclear for H. contortus. Therefore, understanding the resistance mechanism becomes increasingly important for controlling haemonchosis. Recent research showed that the metabolic state of bacteria influences their susceptibility to antibiotics. However, little information is available on the roles of metabolites and metabolic pathways in H. contortus IVM resistance. In this study, comparative analyses of the metabolomics of IVM-susceptible and -resistant adult H. contortus worms were carried out to analyze the role of H. contortus metabolism in IVM resistance. In total, 705 metabolites in 42 categories were detected, and 86 differential metabolites (17 upregulated and 69 downregulated) were identified in the IVM-resistant strain compared to the susceptible one. A KEGG pathway analysis showed that these 86 differential metabolites were enriched in 42 pathways that mainly included purine metabolism; the biosynthesis of amino acids; glycine, serine, and threonine metabolism; and cysteine and methionine metabolism. These results showed that amino acid metabolism may be mediated by the uptake of IVM and related with IVM resistance in H. contortus. This study contributes to our understanding of the mechanisms of IVM resistance and may provide effective approaches to manage infection by resistant strains of H. contortus.

Background: Ivermectin (IVM) is one of the most important and widely used anthelmintics in veterinary medicine. However, its efficacy is increasingly compromised by widespread resistance, and the exact mechanism of IVM resistance remains unclear for most parasitic nematodes including Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants.Methods: In this study, we isolated and assessed an IVM resistant strain from Zhaosu, Xinjiang, China. Subsequently, the comparative analyses on transcriptomics of IVM susceptible and resistant H. contortus adult worms were carried out using RNA sequencing and bioinformatics.Results: In total, 543 and 359 differentially expressed genes (DEGs) were identified in male and female adult worms of the resistant strain compared with the susceptible strain, respectively. The DEGs encode molecules involved in receptor activities, transport, detoxification, lipid metabolism and cuticle collagen formation. In addition, Gene Ontology (GO) analysis revealed that transcriptional changes were dominant in genes associated with ligand-gated channel activity, oxidation-reduction process, lipid metabolic process, and structural constituent of cuticle. The results support previous proposal that the IVM resistant mechanism of H. contortus involved in both neuromuscular and non-neuromuscular pathways. Finally, the quantitative RT-PCR results confirmed that the transcriptional profiles of selected DEGs (male: 8 genes, female: 10 genes) were consistent with those obtained by the RNA-Seq.Conclusions: The findings from this work provided valuable information for further studies on the IVM resistance in H. contortus.