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.

Reconstruction of the TGF-ß signaling pathway of Fasciola gigantica.

In the present study, we reconstructed the transforming growth factor beta (TGF-ß) signaling pathway for Fasciola gigantica, which is a neglected tropical pathogen. We defined the components involved in the TGF-ß signaling pathway and investigated the transcription profiles of these genes for all developmental stages of F. gigantica. In addition, the presence of these components in excretory and secretory products (FgESP) was predicted via signal peptide annotation. The core components of the TGF-ß signaling pathway have been detected in F. gigantica; classical and nonclassical single transduction pathways were constructed. Four ligands have been detected, which may mediate the TGF-ß signaling pathway and BMP signaling pathway. Two ligand-binding type II receptors were detected, and inhibitory Smad7 was not detected. TLP, BMP-3, BMP-1, and ActRIb showed higher transcription in 42-day juvenile and 70-day juvenile, while ActRIIa, Smad1, ActRIIb, Smad8, KAT2B, and PP2A showed higher transcription in egg. TLM, Ski, Smad6, BMPRI, p70S6K, Smad2, Smad3, TgfßRI, Smad4, and p300 showed higher transcription in metacercariae. Four ligands, 2 receptors and 3 Smads are predicted to be present in the FgESP, suggesting their potential extrinsic function. This study should help to understand signal transduction in the TGF-ß signaling pathway in F. gigantica. In addition, this study helps to illustrate the complex mechanisms involved in developmental processes and F. gigantica - host interaction and paves the way for further characterization of the signaling pathway in trematodes.

Secondary infection of Fasciola gigantica in buffaloes shows a similar pattern of serum cytokine secretion as in primary infection.

Background: As a natural host of Fasciola gigantica, buffalo is widely infected by F. gigantica. Its impact on buffalo production has caused great losses to the husbandry sector, and repeat infection is non-negligible. In buffaloes experimentally infected with F. gigantica, primary and secondary infection have yielded the same rate of fluke recovery, indicating a high susceptibility of buffalo to F. gigantica, which contributes to the high infection rate. Determining the immunological mechanism of susceptibility will deepen the understanding of the interaction between F. gigantica and buffalo. Here, we explored the immune response of buffaloes against primary and secondary F. gigantica infection, with a focus on cytokines' dynamics explored through serum cytokine detection. Methods: Buffaloes were assigned to three groups: group A (noninfected, n = 4), group B (primary infection, n = 3), and group C (secondary infection, n = 3). Group B was infected via oral gavage with 250 viable F. gigantica metacercariae, and group C was infected twice with 250 metacercariae at an interval of 4 weeks. The second infection of group C was performed simultaneously with that of group B. Whole blood samples were collected pre-infection (0 weeks) and at 1-6, 10, and 12 weeks after that. The serum levels of seven cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13, TGF-ß, and IL-17) were simultaneously determined using ELISA and further analyzed. Results: In the present study, no significant changes in Th1-type cytokines production were detected in early infection, both in primary and secondary infections, while the Th2-type response was strongly induced. A comparison of primary and secondary infection showed no significant difference in the cytokine secretion, which may indicate that the re-infection at 4 weeks after primary infection could not induce a robust adaptive immune response. The full extent of interaction between buffalo and F. gigantica in re-infection requires further study.

Can Ivermectin kill Sarcoptes scabiei during the molting process?

