Veterinary Biosciences - Research Publications
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ItemH11-induced immunoprotection is predominantly linked to N-glycan moieties during Haemonchus contortus infection.(Frontiers Media SA, 2022)Nematodes are one of the largest groups of animals on the planet. Many of them are major pathogens of humans, animals and plants, and cause destructive diseases and socioeconomic losses worldwide. Despite their adverse impacts on human health and agriculture, nematodes can be challenging to control, because anthelmintic treatments do not prevent re-infection, and excessive treatment has led to widespread drug resistance in nematode populations. Indeed, many nematode species of livestock animals have become resistant to almost all classes of anthelmintics used. Most efforts to develop commercial anti-nematode vaccines (native or recombinant) for use in animals and humans have not succeeded, although one effective (dead) vaccine (Barbervax) has been developed to protect animals against one of the most pathogenic parasites of livestock animals - Haemonchus contortus (the barber's pole worm). This vaccine contains native molecules, called H11 and H-Gal-GP, derived from the intestine of this blood-feeding worm. In its native form, H11 alone consistently induces high levels (75-95%) of immunoprotection in animals against disease (haemonchosis), but recombinant forms thereof do not. Here, to test the hypothesis that post-translational modification (glycosylation) of H11 plays a crucial role in achieving such high immunoprotection, we explored the N-glycoproteome and N-glycome of H11 using the high-resolution mass spectrometry and assessed the roles of N-glycosylation in protective immunity against H. contortus. Our results showed conclusively that N-glycan moieties on H11 are the dominant immunogens, which induce high IgG serum antibody levels in immunised animals, and that anti-H11 IgG antibodies can confer specific, passive immunity in naïve animals. This work provides the first detailed account of the relevance and role of protein glycosylation in protective immunity against a parasitic nematode, with important implications for the design of vaccines against metazoan parasites.
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ItemFirst Evidence of Function for Schistosoma japonicum riok-1 and RIOK-1(MDPI, 2021-07)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.
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ItemMorphometric and molecular characterization of the species of Uncinaria Frolich, 1789 (Nematoda) parasitic in the Australian fur seal Arctocephalus pusillus doriferus (Schreber), with notes on hookworms in three other pinniped hosts(SPRINGER, 2013-05)This study presents morphological and molecular data on hookworms from the Australian fur seal Arctocephalus pusillus doriferus (Schreber) currently identified in Australian waters as Uncinaria hamiltoni Baylis, 1933. Additional specimens from the Australian sea lion Neophoca cinerea (Péron) and the New Zealand fur seal Arctocephalus forsteri (Lesson) from Australia, and the Southern elephant seal Mirounga leonina (Linnaeus) from Antarctica, were included. Using the internal transcribed spacer (ITS), hookworms from A. p. doriferus, N. cinerea and A. forsteri were found to be genetically similar but distinct from Uncinaria spp. found in M. leonina from Antarctica, as well as from Zalophus californianus (Lesson) and Callorhinus ursinus (Linnaeus) from California. Few morphological differences were detected between these taxa.
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ItemCryptosporidium of birds in pet markets in Wuhan city, Hubei, China(ELSEVIER, 2021)Cryptosporidium is a group of protistan parasites of a range of vertebrates including mammals and birds. Stimulated by previous work that revealed "zoonotic" Cryptosporidium meleagridis subtypes (i.e. IIIbA26G1R1b and IIIbA22G1R1c) in diarrhoeic children and domestic chickens in Wuhan city and environs in Hubei Province, China, here we explored whether zoonotic C. meleagridis subtypes might also occur in pet birds in Wuhan city. From 11 bird markets in this city, we collected 322 faecal samples from 48 species of birds (representing six taxonomic orders), isolated genomic DNA and then conducted PCR-based sequencing of genetic markers in the small subunit (SSU) of the nuclear ribosomal RNA and the 60 kDa glycoprotein (gp60) genes of Cryptosporidium. Using SSU, Cryptosporidium was detected in 55 (17%) of the 322 samples. Cryptosporidium avium, C. baileyi, C. meleagridis, C. muris and C. proventriculi were characterised in 18%, 47%, 11%, 2% and 20% of the 55 samples, respectively, and a novel Cryptosporidium galli-like taxon in one sample. Using gp60, only one subtype (IIIeA17G2R1) of C. meleagridis was identified, which had not been detected in a previous study of diarrhoeic children in Wuhan. However, IIIe subtypes have been found in both humans and birds around the world. The relatively high prevalence and genetic diversity of Cryptosporidium recorded here in pet birds raise awareness about possible reservoirs of zoonotic variants of Cryptosporidium in birds in Wuhan, and potentially, other provinces in China.
