Monoclonal antibody 2A6 binds to the surface of the Dictyocaulus viviparus L3 sheath and identifies a molecule which is highly antigenic in both infected and vaccinated cattle. Immunogold electron microscopy reveals that the antibody binds to a highly stable surface coat overlying the epicuticle of the L3 sheath. The binding to the cuticular surface is stage specific, being restricted to the surface of the L3 sheath, although the antigen can be detected on Western blots of parasite homogenates from the L1 stage. Immunofluorescence on live parasites demonstrates that the antibodies also bind to the L3 sheath surface of many other strongylid nematodes. The antigen detected by 2A6 differs in molecular weight between the species; in D. viviparus the antigen is poorly resolved between 29 and 40 kDa by SDS-PAGE whereas in nematodes from the family Trichostrongyloidea a single band or doublet of approximately 20 kDa is detected, suggesting that a related molecule is present in these species. Although infection and vaccination with D. viviparus elicits a strong immune response to the L3 sheath antigen this does not appear to be the case following infection with other strongylid nematodes.

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.

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.

Calves with naturally acquired Dictyocaulus viviparus infection mount an effective immune response. In the search for protection-inducing antigens, we found that several D. viviparus third-stage larval (L3) and adult ES products carry N-glycans. Deglycosylation of the worm antigens using PNGase F resulted in reduced IgA, IgE, IgG1 and IgG2 (but not IgM) reactivities in sera of primary infected animals, suggesting that the carbohydrate moieties contained immunodominant epitopes. Challenge infection resulted in increased specific serum antibody levels against ES and L3 in the re-infected and challenge control groups. Testing of sera by enzyme-linked immunosorbent assay (ELISA) demonstrated a significant increase in IgG1 and IgE (but not IgA or IgG2) reactivity against the deglycosylated antigens in the re-infected group compared with the challenge control group. Sera from calves vaccinated with irradiated larvae showed a strong anti-N-glycan response, but no booster response against the protein backbone after challenge infection, consistent with the absence of a memory response. Together, our results suggest that D. viviparus proteins carry immunodominant N-glycan moieties that elicit a strong but short-lived immune response during infection and after vaccination, whereas the protein backbones effectively induce a memory response which results in a long-lasting, potentially protective immune response in re-infected, but not in vaccinated calves.

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.

Proteomics has come to the forefront in the post-genomic era. The ability to compare and identify proteins expressed in a particular cell type under specific physiological or pathological states requires a range of technologies, including separation of complex protein or peptide mixtures, densitometry-based or isotope-coded methods for comparison of multiple proteomes, and mass spectrometric methods for identification of individual low abundance proteins. Although an emergent technology, thus far, proteomics has provided new perspectives on many problems in biomedical science. In parasitology, proteomics has been used to answer specific biological questions relating to survival and development, and also to identify candidates for vaccines. Here, we describe an ongoing research programme in which proteomics is being used to identify potential vaccine candidates for the bovine lungworm, Dictyocaulus viviparus. This work is focusing on antibody responses to the adult parasite excretory/secretory (ES) products, with selection of candidate antigens based on differential screening with serum from immune versus non-immune animals to simplify the proteome and the ensuing analytical challenges. Thus far, we have identified seven candidate proteins using this strategy. Of these, one protein showed significant identity to a previously cloned gene from D. viviparus, whilst the other six proteins have shown no significant identities. Isolation of further peptide sequences is now warranted to facilitate cloning of the genes encoding these antigens.

Thirty Holstein Friesian heifer calves, 3 months of age, were used to evaluate whether a low primary infection with Dictyocaulus viviparus would result in immunity against challenge infection 5 or 12 weeks later. Fifteen calves were experimentally infected with 30 larvae at day 0, while the other calves remained as uninfected controls. On day thirty-five 6 primary infected (G1) and 6 non-infected calves (G2) were challenged with 2000 larvae. These groups were necropsied on day 70. On day 84 the remaining 9 primary infected (G3) and 9 non-infected (G4) calves were similarly challenged and necropsied on day 119. Respiratory signs developed in most calves from approximately 2 weeks after challenge, but signs were more severe in G2 and G4 than in G1 and G3. The low primary infection resulted in significantly reduced faecal larval excretion (>80%) and worm burdens (>70%). In the primary infected groups the proportion of female worms in the worm burden was increased, suggesting that part of the developed immunity affected survival of the worms after their establishment in the host. Worm lengths and worm fecundity were also significantly reduced. All observed effects were stronger following challenge on day 84 than on day 35, but the effect of the duration of a primary infection (contrast between G1 and G3) was only statistically significant in the case of mean faecal larval excretion on the day of necropsy (P<0.05) and fecundity (P<0.05).

A model simulating Dictyocaulus viviparus infection in calves is described. The present paper only deals with the parasitic phase of the life-cycle. Descriptions are given for establishment, development rate of juvenile stages, mortality rates of both juvenile and adult stages, and fecundity. Literature data were used to to develop parameter functions and to estimate initial values for constants. Development of acquired immunity, defined as the proportional ability of the host to reduce the number of parasite individuals in some stage or about to move into a next stage, against establishment (protection) or affecting mortality rates of juvenile or adult parasite stages has been included. The effect of immunity on one parameter or process is viewed as distinctly separate from the effect on another. Preliminary comparisons between model prediction and observations gives encouraging results, indicating that the model simulates experimental D. viviparus infection in calves reasonably well. Some quantitative discrepancies between prediction and observation make clear however, that not all parts of the model are accurate. Further experimentation is needed to re-evaluate current model description and to improve model simulation.

Parasitic bronchitis is a serious disease of cattle and is caused by the nematode, Dictyocaulus viviparus. For over 30 years, a radiation-attenuated larval vaccine has been used for prevention of this disease. This vaccine has been used with considerable success in the UK and parts of Western Europe, however, it has several disadvantages. It has a short shelf- life and the vaccine has to be produced annually necessitating the use of donor calves. Following vaccination, calves must receive further boosting from natural challenge to maintain protective immunity. Sales of the irradiated larval vaccine have decreased dramatically since the 1970s. This is thought to be due to increased reliance of farmers on anthelmintic programmes to control lungworm infection. It is possible that, under certain circumstances, these programmes do not allow sufficient parasite exposure to stimulate protective immunity to further Dictyocaulus challenge. This is borne out by the recent documented increase in the number of outbreaks of parasitic bronchitis in the UK. A stable vaccine against D. viviparus that is capable of stimulating a more prolonged immunity would be beneficial. Recent research has been directed at identification and isolation of components thought to be involved in parasite survival in the host and examination of their potential as vaccine candidates. One of these components is acetylcholinesterase (AChE), an enzyme secreted by adult worms. This review describes the development of the secreted AChE as a vaccine candidate, as well as documenting recent developments in the immunodiagnosis of D. viviparus.