A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

Extracellular and cytoplasmic Cu/Zn superoxide dismutases from Haemonchus contortus.

Full-length cDNAs encoding cytosolic (SODc) and putative extracellular (SODe) Cu/Zn superoxide dismutases (SODs) from the ovine gastrointestinal parasitic nematode Haemonchus contortus have been isolated and characterized. The predicted sequences of the H. contortus SODs showed strong homology to other helminth SODs, the highest level of sequence similarity was with those of the free-living nematode Caenorhabditis elegans++. The predicted amino acid sequence of the putative extracellular form contained an N-terminal extension with the characteristics of a signal sequence including a potential signal peptidase cleavage site. Transcripts of both classes of Cu/Zn SOD were detected in all life-cycle stages examined. The cytosolic SOD mRNA was approximately 6-fold more abundant than that of the extracellular enzyme in adult parasites. Immunoblotting with antisera raised to in vitro-expressed parasite SODs revealed the presence of 2 proteins in extracts of adult H. contortus, with molecular masses of approximately 19.8 and 18 kDa. An additional protein of approximately 16.8 kDa was detected in adult ES material. Immunofluorescent staining showed Cu/Zn SOD was localized in the body wall musculature and the pharynx in adult worms and in the uterine tract of adult females. The immunogenic properties of recombinant H. contortus Cu/Zn SODs was assessed in a challenge infection experiment in lambs.

Molecular cloning and characterisation of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus.

Antisera from lambs immunised with the Haemonchus contortus integral membrane protein complex, Haemonchus galactose-containing glycoprotein (H-gal-GP), the lambs being refractory to subsequent challenge, were used to identify several clones from an adult H. contortus lambda gt11 cDNA library. Using gene-specific oligonucleotide primers in conjunction with primers directed to a conserved nematode Spliced Leader (SL) sequence and to the polyA+ tail of mRNA, the remaining 5' and 3' sequences of one of these clones, metallopeptidase-1 (MEP1), were amplified. The 2.4 kb full-length coding sequences was subsequently amplified in a single reaction. Sequence analysis identified MEP1 as encoding a putative zinc metallopeptidase, which shared limited homology with the mammalian type II integral membrane protein neutral endopeptidase (NEP). Southern blotting indicated that MEP1 belonged to a multigene family. MEP1 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and a specific antiserum raised in sheep. This antiserum recognised several polypeptide components of H-gal-GP. Immunolocalisation studies showed that MEP1 encoded a protein located on the luminal surface of the nematode gut. Both MEP1 mRNA and protein are developmentally regulated with expression being limited to the blood-feeding stages of H. contortus.

Immunisation of guinea pigs against Dictyocaulus viviparus using adult ES products enriched for acetylcholinesterases.

The adult ES products of Dictyocaulus viviparus are a source of protective antigens against challenge in the guinea pig laboratory model. High levels of acetylcholinesterase (AChE) activity are present in these products and these enzymes are immunogenic in infected cattle. Here, the potential role of these enzymes in protective immunity was investigated using a fraction enriched for AChE to immunise guinea pigs. The antibody response stimulated by immunisation with AChE-enriched ES products and the worm burdens obtained following challenge with infective larvae were compared with those in animals immunised with whole ES products and challenge controls. The AChE-enriched preparation stimulated high levels of enzyme-specific antibody in immunised animals, which was not the case for those which received unfractionaed ES products. Worm burdens of guinea pigs which received the AChE-enriched fraction were significantly lower than those obtained in adjuvant controls. The animals which received the unfractionated ES products were not significantly protected against challenge. These results suggest that AChEs may be potential candidates for incorporation in a sub-unit vaccine against D. viviparus.

Effects of dietary molybdenum on nematode and host during Haemonchus contortus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry-matter) to the diet of lambs given a trickle infection of Haemonchus contortus larvae (500 third stage larvae d-1 over six weeks) reduced mean faecal egg counts (epg) from 3952 to 2312 +/- 402 by 32 days (P less than 0.02) and greatly reduced the mean number of worms recovered from the abomasum 14 days after infection ceased (907 compared with 4167: P less than 0.01). Infection reduced haemoglobin concentrations less in lambs given molybdenum although the difference was small relative to the reduction in worm burden. Lambs not given molybdenum had low intraepithelial mast cell counts in the abomasal mucosa and less abomasal hypertrophy than expected from abomasal parasitism. Molybdenum did not consistently reduce the copper status of the host or the parasite. Previous exposure to molybdenum greatly reduced protein but not proteinase activity in, or secreted by, adult worms cultured for eight hours. It is suggested that molybdenum either increased the inflammatory response which preceded worm rejection or that it indirectly enhanced that reaction by reducing the effectiveness of copper-dependent, anti-inflammatory enzymes in the gastrointestinal mucosa.

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.