Paul Ja Presidente

Differences in transcription between free-living and CO2-activated third-stage larvae of Haemonchus contortus

BackgroundThe disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development.ResultsTotals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development.ConclusionThe present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention.

Analysis of developmentally regulated genes of the parasite Haemonchus contortus

Differential regulation of gene expression in the development of Haemonchus contortus was analysed using RNA arbitrarily-primed PCR. A study of third-stage larval and adult H. contortus revealed large differences between the two stages; 32 and 30% unique third-stage larval and adult RNA arbitrarily-primed PCR products, respectively. This finding is consistent with a high degree of differential gene expression between these developmental stages. A number of adult products were sequenced, revealing 11 molecules to be similar to deposits within sequence databases. Four other molecules that did not have significant similarity to sequences in the databases may represent developmentally regulated genes specific to H. contortus. Northern analysis of the putative adult-expressed molecules with homologues in the databases confirmed that four were expressed only in adults, while four were expressed in both stages, but had different sized transcripts. This may reflect differential splicing, or expression of closely related but different molecules at different life cycle stages. Two molecules were present in mRNA populations from both stages, suggesting these were false stage-associated molecules. No transcript was detected for one molecule by Northern analysis, probably due to low level of expression. In situ hybridisation analysis was used to localise expression of transcripts in the adult parasite, in particular, to gain some insight into the nature of those molecules with no known predicted function.

HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus

A putative serine/threonine protein kinase (HcSTK) from the parasitic nematode Haemonchus contortus was characterised at the mRNA and amino acid levels. HcSTK displays a high level of identity (85-93% in the catalytic domain) with proteins of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily, which represent signal transduction molecules involved in establishing and maintaining polarity in proliferating and differentiating cells. The transcript of hcstk is expressed in different developmental stages (second-, third-, fourth-stage larvae and adults) and various organs (muscle, intestine and reproductive) of H. contortus. In addition, there are several isoforms which appear to relate to a single gene. The expression profile of hcstk is similar to that of Caenorhabditis elegans PAR-1, and the level of sequence identity among members of the PAR-1/MARK STK subfamily, representing a range of species of vertebrates (e.g. humans and rodents), invertebrates (e.g. insects and C. elegans) and yeast, suggests that HcSTK may be involved in a conserved signal transduction pathway.

Haemonchus contortus: molecular characterisation of a small heat shock protein

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development. Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein

Rugopharynx rosemariae new species (nematoda: Pharyngostrongylidae) from grey kangaroos (Macropus gig anteus and M. fuliginosus) with life cycle stages and associated pathology

Abstract Beverdge I. and Ppresidente P. J. A. 1978. Rugopharynx rosemarie sp. nov. (Nematoda: Pharyngostrongylidae) from grey kangaroos (Macropus giganteus and M. fuliginosus) with life cycle stages and associated pathology. International Journal for Parasitology8: 379–387. Rugopharynx rosemariae new species is described from the stomachs of grey kangaroos, Macropus giganteus Shaw, 1790 and Macropus fuliginosus (Desmarest, 1817) from south-eastern Australia. The new species differs from other species of the genus in spicule length and in the length and shape of the oesophagus. Parasitic life-cycle stages are described from natural infections; free-living life-cycle stages were obtained by culturing eggs from gravid females. Third stage larvae burrow into the gastric mucosa producing small elevated nodules resulting from a localized fibroplastic and inflammatory reaction in the lamina propria and submucosa. Fourth stage and adult worms occur in the stomach lumen. Rugopharynx brevis (Canavan, 1931) is made a synonym of Rugopharynx australis (Monnig, 1926).

Isolation and characterization of class II myosin genes from Haemonchus contortus

In this study, cDNAs encoding myosin from the parasitic nematode Haemonchus contortus were isolated and characterized. Several exhibited a considerable degree of sequence variation at the nucleotide and limited divergence at the amino acid levels within the various functional domains. The results suggest that the cDNAs isolated represented a single myosin heavy chain, which, by comparison with a number of other myosins, is inferred to represent a homologue of a muscle myosin (CeMHCA) of the free-living nematode Caenorhabditis elegans. The findings could have implications for investigating cytoskeletal dynamics and/or signalling pathways.

Liver lesions in the common wombat associated with migrating Taenia hydatigena larvae

Abstract Four mature female wombats (Vombatus ursinus) were experimentally infected per os with 10,000 viable eggs of Taenia hydatigena. One wombat was killed and examined on each of days 7, 14, 28 and 56 post-inoculation (PI). Subcapsular haemorrhagic and healing tracks were found in the livers on each day, and on day 56 PI a granuloma, probably a reaction to a degenerating larvae, was evident. There were no signs of further development in the peritoneal cavity and it was concluded that the wombat is probably an aberrant host for this cestode. Similar lesions in the livers of three of seven wombats examined in South Gippsland, Victoria suggested strongly that these had been caused by migrating T. hydatigena larvae.