Structural integrity of surface antigens determines complement-dependent protective immunity against Philasterides dicentrarchi in turbot.

Scuticociliatosis caused by Philasterides dicentrarchi is a major constraint for turbot (Scophthalmus maximus) aquaculture, yet the molecular identity and immunological relevance of parasite surface antigens remain poorly defined. This study evaluates the role of antigen structural integrity in shaping humoral immune responses and protective immunity, integrating immunological assays with proteomic characterization of a phospholipase C-released surface antigen fraction (SAF). Immunization experiments in turbot showed that native SAF induces significantly higher antibody titers in serum and peritoneal fluid, as well as stronger complement-dependent cytotoxic activity, compared to its denatured counterpart (SAF-D). Epitope profiling by ELISA revealed a clear predominance of conformational epitopes in native SAF, with a marked decrease in the conformational dominance index (CDI) following denaturation. In contrast, control antigens such as ovalbumin exhibited minimal differences between native and denatured forms, confirming that the observed effects are antigen specific. Functional assays further demonstrated that disruption of antigen structure compromises protective immunity, as reflected by increased cumulative mortality in fish immunized with denatured antigens. Proteomic analysis of the SAF fraction (55-100 kDa) identified a limited number of highly abundant proteins, predominantly uncharacterized but supported by multiple unique peptides, indicating their relevance as major antigenic targets. Notably, no variant surface proteins (VSP-like) or predicted GPI-anchored proteins were detected, and no proteins with transmembrane domains were identified, suggesting that the dominant immunogenic components correspond to previously uncharacterized surface-associated proteins. These findings demonstrate that preservation of antigen structural integrity is essential for effective humoral and protective immune responses in turbot and identify novel candidate antigens for vaccine development against scuticociliatosis.