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.

A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida).

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.

Stimulation of the in vitro migration of ovine eosinophils by factors derived from the sheep scab mite, Psoroptes ovis.

The ectoparasitic astigmatid mite Psoroptes ovis causes sheep scab, a highly contagious, severe allergic dermatitis associated with damage to the fleece and hide, loss of condition and occasional mortality. The scab lesion is characterized by a massive infiltration of eosinophils that begins very rapidly after infection. This paper reports the finding that mite-derived factors directly enhance the migration of ovine eosinophils in vitro. Significant (p < 0.01) and dose-dependent (r = 0.972 +/- 0.018 (SD)) activity was initially identified in whole mite extracts, by comparison with medium controls in an assay based on modified Boyden chambers and ovine bone marrow target cells. Similar pro-migratory activity (p < 0.005; r = 0.928 +/- 0.069 (SD)) was detected in washes containing mite excretory/secretory material. By direct comparison with migration ratios (n = 3) for defined chemotactic (rmeotaxin = 3.430 +/- 0.360 (SD)) and chemokinetic (rminterleukin-5 = 0.982 +/- 0.112 (SD)) stimuli it was determined that the activity in both mite extracts (0.992 +/- 0.038 (SD)) and mite washes (0.969 +/- 0.071 (SD)) was chemokinetic. Subsequent experiments (n = 3) in which live mites were incorporated directly into the in vitro assay system indicated that they produced factors that significantly (p < 0.001) enhanced eosinophil migration to a degree directly related to mite numbers (r = 0.993 +/- 0.005 (SD)). The identity of the factor(s) responsible is uncertain, but their presence suggests that mites may be capable of directly activating eosinophils in vivo, and raises the possibility that mites could directly influence, perhaps even initiate, the rapid early tissue eosinophilic response observed in experimental sheep scab infections.

Early immunopathological events in experimental ovine paratuberculosis.

An experimental oral infection of neonatal (< 2 weeks old) lambs with a cervine isolate of Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis), the causal agent of ruminant paratuberculosis (Johne's disease) was used to investigate bacteriological, histopathological and immunological changes during the early (up to 8 weeks) post-infection phase. In vitro culture for mycobacteria was positive in one faecal and three mesenteric lymph node (MLN) samples from the eight infected lambs. All mycobacterial isolates from MLN were identified as M.a. paratuberculosis by polymerase chain reaction (PCR). Small-to-medium sized focal granulomata were observed in jejunal (JPP) and ileal Peyer's patches (IPP) from four of the eight infected lambs. Compared with controls, JPP from all infected lambs had significantly (p < 0.05) higher proportions of CD8+ and CD2+ lymphocytes, and there were significantly (p < 0.05) fewer cells expressing B lymphocyte-associated markers in IPP and MLN. The T/B cell ratio was significantly (p < 0.05) increased in both JPP and MLN from infected lambs. The expression of a range of genes for cytokines was examined using specific reverse transcriptase PCR (RT-PCR) amplification of messenger RNA (mRNA) template isolated from MLN, JPP and IPP from both groups of animals. Densitometric analyses indicated that, in infected animals, MLN expressed significantly (p < 0.05) more mRNA for TNF-alpha: JPP had significantly increased (p < 0.05) mRNA for GM-CSF and significantly decreased (p < 0.05) mRNA for IL-4 and IFN-gamma. Infected lambs had significantly (p < 0.05) decreased titres of both circulating IgG and gut mycobacteria-associated IgG antibody. Infection was not associated with any consistent changes in lymphocyte reactivity to specific mycobacterial antigens, IFN-gamma release into supernatants from in vitro intestinal lymphocyte cultures or gut IgA antibody levels.