Non-coding RNA in the gut of the blood-feeding parasitic worm, Haemonchus contortus.

The intestine of Haemonchus contortus is an essential tissue that has been indicated to be a major target for the prevention of haemonchosis caused by this parasitic nematode of small ruminants. Biological peculiarities of the intestine warrant in-depth exploitation, which can be leveraged for future disease control efforts. Here, we determined the intestinal ncRNA (lncRNA, circRNA and miRNA) atlas using whole-transcriptome sequencing and bioinformatics approaches. In total, 4846 novel lncRNA, 982 circRNA, 96 miRNA (65 known and 31 novel) and 8821 mRNA were identified from the H. contortus intestine. The features of lncRNA, circRNA and miRNA were fully characterized. Comparison of miRNA from the intestines and extracellular vesicles supported the speculation that the miRNA from the latter were of intestinal origin in H. contortus. Further function analysis suggests that the cis-lncRNA targeted genes were involved in protein binding, intracellular anatomical structure, organelle and cellular process, whereas the circRNA parental genes were mainly enriched in molecular function categories, such as ribonucleotide binding, nucleotide binding, ATP binding and carbohydrate derivative binding. The miRNA target genes were related to the cellular process, cellular response to stimulus, cellular protein modification process and signal transduction. Moreover, competing endogenous RNA network analysis revealed that the majority of lncRNA, circRNA and mRNA only have one or two binding sites with specific miRNA. Lastly, randomly selected circRNA, lncRNA and miRNA were verified successfully using RT-PCR. Collectively, these data provide the most comprehensive compilation of intestinal transcripts and their functions, and it will be helpful to decipher the biological and molecular complexity of the intestine and lay the foundation for further functional research.

Comparative Metabolome Analyses of Ivermectin-Resistant and -Susceptible Strains of Haemonchus contortus.

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 IVM resistance of H. contortus. In this study, comparative analyses of the metabolomics of IVM-susceptible and -resistant adult H. contortus worms were carried out to explore the role of H. contortus metabolism in IVM resistance. In total, 705 metabolites belonging to 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.

Preparation and activity study of Ruoqiang jujube polysaccharide copper chelate.

Background: Polysaccharide metal chelate exhibit both immunoregulatory activity and metal element supplementation effects. Methods: In this study, Ruoqiang jujube polysaccharide copper chelate (RJP-Cu) was prepared and the preparation conditions were optimized using the response surface method. Subsequently, RJP-Cu was administered to lambs to evaluate its impact on growth performance, copper ion (Cu2+) supplementation, immune enhancement, and intestinal flora was evaluated. Results: The results indicated that optimal RJP-Cu chelation conditions included a sodium citrate content of 0.5 g, a reaction temperature of 50°C, and a solution pH of 8.0, resulting in a Cu2+ concentration of 583°mg/kg in RJP-Cu. Scanning electron microscopy (SEM) revealed significant structural changes in RJP before and after chelation. RJP-Cu displaying characteristic peaks of both polysaccharides and Cu2+ chelates. Blood routine indexes showed no significant differences among the RJP-Cu-High dose group (RJP-Cu-H), RJP-Cu-Medium dose group (RJP-Cu-M), RJP-Cu-low dose group (RJP-Cu-L) and the control group (p > 0.05). However, compared with the control group, the RJP-Cu-H, M, and L dose groups significantly enhanced lamb production performance (p < 0.05). Furthermore, RJP-Cu-H, M, and L dose groups significantly increased serum Cu2+ concentration, total antioxidant capacity (T-AOC), catalase (CAT), and total superoxide dismutase (T-SOD) contents compared with control group (p < 0.05). The RJP-Cu-H group exhibited significant increases in serum IgA and IgG antibodies, as well as the secretion of cytokines IL-2, IL-4, and TNF-α compared to the control group (p < 0.05). Furthermore, RJP-Cu-H group increased the species abundance of lamb intestinal microbiota, abundance and quantity of beneficial bacteria, and decrease the abundance and quantity of harmful bacteria. The RJP-Cu-H led to the promotion of the synthesis of various Short Chain Fatty Acids (SCFAs), improvements in atrazine degradation and clavulanic acid biosynthesis in lambs, while reducing cell apoptosis and lipopolysaccharide biosynthesis. Conclusion: Thus, these findings demonstrate that RJP-Cu, as a metal chelate, could effectively promote lamb growth performance, increase Cu2+ content, and potentially induce positive immunomodulatory effects by regulating antioxidant enzymes, antibodies, cytokines, intestinal flora, and related metabolic pathways.

Comparative analysis on transcriptomics of ivermectin resistant and susceptible strains of Haemonchus 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, 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.

Genome-Wide Identification of CircRNAs of Infective Larvae and Adult Worms of Parasitic Nematode, Haemonchus contortus.

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.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of the E198A SNP in the isotype-1 ß-tubulin gene of Haemonchus contortus populations in China.

Haemonchus contortus is one of the most important gastrointestinal nematodes of small ruminants around the world, seriously hampering the healthy development of the sheep industry. The control of this parasite mainly depends on anthelmintics, however, drug resistance of H. contortus has become a serious problems worldwide. Previous studies demonstrated that the E198A (GAA to GCA), a single nucleotide polymorphism (SNP) in the isotype-1 ß-tubulin gene is associated with benzimidazole resistance in H. contortus. However, only PCR-RFLP and ARMS-PCR methods have been previously used for the detection of the E198A mutation. In the present study, a loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of the E198A SNP in H. contortus. The results showed that optimization of LAMP reaction reagents and conditions could achieve this. The resulting amplicons were visualized by adding hydroxynaphthol blue dye (HNB) prior to amplification. The color of LAMP products amplified without DNA or from DNA from worms with the E198A homozygous susceptible genotype was still violet, but the products with DNA from worms with the E198A heterozygous genotype or the E198A resistant homozygous genotype changed to sky blue. The specificity of this method was further verified by sequencing, which confirmed the successful LAMP detection of the E198A mutation with high specificity. In conclusion, the developed LAMP method has high specificity and good reproducibility for screening the E198A SNP of isotype-1 ß-tubulin gene of H. contortus of field samples without using sophisticated equipment, providing useful technique for the rapid detection and thus prevention and control of benzimidazole resistant H. contortus infections.