Biological function of Dictyocaulus viviparus asparaginyl peptidase legumain-1 and its suitability as a vaccine target.

The present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35-39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

A tetrameric acetylcholinesterase from the parasitic nematode Dictyocaulus viviparus associates with the vertebrate tail proteins PRiMA and ColQ.

Dictyocaulus viviparus causes a serious lung disease of cattle. Similar to other parasitic nematodes, D. viviparus possesses several acetylcholinesterase (AChE) genes, one of which encodes a putative neuromuscular AChE, which contains a tryptophan (W) amphiphilic tetramerization (WAT) domain at its C-terminus. In the current study, we describe the biochemical characterization of a recombinant version of this WAT domain-containing AChE. To assess if the WAT domain is biologically functional, we investigated the association of the recombinant enzyme with the vertebrate tail proteins, proline-rich membrane anchor (PRiMA) and collagen Q (ColQ), as well as the synthetic polypeptide poly-l-proline. The results indicate that the recombinant enzyme hydrolyzes acetylthiocholine preferentially and exhibits inhibition by excess substrate, a characteristic of AChEs but not butyrylcholinesterases (BChEs). The enzyme is inhibited by the AChE inhibitor, BW284c51, but not by the BChE inhibitors, ethopropazine or iso-OMPA. The enzyme is able to assemble into monomeric (G(1)), dimeric (G(2)), and tetrameric (G(4)) globular forms and can also associate with PRiMA and ColQ, which contain proline-rich attachment domains (PRADs). This interaction is likely to be mediated via WAT-PRAD interactions, as the enzyme also assembles into tetramers with the synthetic polypeptide poly-l-proline. These interactions are typical of AChE(T) subunits. This is the first demonstration of an AChE(T) from a parasitic nematode that can assemble into heterologous forms with vertebrate proteins that anchor the enzyme in cholinergic synapses. We discuss the implications of our results for this particular host/parasite system and for the evolution of AChE.

GTP-cyclohydrolase and development in Teladorsagia circumcincta and Dictyocaulus viviparus (Nematoda: Strongylida).

GTP-Cyclohydrolase (GTP-CH) is necessary for the production of tetrahydrobiopterin, a required cofactor for the three aromatic amino acid hydroxylases and nitric oxide synthases. The gene encoding GTP-CH is transcribed at high levels in infective third larval stages of a number of parasitic trichostrongylid nematodes. We explore the potential role of GTP-CH within the processes of nematode development and environmentally-induced hypobiosis. For two species of parasitic nematode that are of major economic and welfare importance to livestock in temperate regions, Teladorsagia circumcincta and Dictyocaulus viviparus, we have demonstrated that each of the pre-parasitic larval stages transcribe high mean levels of cat-4 (the gene encoding GTP-CH). Using quantitative real-time polymerase chain reaction analysis and two different isolates of D. viviparus, only one of which is capable of entering hypobiosis, we have shown that there were only minor differences between these isolates in mean cat-4 transcript levels, both during the parasitic stages and during the earlier environmental life cycle stages (L(1)-L(3)). Taken together, these data indicate that, although both species of nematode produce high levels of cat-4 transcript in pre-parasitic larval stages, GTP-CH levels are unlikely to be involved in the induction of parasite hypobiosis. Alternative roles for GTP-CH in larval development are discussed.

A tryptophan amphiphilic tetramerization domain-containing acetylcholinesterase from the bovine lungworm, Dictyocaulus viviparus.

Acetylcholine (ACh) is one of an array of neurotransmitters used by invertebrates and, analogous to vertebrate nervous systems, acetylcholinesterase (AChE) regulates synaptic levels of this transmitter. Similar to other invertebrates, nematodes possess several AChE genes. This is in contrast to vertebrates, which have a single AChE gene, transcripts of which are alternatively spliced to produce different types of the enzyme which vary at their C-termini. Parasitic nematodes have a repertoire of AChE genes which include those encoding neuromuscular AChEs and those genes which code for secreted AChEs. The latter proteins exist as soluble monomers released by the parasite during infection and these AChE are distinct from those enzymes which the nematodes use for synaptic transmission in their neuromuscular system. Thus far, Dictyocaulus viviparus is the only animal-parasitic nematode for which distinct genes that encode both neuromuscular and secreted AChEs have been defined. Here, we describe the isolation and characterization of a cDNA encoding a putative neuromuscular AChE from D. viviparus which contains a tryptophan amphiphilic tetramerization (WAT) domain at its C-terminus analogous to the common 'tailed' AChE form found in the neuromuscular systems of vertebrates and in the ACE-1 AChE from Caenorhabditis elegans. This enzyme differs from the previously isolated, D. viviparus neuromuscular AChE (Dv-ACE-2), which is a glycosylphosphatidylinositol-anchored variant analogous to vertebrate 'hydrophobic' AChE.

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products.

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freund's incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.

Malic dehydrogenase isoenzymatic pattern in lung-nematode parasite species.

The malic dehydrogenase (MDH) isoenzymatic pattern of Dictyocaulus viviparus, Protostrongylus rufescens, and Slovakian and Spanish isolates of D. filaria was studied. The MDH isoenzymatic pattern in both isolates of D. filaria was characterized by the presence of three phenotypes: (1) a single, slow anodic band; (2) a single, fast anodic band; and (3) a large spot matching its migration with bands 1 and 2. These three phenotypes may be explained as the existence of only one gene locus for the MDH in D. filaria. Allelic frequencies and the Hardy-Weinberg test were determined for Slovakian and Spanish isolates of D. filaria. This test indicated that the population was not in Hardy-Weinberg equilibrium. The MDH isoenzymatic pattern of D. viviparus displayed the same phenotypes 1 and 2 observed in D. filaria. Furthermore, the MDH isoenzymatic pattern of P. rufescens was characterized by the presence of two bands with anodic and cathodic migration. The isoenzyme with anodic migration appeared more intensively stained than did that with cathodic migration. This last isoenzyme was not observed when the samples had been stored for 1 month.