BACKGROUND: Sarcoptes scabiei is a permanent obligate ectoparasite that lives and reproduces in the epidermis of humans and other mammals worldwide. There is a lack of information on the molting process of Sarcoptes scabiei. Ivermectin is widely used to treat Sarcoptes infection in humans and animals, while the survival of molting Sarcoptes mites in the presence of ivermectin is unknown. The aim of the present study is to investigate the molting process of Sarcoptes mites and assess the activity of ivermectin during the molting process of Sarcoptes mites. METHODOLOGY/PRINCIPAL FINDINGS: molting Sarcoptes mites were incubated at 35°C and 80% relative humidity and observed hourly until complete molt. Of the 192 molting mites recorded, the longest molt periods for larvae and nymphs were 23 and 30 h, respectively. The activity of ivermectin on molting Sarcoptes mites was also assessed using two concentrations of the drug (0.1 and 0.05 mg/ml). The exposure time for molting mites was determined by 100% mortality of female mites exposed to the solution of ivermectin. While all female mites were killed after exposure to 0.1 mg/ml ivermectin for 2 h and and 0.05 mg/ml for 7 h, 32% and 36% of molting mites survived and successfully molted, respectively. CONCLUSIONS/SIGNIFICANCE: The present study demonstrated that molting Sarcoptes mites are less susceptible to ivermectin than active mites. As a consequence, mites may survive after two doses of ivermectin given 7 days apart due not only to hatching eggs but also to the resistance of mites during their molting process. Our results provide insight into the optimal therapeutic regimens for scabies and highlight the need for further research on the molting process of Sarcoptes mites.

A proteasomal ß5 subunit of Haemonchus contortus with a role in the growth, development and life span.

BACKGROUND: The proteasome in eukaryotic cells can degrade a variety of proteins and plays an important role in regulating the cell cycle, cell survival and apoptosis. The proteasome receives much attention as a potential chemotherapeutic target for treatment of a variety of infectious parasitic diseases, but few studies of proteasomes have been done on parasitic nematodes. METHODS: A proteasomal ß5 subunit encoding gene (named Hc-pbs-5) and its inferred product (Hc-PBS-5) in Haemonchus contortus were identified and characterized in this study. Then, the transcriptional profiles and anatomical expression were studied using an integrated molecular approach. Finally, a specific proteasome inhibitor bortezomib (BTZ), together with RNA interference (RNAi), was employed to assess the function of Hc-PBS-5. RESULTS: Bioinformatic analysis revealed that the coding sequence of Hc-pbs-5 was 855 bp long and encoded 284 amino acids (aa). The predicted protein (Hc-PBS-5) had core conservative sequences (65-250 aa) belonging to N-terminal nucleophile (Ntn) family of hydrolases. Real-time PCR results revealed that Hc-pbs-5 was continuously transcribed in eight developmental stages with higher levels at the infective third-stage larvae (L3s) and adult males of H. contortus. Immunohistochemical results revealed that Hc-PBS-5 was expressed in intestine, outer cuticle, muscle cells under the outer cuticle, cervical glands and seminal vesicles of male adults and also in intestine, outer cuticle, cervical glands, uterine wall, eggs and ovaries of female adults of H. contortus. BTZ could reduce proportions of egg hatching, and the fourth-stage larvae (L4s) developed from the exsheathed L3s (xL3s) of H. contortus. In addition, silencing Hc-pbs-5 by soaking the specific double-stranded RNA (dsRNA) could decrease the transcription of Hc-pbs-5 and result in fewer xL3s developing to L4s in vitro. CONCLUSIONS: These results indicate that proteasomal ß5 subunit plays an important role in the growth, development and life span of H. contortus.

The 22nd Chromatography Component of the Fasciola gigantica Excretory-Secretory Products Decreased the Proliferation of Peripheral Blood Mononuclear Cells from Buffalo.

The 22nd chromatography component (F22) of the Fasciola gigantica excretory-secretory products (FgESP) shows better diagnostic value than the FgESP, and diagnostic methods based on F22 have also been established. Thus, exploring its immunomodulatory function and potential as a molecular vaccine candidate is attractive. In the present study, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) in the innate immune response was preliminarily studied using the FgESP as a control. PBMCs were incubated with concanavalin A (ConA) and phytohemagglutinin (PHA) at optimal (1 µg/well) or suboptimal (0.25 µg/well) doses coupled with FgESP and F22 at different doses (1-16 µg/well). Cell proliferation was then assessed by microenzyme reaction colorimetry (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay). In addition, the components of F22 were also explored by mass spectrometry and then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to infer their functions. The results indicated that FgESP decreased the proliferation of PBMCs stimulated with ConA and PHA at specific doses, whereas F22 significantly decreased the proliferation of PBMCs stimulated with ConA and PHA at both optimal and suboptimal doses (p < 0.05). Two hundred and sixteen proteins were identified in F22, and these included 86 proteins that could be assigned to more than one pathway and some with robust immunomodulatory ability. Further studies should be performed to investigate the immunomodulatory function of F22 in the adaptive immune response, and the components of F22 can be further studied as potential vaccine candidate molecules.

