Administration of recombinant attachment protein (r22C03) of Neoparamoeba perurans induces humoral immune response against the parasite in Atlantic salmon (Salmo salar)

• We tested a recombinant protein from N. perurans as an immunogen against the parasite.
• We measured the antibody levels in serum and skin mucus over 12 weeks using ELISA.
• Antibodies produced were characterized by ELISA, Western blot, dot blot and immunocytochemistry.
• Systemic and mucosal antibody levels increased significantly after immunization.
• Antibodies produced were able to bind the surface of the parasite.

This study investigated the use of a recombinant protein of Neoparamoeba perurans, the causative agent of Amoebic gill disease (AGD), as an immunogen to generate systemic and mucosal antibody responses against the parasite. Genes encoding N. perurans homologs of mannose-binding protein (MBP) from Acanthamoeba spp. have been identified. From these, a Neoparamoeba MBP – like EST has been identified and produced as a recombinant fusion protein. Attachment of N. perurans to the gill might be reduced by antibody-mediated interference of this protein, but this is dependent on the presence and level of functional antibodies in the mucus. Fish were immunized with the protein via i.p. injection with Freund's complete adjuvant (FCA); and serum and skin mucus samples were collected before and after immunization. Antibodies (IgM) present in samples were characterized via Western blot and their levels measured with an ELISA. The immunization was able to induce a systemic IgM response 8 weeks after primary exposure and a mucosal response 4 weeks post initial immunization, which were specific to the recombinant protein but not to antigens obtained from crude amoebic preparations. However, adherence of the antibodies to the parasite was observed using immunocytochemistry, and both, serum and skin mucus IgM, were able to bind the surface of formalin-fixed N. perurans. This finding may contribute to further research into the development of a vaccine for AGD.