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The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

ABSTRACT: The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

SUBMITTER: Nguyen LT

PROVIDER: S-EPMC8482649 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

Nguyen Linh Thuy LT Zajíčková Markéta M Mašátová Eva E Matoušková Petra P Skálová Lenka L

Veterinary research 20210930 1

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for t ...[more]

PMID: 34593042

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Project description: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.

Project description:BackgroundHaemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant's production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators.MethodsThe recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes.ResultsWe cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm's gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes.ConclusionsOur results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.

Dataset Information

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

Publications

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

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