Development of a colloidal gold immunochromatographic strip for the rapid detection of antibodies against Fasciola gigantica in buffalo.

Background: Fasciola gigantica, a tropical liver fluke, infects buffalo in Asian and African countries, causing significant economic losses and posing public health threats. The diagnostic of buffalo fascioliasis caused by F. gigantica is vital in fascioliasis control and preventation. The 22nd gel filtration chromatography fraction of F. gigantica Excretory-Secretory Products (FgESP), namely Fasciola 22 (F22), which was used as a diagnostic antigen in indirect ELISA, has demonstrated great potential for fascioliasis diagnosing. In the absence of rapid diagnostic methods, the use of a colloidal gold immunochromatographic strip based on F22 was applied to detect F. gigantica infection in buffalo. Methods: In the present study, the 22nd gel filtration chromatography fraction of FgESP (F22) was used as an antigen to establish the colloidal gold-based immunochromatographic strip (ICS). The nitrocellulose membrane was incubated with F22 at the test line (T line) and goat anti-mouse secondary antibody at the control line (C line). The mouse anti-buffalo secondary antibody 2G7 conjugated to colloidal gold particles was used as the detection system for line visualization. The strip was assembled and developed by optimizing reaction conditions. The sensitivity, specificity, stability, and early diagnostic value of the strip were evaluated employing buffalo-derived sera. Results: An immunochromatographic strip for the rapid detection of antibodies against F. gigantica-FgICS was developed. The strip demonstrated high sensitivity and specificity. Sensitivity tests confirmed positive results even when the positive reference serum was diluted 4,096 times. Except for one Schistosoma japonicum-positive serum that tested positive via FgICS, specificity tests confirmed no cross-reactivity with other positive sera of Schistosoma japonicum and Babesia bovis. The strip remained stable after storage at 4°C for up to 3 months. In infected buffalo, antibodies could be detected as early as 14-21 days post-infection. The detection of 17 positive sera yielded an 82.4% positive rate via FgICS vs. a 100.0% positive rate via ELISA based on FgESP. For FgICS, the 95% confidence interval of sensitivity was 84.8-95.4%, while specificity was 4.2-14.7%. Conclusion: The immunochromatographic strip FgICS developed in this study provides a simple and rapid method of F. gigantica antibody detection and infected buffalo monitoring in the field.

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.

Effect of primary and secondary Fasciola gigantica infection on specific IgG responses, hepatic enzyme levels and weight gain in buffaloes.

Buffaloes, as highly susceptible definitive hosts of Fasciola gigantica, suffer from a high infection rate of fasciolosis, which causes enormous economic losses. Repeat infection is responsible for this high rate; thus, elucidating the protective immunity mechanism in repeat infection is decisive in fasciolosis prevention. Herein, a secondary experimental infection model was established to preliminarily reveal the protective immunity that occurs in repeat infection. In brief, animals were assigned to three groups: group A (uninfected control), group B (primary infection) and group C (secondary infection). Buffaloes were autopsied 20 weeks post-infection for measurements of the recovered flukes and hepatic examination. In addition, the detection of specific antibody (IgG) responses to F. gigantica excretory-secretory product (FgESP) throughout the whole period and weight gain throughout the first 4 months as a percentage (%) of the starting weight were also determined. The serum hepatic enzyme gamma glutathione transferase (GGT) levels were monitored to assess hepatic damage throughout the study period. Infection establishment was compared between group B and group C. Similar specific IgG patterns were observed between group B and group C, and hepatic damage was more severe in group C than group B. Significant differences in weight gain as a percentage of the start weight were observed between group A and group B at the 3rd and 4th months postprimary infection, while significant differences were not observed between group A and group C or group B and group C. Our results suggest that challenge infection cannot induce resistance against F. gigantica in buffaloes, which is consistent with the protective immunity against Fasciola hepatica reinfection observed in sheep and goats.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) co-immunoprecipitated using a time course of infected buffalo sera.

