The recombinant Echinococcus multilocularis antigen ll/3–10 is one of the most promising tools for immunodiagnosis of alveolar echinococcosis in human patients. Its nucleic acid sequence represents a part of the E. multilocularis gene encoding the metacestode antigen II/3, the former being basically present and expressed in both E. multilocularis and E. granulosus. Most (94%) patients with alveolar echinococcosis respond to infection with a marked anti-II/3–10 IgG synthesis; in contrast, most of the cystic echinococcosis patients do not, for some reason, recognize the recombinant antigen. We tackled this problem by generating cDNA derived from both E. granulosus and E. multilocularis full length II/3 genes, performed by reverse transcription and PCR amplification. Sequence analysis revealed a very high degree of conservation of the primary sequence of the antigen II/3 in both Echinococcus species. cDNA fragments were subcloned and expressed in E. coli as fusion proteins with Schistosoma japonicum glutathione S-transferase. Recombinant proteins were affinity purified and comparatively assessed by ELISA with respect to antibody-binding characteristics. Sera from patients suffering from cystic echinococcosis showed no significant differences in reactivity with the antigens derived from either E. multilocularis or E. granulosus. Therefore, parameters other than some minor differences in the primary sequence seem to be responsible for the lack of antigen II/3 recognition in cystic echinococcosis.

Note

Nucleotide sequence data reported in this paper have been submitted to the GenBank® data base with the accession numbers U05573 and U05574.

A recombinant Echinococcus multilocularis antigen (II/3–10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3–10 antibodies identified the corresponding epitopes on bands with identical Mr in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3–10 antibodies reacted with antigens of different Mr from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.