Cloning and sequence analysis of the candidate nicotinic acetylcholine receptor alpha subunit gene tar-1 from Trichostrongylus colubriformis.

A T. colubriformis genomic library in lambda EMBL3 was screened for sequences homologous to the Caenorhabditis elegans unc-38 nicotinic acetylcholine receptor (nAChR) alpha-subunit gene. The candidate gene tar-1 (for Trichostrongylus acetylcholine receptor subunit gene 1) comprising 13704 base pairs was thus identified. BLAST comparison of the sequenced clone with GenBank, followed by comparison of translated regions in six reading frames with protein databases, identified clearly defined tracts corresponding to 12 putative exons sharing high sequence homology to other nAChR genes and able to code for sequential regions of a putative nAChR alpha-subunit protein (tar-1). Tar-1 shares sequence similarities with over 40 nAChR subunit proteins. The highest similarity (91.6%) is with unc-38, suggesting that nAChR sequences from nematodes are closely related. The sequence includes motifs typical of these molecules including adjacent cysteine residues at the ACh binding site and four transmembrane regions. The DNA sequence presents the longest genomic tract described for this organism and should prove useful as a probe source in the search for nAChR genes from this and other nematodes and for studying the molecular mechanism of resistance to levamisole, a drug which is known to act on nAChRs of worms and which is widely used for parasite control.

Stimulation of HT29-D4 cell growth by excretory/secretory products of the parasite nematode Trichostrongylus colubriformis.

The presence of the nematode parasite Trichostrongylus colubriformis in the small intestine is associated with an increase in epithelial renewal. To assess the possible role of excretory/secretory products from the worm on cell proliferation, adult Trichostrongylus colubriformis were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h and the conditioned medium was added to the culture medium of the transformed epithelial cell line HT29-D4. A stimulation of the HT29-D4 cell growth was ascertained at concentrations of 0.25-1.0 micrograms protein/ml using counts of cell numbers, the MTT method and incorporation of tritiated thymidine. An increased incorporation of tritiated thymidine was also observed with the excretory/secretory products from T. colubriformis fourth stage larvae at 1.0 microgram/ml. Dialysis of the medium conditioned by the worms indicated that the molecular weight of the factor is greater than 8000 Daltons in size. Heat treatment, acid hydrolysis and precipitation by trichloracetic acid of the conditioned medium resulted in the disappearance of the proliferative effect while treatment with trypsin partially depleted the stimulative activity. These results suggest that T. colubriformis produce some protein factor which could increase the epithelial regeneration in the host small intestine.

Effects of dietary molybdenum on nematode and host during Trichostrongylus vitrinus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry matter) to the diet of lambs exposed for four weeks to a trickle (2500 third stage larvae per day) infection with Trichostrongylus vitrinus reduced the number and length of adult worms retrieved from the small intestine 11 days later: both effects were particularly marked in female worms from female lambs (P less than 0.01). Worms from lambs given molybdenum contained less proteinase enzyme activity and secreted less proteinases in culture irrespective of the sex of the host. Pathogenicity was not attenuated by molybdenum. Damage to the intestinal mucosa was severe in both dietary groups but infected females given molybdenum developed lower plasma albumin concentrations and lighter dressed carcases than those not given molybdenum. Neither the effects on the parasite nor those on the host could be attributed simply to molybdenum-induced copper depletion, using conventional measures of copper status. Molybdenum may be toxic to T vitrinus but may also facilitate or enhance the inflammatory process limiting larval establishment or increasing parasite rejection.