Immunochemical Analysis of Antigens of the Bovine Lungworm Dictyocaulus viviparus

A successful irradiated larval vaccine against the cattle lungworm, Dictyocaulus viviparus, has been available for over thirty years. At the outset of the project, however, little was known of antigens of the parasite or of the mechanism of vaccine-induced immunity. This study aimed to characterise antigens of this parasite which may be involved in protective immunity and which could eventually lead to the development of a molecularly-defined vaccine against lungworm infection. The antibody responses to surface, somatic and excretory-secretory (ES) products of larval and adult stage parasites were examined following infection or vaccination of bovine hosts and of the guinea-pig model host in which infection proceeds only to the L5 stage. No parasite proteins could be detected in ES products of infective stage larvae, despite containing substantial levels of proteinase activity. In contrast, a complex range of proteins were detected in the secretions of adult worms. Immunoprecipitation studies of radioiodinated adult ES products revealed that all of these, with the exception of two components, one of which was identified as bovine serum albumin, are antigenic to infected bovine hosts. Calves vaccinated with irradiated larvae, and therefore, not exposed to patent lungworm infection showed restricted recognition of adult ES products, thus demonstrating the stage-specific nature of D. viviparus released products. Significant heterogeneity in the specificity of the antibody response to adult ES products was observed between individual bovine and guinea-pig hosts. This individual variability was examined in the model system using inbred strains of guinea-pig and was shown to have a genetic basis, possibly being controlled by the MHC class II region. Examination of the antibody response to surface-exposed antigens of the egg, L1, L3 and adult stages of D. viviparus demonstrated both the antigenicity and stage-specificity of surface components. Immunofluorescence studies revealed significant recognition of the L3 sheath by antibody from infected and vaccinated bovine and guinea-pig hosts. In contrast, surface-exposed antigens of the adult, egg and L1 stages were recognised uniquely by calves infected with normal larvae and , therefore, exposed to patent lungworm infection. No binding of parasite specific IgG antibody was observed on the exposed surface of exsheathed L3 (i. e. the L3 cuticle) with serum from infected calves. All bovine pre-infection sera examined showed a substantial degree of IgM antibody binding to the L3 cuticle and it is proposed that this non-specific IgM antibody may block immune recognition of parasite-specific surface antigens. IgG antibody recognition of exposed L3 cuticular antigens was observed, however, with sera from hosts exposed to irradiated larvae suggesting that this immunoevasive mechanism may be overcome to some extent by vaccination. As well as differing antigenically, the L3 cuticle was found to differ biophysically from that of other stages of D. viviparus as demonstrated by its inability to bind the fluorescent lipid analogue 5-(N-octadecanoyl)aminofluorescein and to incorporate Iodine-125 into cuticular proteins. These findings may reflect changes in the surface properties of the parasite associated with host infection. Radioiodination of intact sheathed larvae identified a restricted set of proteins while a complex set of labelled proteins was observed following radioiodination of intact adult parasites. Many more adult components were labelled by the Bolton-Hunter than by the lodogen technique, probably reflecting that labelling by the latter method is more surface-restricted. There was no turnover of the major adult surface-associated antigens suggesting that surface components do not contribute to adult ES products of this parasite. Examination of the biological functions of larval and adult extracts and ES products revealed the presence of superoxide dismutase (SOD) and proteinase activities. Characterisation of the latter by pH optima, substrate specificity, inhibitor sensitivity and substrate gel electrophoresis identified multiple proteolytic activities. These enzymes may be involved in parasite invasion and survival within the host. The significant inhibition of both proteinase and SOD activities observed following incubation with immunoglobulin from immune calves may, therefore, be important in limiting parasite survival and consequently such enzymes may be of value as potential vaccine candidates. Finally, a comparison of 35S-methionine labelled polypeptides of normal and irradiated third stage larvae revealed no qualitative nor quantitative differences. It is, therefore, proposed that vaccine-induced immunity to D. viviparus may not depend on the expression of novel parasite antigens but on an enhanced immune recognition of larval stage antigens