Introduction: Widespread Fasciola gigantica infection in buffaloes has caused great economic losses in buffalo farming. Studies on F. gigantica excretory and secretory products (FgESP) have highlighted their importance in F. gigantica parasitism and their potential in vaccine development. Identifying FgESP components involved in F. gigantica-buffalo interactions during different periods is important for developing effective strategies against fasciolosis. Methods: Buffaloes were assigned to non-infection (n = 3, as control group) and infection (n = 3) groups. The infection group was orally administrated 250 metacercariae. Sera were collected at 3, 10, and 16 weeks post-infection (wpi) for the non-infection group and at 0 (pre-infection), 1, 3, 6, 8, 10, 13, and 16 wpi for the infection group. FgESP components interacting with sera from the non-infection and infection groups assay were pulled down by co-IP and identified using LC-MS/MS. Interacting FgESP components in infection group were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway and gene ontology (GO) functional annotation to infer their potential functions. Results and discussion: Proteins of FgESP components identified in the non-infection group at 3, 10, and 16 wpi accounted for 80.5%, 84.3%, and 82.1% of all proteins identified in these three time points, respectively, indicating surroundings did not affect buffalo immune response during maintenance. Four hundred and ninety proteins were identified in the infection group, of which 87 were consistently identified at 7 time points. Following GO analysis showed that most of these 87 proteins were in biological processes, while KEGG analysis showed they mainly functioned in metabolism and cellular processing, some of which were thought to functions throughout the infection process. The numbers of specific interactors identified for each week were 1 (n = 12), 3 (n = 5), 6 (n = 8), 8 (n = 15), 10 (n = 23), 13 (n = 22), and 16 (n = 14) wpi, some of which were thought to functions in specific infection process. This study screened the antigenic targets in FgESP during a dense time course over a long period. These findings may enhance the understanding of molecular F. gigantica-buffalo interactions and help identify new potential vaccine and drug target candidates.

Identification of Fasciola spp. based on ITS-2 reveals the Fasciola gigantica infection in buffaloes in Nanning city, South China.

Fasciolosis is harmful to ruminant husbandry worldwide. Given the superficial survey on Fasciolosis infection in Guangxi, the main buffalo breeding area in China, an in-depth investigation in the infection of buffaloes in Nanning, the capital of Guangxi Zhuang Autonomous Region, with Fasciola (Platyhelminthes: Trematoda: Digenea) species will provide a theoretical support for the control and prevention of Fasciolosis infection in buffaloes. Five water buffalo livers were collected from an abattoir in Nanning every 2 weeks from June 2018 to April 2019, and a total of 101 livers were obtained. All livers were then dissected to observe the liver lesions caused by the flukes. Afterwards, Fasciola spp. collected from Fasciolosis-infected livers were numbered and measured. Then, the livers infected with more than 3 flukes were marked, and 3 flukes were picked from each liver according to their morphological differences, such as body length (BL), body maximum width (BW) and length-width ratio (BL/BW). Moreover, these Fasciola spp. worms were selected for molecular biological analysis. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR) and sequenced. Finally, sequential and phylogenetic analyses were also performed. The infection rate was 38.6 % according to anatomic examination, and the livers infected by Fasciola spp. were damaged seriously. The principal manifestations were the enlargement of the liver and protrusion of the bile ducts. In some cases, the bile duct wall became inflamed and rough, in which some sediment such as phosphate could be easily found. After dissection, 1243 Fasciola spp. flukes were collected from 39 out of 101 livers. The morphometric measurements obtained from the present study showed that the BL/BW ranged from 1.42-10.25. However, it might vary considerably among different geographical locations and could not be used as an accurate method for the identification of Fasciola spp.. Analysis of the ITS-2 sequences showed that 83 out of 87 flukes had 100 % homology with each other, and the other 4 flukes with 99.3 % homology possessed a nucleotide polymorphism. A unique position (271) was detected in flukes in Nanning isolates. Phylogenetic analysis indicated that all the flukes were Fasciola gigantica, and no Fasciola hepatica or the intermediate form was found in this study.