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ItemThe RIO protein kinase-encoding gene Sj-riok-2 is involved in key reproductive processes in Schistosoma japonicum(BMC, 2017-12-12)BACKGROUND: Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is caused by parasitic trematodes of the genus Schistosoma. The pathogenesis of schistosomiasis is caused by eggs whose production is the consequence of the pairing of schistosomes and the subsequent sexual maturation of the female. Previous studies have demonstrated that protein kinases are involved in processes leading to the male-induced differentiation of the female gonads, ovary and vitellarium. Right open reading frame protein kinase 2 (RIOK-2) is a member of the atypical kinase family and shown in other organisms to be responsible for ribosomal RNA biogenesis and cell-cycle progression, as well as involves in nematode development. However, nothing is known about its functions in any trematode including schistosome. METHODS: We isolated and characterized the riok-2 gene from S. japonicum, and detected the transcriptional profiles of Sj-riok-2 by using real-time PCR and in situ hybridization. RNAi-mediated knockdown of Sj-riok-2 was performed, mitotic activities were detected by EdU incorporation assay and morphological changes on organs were observed by confocal laser scanning microscope (CLSM). RESULTS: In silico analyses of the amino acid sequence of Sj-RIOK-2 revealed typical features of this class of kinases including a winged helix (wHTH) domain and a RIO kinase domain. Sj-riok-2 is transcribed in different developmental stages of S. japonicum, with a higher abundance in adult females and eggs. Localization studies showed that Sj-riok-2 was mainly transcribed in female reproductive organs. Experiments with adult schistosomes in vitro demonstrated that the transcriptional level of Sj-riok-2 was affected by pairing. Knocking down Sj-riok-2 by RNAi reduced cell proliferation in the vitellarium and caused the increased amount of mature oocytes in ovary and an accumulation of eggs within the uterus. CONCLUSIONS: Sj-riok-2 is involved in the reproductive development and maturation of female S. japonicum. Our findings provide first evidence for a pairing-dependent role of Sj-riok-2 in the reproductive development and maturation of female S. japonicum. Thus this study contributes to the understanding of molecular processes controlling reproduction in schistosomes.
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ItemA TGF-β type II receptor that associates with developmental transition in Haemonchus contortus in vitro.(Public Library of Science, 2019-12-02)BACKGROUND: The TGF-β signalling pathway plays a key role in regulating dauer formation in the free-living nematode Caenorhabditis elegans, and previous work has shown that TGF-β receptors are involved in parasitic nematodes. Here, we explored the structure and function of a TGF-β type II receptor homologue in the TGF-β 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-β 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-β 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-β signalling pathway, particularly in the transition from the free-living to the parasitic stages.
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ItemIdentification and characterization of an R-Smad homologue (Hco-DAF-8) from Haemonchus contortus(BMC, 2020-04-03)BACKGROUND: Smad proteins are essential cellular mediators within the transforming growth factor-β (TGF-β) 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-β 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-β signalling pathway. They provide a basis for future explorations of Hco-DAF-8 and associated pathways in H. contortus and other important parasitic nematodes.