Recombinant fusion subunit vaccine provides enhanced immune protection against Cryptocaryon irritans infection in grouper.

Cryptocaryon irritans causes severe losses in marine aquaculture, yet effective vaccines remain limited. In this study, we developed a novel fusion vaccine composed of an immobilization antigen (IA8) and the 26S proteasome, and evaluated its immunogenicity and protective efficacy in Epinephelus coioides. The recombinant fusion protein was successfully expressed in Escherichia. coli and exhibited strong immunoreactivity. Following parasite challenge, the fusion vaccine conferred the highest protection, reducing parasite abundance by 81.2%, compared with 44.6% and 22.6% for IA8 and 26S proteasome alone, respectively. Tomont diameters were also reduced by 27.8% in the fusion group, indicating inhibited parasite development. Consistently, fusion vaccination elicited markedly elevated IgM responses in both serum and mucus. Comparative evaluation of administration routes revealed that intraperitoneal injection induced the strongest protective efficacy (67.2% reduction in parasite abundance) and the highest systemic IgM levels, whereas intramuscular injection conferred moderate protection (41.2%) and oral vaccination produced negligible immunity. Immunofluorescence analysis further revealed an increase in IgM+ B cells in the head kidney following injectable immunization, supporting enhanced systemic immune activation. Overall, these findings identify the IA8-26S proteasome fusion protein as a promising vaccine candidate and highlight intraperitoneal administration as the most effective delivery strategy against C. irritans.