A transcription factor DAF-5 functions in Haemonchus contortus development.

BACKGROUND: Abnormal dauer formation gene (daf-5), located downstream of the DAF-7 signalling pathway, mainly functions in dauer formation and reproductive processes in the free-living nematode Caenorhabditis elegans. Although the structure and function of daf-5 have been clarified in C. elegans, they still remain totally unknown in Haemonchus contortus, a socio-economically important parasitic nematode of gastric ruminants. METHODS: A homologue of daf-5, Hc-daf-5, and its inferred product (Hc-DAF-5) in H. contortus were identified and characterized in this study. Then the transcriptional profiles of Hc-daf-5 and the anatomical expression of Hc-DAF-5 in H. contortus were studied using an integrated molecular approach. RNA interference (RNAi) was performed to explore its function in transition from the exsheathed third-stage larvae (xL3s) to the fourth-stage larvae (L4s) in vitro. Finally, the interaction between Hc-DAF-5 and Hc-DAF-3 (a co-Smad) was detected by bimolecular fluorescence complementation (BiFc) in vitro. RESULTS: It was shown that Hc-DAF-5 was a member of the Sno/Ski superfamily. Hc-daf-5 was transcribed in all developmental stages of H. contortus, with significant upregulation in L3s. Native Hc-DAF-5 was localized in the reproductive organs, cuticle, and intestine via immunohistochemistry. RNAi revealed that specific small interfering RNAs (siRNAs) could retard xL3 development. In addition, the interaction between Hc-DAF-5 and Hc-DAF-3 indicated that the SDS box of Hc-DAF-5 was dispensable for the binding of Hc-DAF-5 to Hc-DAF-3, and the MH2 domain was the binding region between Hc-DAF-3 and Hc-DAF-5. CONCLUSIONS: In summary, these findings show that Hc-daf-5 functions in the developmental processes of H. contortus, and this study is the first attempt to characterize the daf-5 gene in parasitic nematodes.

First Evidence of Function for Schistosoma japonicumriok-1 and RIOK-1.

Protein kinases are known as key molecules that regulate many biological processes in animals. The right open reading frame protein kinase (riok) genes are known to be essential regulators in model organisms such as the free-living nematode Caenorhabditis elegans. However, very little is known about their function in parasitic trematodes (flukes). In the present study, we characterized the riok-1 gene (Sj-riok-1) and the inferred protein (Sj-RIOK-1) in the parasitic blood fluke, Schistosoma japonicum. We gained a first insight into function of this gene/protein through double-stranded RNA interference (RNAi) and chemical inhibition. RNAi significantly reduced Sj-riok-1 transcription in both female and male worms compared with untreated control worms, and subtle morphological alterations were detected in the ovaries of female worms. Chemical knockdown of Sj-RIOK-1 with toyocamycin (a specific RIOK-1 inhibitor/probe) caused a substantial reduction in worm viability and a major accumulation of mature oocytes in the seminal receptacle (female worms), and of spermatozoa in the sperm vesicle (male worms). These phenotypic alterations indicate that the function of Sj-riok-1 is linked to developmental and/or reproductive processes in S. japonicum.

A daf-7-related TGF-ß ligand (Hc-tgh-2) shows important regulations on the development of Haemonchus contortus.

