Haemonchus contortus: molecular characterisation of a small heat shock protein☆
Introduction
Parasitism by worms is one of the most challenging problems to livestock industries, with an estimated cost of million per annum attributed to parasitism (Newton and Munn, 1999). The abomasal nematode, Haemonchus contortus, is predominantly a parasite of sheep, with the pathological effects of infection being largely due to the worm’s blood feeding behaviour. The transition from free living larval stages to parasitism of a mammalian host describes a period where parasites encounter stress due to dramatic environmental changes. Small heat shock proteins (sHSPs), with molecular weights ranging from 12 to 43 kDa, have been identified in virtually all eukaryotes and are believed to interact within cells as molecular chaperones, with the level of expression being inducible through exposure to particular environments including stress (reviewed in Arrigo and Landry, 1994). Some sHSP genes are developmentally regulated, including those of parasitic nematodes, where sHSPs may play a role in transition from free-living or insect vector stages to the mammalian host (Raghavan et al., 1999; Thompson et al., 1996; Tweedie et al., 1993). In this study, a small heat shock protein from the parasitic nematode H. contortus was identified and characterised at the molecular level. The transcript and protein were constitutively expressed in the parasitic stages, and expression levels were not elevated by heat shock treatment.
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Materials and methods
Infective larvae (L3), exsheathed L3 (xL3), in vitro cultured early L4 (eL4), and adult H. contortus were prepared by methods previously described (Nikolaou et al., 2002). Trichostrongylus colubriformis were provided by Novartis Animal Health; Oesophagostomum dentatum, Ancylostoma caninum, and Toxocara canis were provided by Peter Boag (Victorian Institute of Animal Science); and Dictyocaulus viviparus were obtained from an infected bovine calf at postmortem examination.
Total RNA for cDNA
Results
Clone G0056 was identified from an adult H. contortus intestinal cDNA library as part of an expressed sequence tag (EST) sequencing project. The full-length DNA sequence of the G0056 insert was determined in both directions (refer to Genbank Accession No. AY130968). The cDNA insert of 558 bp included an open reading frame of 468 nucleotides, encoding a predicted protein of 156 amino acids with a predicted molecular weight of 18.35 kDa. The sequence surrounding the predicted initiating methionine
Discussion
Small HSPs have been described previously for the rat intestinal nematode N. brasiliensis (Tweedie et al., 1993), canine heartworm, Dirofilaria immitis (Lillibridge et al., 1996), and the human filarioids Brugia malayi (Raghavan et al., 1999) and B. pahangi (Thompson et al., 1996), but this is the first report of a sHSP from a gastrointestinal nematode of ruminants.
With the advent of EST sequencing programs, sequences for putative sHSPs have now been deposited in the databases for a wide range
Acknowledgements
Project support was provided by Novartis Animal Health Australasia, the Department of Natural Resources and Environment, Victoria and Agriculture Victoria. We thank the Molecular Vaccines team for dissecting the nematode intestines, preparation of the Northern Blots, and parasite extracts. We also thank Tamarae Griffiths and Toby Williams (Novartis Animal Health Australia) for immunisation of sheep, preparation of antisera and provision of L3 H. contortus and adult T. colubriformis; and Peter
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The sequence data reported herein have been submitted to the Genbank, EMBL, and DDBJ databases and assigned Accession No. AY130968.
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Present address: School of Engineering and Science, Swinburne University of Technology, Hawthorn, Vic. 3122, Australia.