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.

Superoxide dismutase (SOD) activity of Dictyocaulus viviparus and its inhibition by antibody from infected and vaccinated bovine hosts.

The presence of superoxide dismutase (SOD) activity in the bovine lungworm Dictyocaulus viviparus was examined using the xanthine-xanthine oxidase assay system and by non-denaturing PAGE followed by specific enzyme staining. High levels of activity were detected in excretory-secretory (ES) products of adult worms and in soluble extracts of both the L3 and adult stages of the parasite. Stage-specific and ES-specific activities were indicated by differences in SOD isoenzyme profiles between adult and larval parasite extracts and between adult extract and ES products, with a fast migrating activity being specific to ES products. All isoenzymes were sensitive to cyanide, indicating copper/zinc dependency. The antigenicity of ES SOD was demonstrated by a reduction in SOD activity in both the chemical assay and non-denaturing PAGE following incubation of parasite ES products with IgG antibody purified from serum of infected or vaccinated bovine hosts. The high level of SOD activity released by adult D. viviparus may be a reflection of the problems faced by a parasite occupying an oxygen-rich environment. Antibody inhibition of SOD may, therefore, be an important target of protective immunity.

The immunogenicity of the acetylcholinesterases of the cattle lungworm Dictyocaulus viviparus.

Somatic extracts and excretory/secretory (ES) products of the adult stage of the cattle nematode, Dictyocaulus viviparus, were examined for acetylcholinesterase (AChE) activity. Both were found to contain activity which had an optimum pH of 9.5, however, the adult ES products contained over 200 times more AChE activity per unit protein. Gel electrophoresis and specific enzyme staining revealed 5 migratory isoforms of AChE which were common to adult ES products and adult homogenates. Comparison of L3 with L4 and adult extracts indicated that the AChE were only produced by later developmental stages of this parasite. The antigenicity of D. viviparus AChE was demonstrated by binding to serum IgG from naturally and experimentally infected calves but the enzymes were not recognized by calves vaccinated twice with 400 Gy-irradiated larvae. This is the first report of helminth AChE release by a parasitic nematode in a pulmonary location. The presence of these enzymes in such high amounts in the ES products, along with their immunogenicity, suggests that they might have an important role to play in the immunobiology of D. viviparus in the lungs.

Genetic control of the antibody repertoire against excretory/secretory products and acetylcholinesterases of Dictyocaulus viviparus.

Outbred Dunkin-Hartley and inbred strain 2 and strain 13 guinea pigs were immunized with Dictyocaulus viviparus adult ES products prior to challenge with third stage larvae. Antibody responses of the three strains to adult ES products and the acetylcholinesterase (AChE) isoforms which they contain were examined. Using immunoprecipitation and ELISA, it was observed that responses in the three strains to adult ES products were distinct: considerable heterogeneity in the antibody repertoire was observed between outbred Dunkin-Hartley animals, with only slight variation occurring amongst the inbred individuals. Responses to the AChE isoforms were heterogeneous amongst individual outbred guinea pigs but were more consistent in inbred strain 2 and 13 animals in which strain-specific patterns of recognition were observed. Previous studies with nematode infections have indicated a role for the major histocompatibility complex in determining the nature and level of the immune response. As the inbred strains bear different alleles at the Class II region but are identical at the Class I region, the differences observed are likely to be due to genes mapping to the Class II locus. This is therefore the first report of genetic restriction of the antibody repertoire to secreted AChEs of a parasitic nematode.

The secreted and somatic proteinases of the bovine lungworm Dictyocaulus viviparus and their inhibition by antibody from infected and vaccinated animals.

Proteinase activities were examined in extracts and excretory-secretory (ES) products of the infective and adult stages of the cattle lungworm, Dictyocaulus viviparus. Multiple enzyme activities were identified from each source, as defined by pH optima, substrate specificities, inhibitor effects and substrate gel electrophoresis. Serine-, cysteine- and metalloproteinases were identified, secreted materials being more active against protein substrates per unit protein than were extracts, and the particular proteinases produced varied with the developmental stage of the parasite. The antigenicity of these parasite proteinases was demonstrated by the inhibition of enzymic activity with Protein G-purified serum IgG antibody from both infected and vaccinated hosts and in the retardation of enzyme migration on electrophoresis of enzyme-antibody complexes. For the adult products, this confirmed that the enzymes concerned were of parasite origin, and not host-derived. These results argue for investigation of D. viviparus proteinases as targets for the antibody response in the limitation of parasite-mediated tissue damage and as the active principle behind the anti-D. viviparus vaccine.

Isolation and partial characterisation of a potentially pathogenic cysteine proteinase from adult Dictyocaulus viviparus.

Dictyocaulus viviparus adult worms feed on pulmonary tissues and cause significant pathology in the bovine host. In this report, acidic extracts of these organisms were examined for cysteine proteinase activity. A soluble thiol-dependent proteinase with native molecular weight of approximately 25 kDa was isolated and partially purified. This enzyme had maximal activity at acidic pH and showed inhibitor susceptibilities similar to the vertebrate acidic cysteine proteinases. When stored at 4 degrees C, it was stable at pH 5.0 for at least 10 days and at pH 7.0 for at least 24 h. The D. viviparus cysteine proteinase was capable of degrading type I collagen and hemoglobin. It is suggested that this enzyme may be involved in the nutrition of this parasite and/or have the potential to contribute to bronchial pathology in cattle.