BACKGROUND: In most multicellular organisms, the transforming growth factor-ß (TGF-ß) signalling pathway is involved in regulating the growth and stem cell differentiation. Previous studies have demonstrated the importance of three key molecules in this pathway in the parasitic nematode Haemonchus contortus, including one TGF-ß type I receptor (Hc-tgfbr1), one TGF-ß type II receptor (Hc-tgfbr2), and one co-Smad (Hc-daf-3), which regulated the developmental transition from the free-living to the parasitic stages of this parasite. However, almost nothing is known about the function of the TGF-ß ligand (Hc-tgh-2) of H. contortus. METHODS: Here, the temporal transcription profiles of Hc-tgh-2 at eight different developmental stages and spatial expression patterns of Hc-TGH-2 in adult female and male worms of H. contortus have been examined by real-time PCR and immunohistochemistry, respectively. In addition, RNA interference (RNAi) by soaking was employed to assess the importance of Hc-tgh-2 in the development from exsheathed third-stage larvae (xL3s) to fourth-stage larvae (L4s) in H. contortus. RESULTS: Hc-tgh-2 was continuously transcribed in all eight developmental stages of H. contortus studied with the highest level in the infective third-stage larvae (iL3) and Hc-TGH-2 was located in the muscle of the body wall, intestine, ovary of adult females and testes of adult males. Silencing Hc-tgh-2 by the specific double-stranded RNA (dsRNA), decreased the transcript level of Hc-tgh-2 and resulted in fewer xL3s developing to L4s in vitro. CONCLUSIONS: These results suggested that the TGF-ß ligand, Hc-TGH-2, could play important roles in the developmental transition from the free-living (L3s) to the parasitic stage (L4s). Furthermore, it may also take part in the processes such as digestion, absorption, host immune response and reproductive development in H. contortus adults.

Identification and characterization of an R-Smad homologue (Hco-DAF-8) from Haemonchus contortus.

BACKGROUND: Smad proteins are essential cellular mediators within the transforming growth factor-ß (TGF-ß) superfamily. They directly transmit incoming signals from the cell surface receptors to the nucleus. In spite of their functional importance, almost nothing is known about Smad proteins in parasitic nematodes including Haemonchus contortus, an important blood-sucking nematode of small ruminants. METHODS: Based on genomic and transcriptome data for H. contortus and using bioinformatics methods, a Smad homologue (called Hco-daf-8) was inferred from H. contortus and the structural characteristics of this gene and its encoded protein Hco-DAF-8 established. Using real-time PCR and immunofluorescence assays, temporal transcriptional and spatial expression profiles of Hco-daf-8 were studied. Gene rescue in Caenorhabditis elegans was then applied to assess the function of Hco-daf-8 and a specific inhibitor of human Smad3 (called SIS3) was employed to evaluate the roles of Hco-DAF-8 in H. contortus development. RESULTS: The features of Hco-DAF-8 (502 amino acids), including conserved R-Smad domains and residues of the L3-loop that determine pathway specificity, are consistent with a TGF-ß type I receptor-activated R-Smad. The Hco-daf-8 gene was transcribed in all developmental stages of H. contortus studied, with a higher level of transcription in the fourth-stage larval (L4) females and the highest level in adult males. Hco-DAF-8 was expressed in the platymyarian muscular cells, intestine and reproductive system of adult stages. Gene rescue experiments showed that Hco-daf-8 was able to partially rescue gene function in a daf-8 deficient mutant strain of C. elegans, leading to a resumption of normal development. In H. contortus, SIS3 was shown to affect H. contortus development from the exsheathed third-stage larvae (L3s) to L4s in vitro. CONCLUSIONS: These findings suggest that Hco-DAF-8, encoded by the gene Hco-daf-8, is an important cellular mediator of H. contortus development via the TGF-ß signalling pathway. They provide a basis for future explorations of Hco-DAF-8 and associated pathways in H. contortus and other important 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.

A serine/threonine-specific protein kinase of Haemonchus contortus with a role in the development.

In the free-living nematode Caenorhabditis elegans, the serine/threonine-specific protein kinase, AKT, is known to play a key role in dauer formation, life-span, and stress-resistance through the insulin-like signaling pathway. Although the structure and function of AKT-coding genes of C. elegans are understood, this is not the case for homologous genes in parasitic nematodes. In the present study, we explored a C. elegans akt-1 gene homolog in the parasitic nematode Haemonchus contortus, investigated its transcript isoforms (Hc-akt-1a and Hc-akt-1b), and studied expression and function using both homologous and heterologous functional genomic tools. In C. elegans, we showed that the predicted promoter of Hc-akt-1 drives substantial expression in ASJ neurons of the N2 (wild-type) strain. In H. contortus (Haecon-5 stain), RNAi (soaking) led to a significantly decreased transcript abundance for both Hc-akt-1a and Hc-akt-1b, and reduced larval development in larval stages in vitro. Chemical inhibition was also shown to block larval development. Taken together, the evidence from this study points to a key functional role for Hc-akt-1 in H. contortus.

A TGF-ß type II receptor that associates with developmental transition in Haemonchus contortus in vitro.

BACKGROUND: The TGF-ß signalling pathway plays a key role in regulating dauer formation in the free-living nematode Caenorhabditis elegans, and previous work has shown that TGF-ß receptors are involved in parasitic nematodes. Here, we explored the structure and function of a TGF-ß type II receptor homologue in the TGF-ß signalling pathway in Haemonchus contortus, a highly pathogenic, haematophagous parasitic nematode. METHODOLOGY/PRINCIPAL FINDINGS: Amino acid sequence and phylogenetic analyses revealed that the protein, called Hc-TGFBR2 (encoded by the gene Hc-tgfbr2), is a member of TGF-ß type II receptor family and contains conserved functional domains, both in the extracellular region containing cysteine residues that form a characteristic feature (CXCX4C) of TGF-ß type II receptor and in the intracellular regions containing a serine/threonine kinase domain. The Hc-tgfbr2 gene was transcribed in all key developmental stages of H. contortus, with particularly high levels in the infective third-stage larvae (L3s) and male adults. Immunohistochemical results revealed that Hc-TGFBR2 was expressed in the intestine, ovary and eggs within the uterus of female adults, and also in the testes of male adults of H. contortus. Double-stranded RNA interference (RNAi) in this nematode by soaking induced a marked decrease in transcription of Hc-tgfbr2 and in development from the exsheathed L3 to the fourth-stage larva (L4) in vitro. CONCLUSIONS/SIGNIFICANCE: These results indicate that Hc-TGFBR2 plays an important role in governing developmental processes in H. contortus via the TGF-ß signalling pathway, particularly in the transition from the free-living to the parasitic stages.

A DAF-3 co-Smad molecule functions in Haemonchus contortus development.

BACKGROUND: The Smad proteins function in TGF-ß signalling transduction. In the model nematode Caenorhabditis elegans, the co-Smad, DAF-3 mediates R-Smads and performs a central role in DAF-7 signal transduction, regulating dauer formation and reproductive processes. Considering the divergent evolutionary patterns of the DAF-7 signalling pathway in parasitic nematodes, it is meaningful to explore the structure and function of DAF-3 in parasitic nematodes, such as Haemonchus contortus. METHODS: A daf-3 gene (Hc-daf-3) and its predicted product (Hc-DAF-3) were identified from H. contortus and characterised using integrated genomic and genetic approaches. In addition to immunohistochemistry employed to localise Hc-DAF-3 within adult worm sections, real-time PCR was conducted to assess the transcriptional profiles in different developmental stages of H. contortus and RNA interference (RNAi) was performed in vitro to assess the functional importance of Hc-daf-3 in the development of H. contortus. RESULTS: Hc-DAF-3 sequences predicted from Hc-daf-3 displayed typical features of the co-Smad subfamily. The native Hc-DAF-3 was localised to the gonad and cuticle of adult parasites. In addition, Hc-daf-3 was transcribed in all developmental stages studied, with a higher level in the third-stage larvae (L3) and adult females. Moreover, silencing Hc-daf-3 by RNAi retarded L4 development. CONCLUSION: The findings of the present study demonstrated an important role of Hc-DAF-3 in the development of H. contortus larvae.

A TGF-ß type I receptor-like molecule with a key functional role in Haemonchus contortus development.

Here we investigated the gene of a transforming growth factor (TGF)-ß type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-ß family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-ß type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-ß type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.