Immune responses and cross-protection elicited by parasitic versus free-living ciliate vaccines in koi carp (Cyprinus carpio).

Ciliated protozoans such as Ichthyophthirius multifiliis and Chilodonella uncinata pose severe threats to freshwater aquaculture, yet the antigenic relationships and cross-protective potential among different ciliate species remain poorly understood. This study systematically evaluated the immune responses and protective efficacy induced by inactivated whole-ciliate vaccines prepared from three ciliates - I. multifiliis (I.M), C. uncinata (C.U) and Tetrahymena thermophila (T.T)-in koi carp (Cyprinus carpio). Fish were intraperitoneally immunized with four doses (1×104-6×104 inactivated ciliates/fish) and boosted at 14 days post-primary vaccination. Results showed that all three vaccines significantly elevated serum levels of IgM, complement C3/C5 and hepatic antioxidant enzymes (SOD, CAT) (P < 0.05). Transcriptional upregulation of key immune genes (IgM, MHC IIβ, IL-1β, IFN-γ, TNF-α, IL-8) was observed in head kidney, spleen and skin, accompanied by increased proportions of IgM+ B cells in primary and secondary lymphoid organs. Challenge trials at 28 days post-immunization revealed strong dose-dependent protection against both I. multifiliis and C. uncinata. Notably, the I.M and C.U vaccines conferred robust homologous protection and exhibited substantial bidirectional cross-protection, while the T.T vaccine provided moderate, dose-dependent cross-protection. Overall, vaccines derived from parasitic ciliates elicited stronger immune responses than those derived from the free-living ciliate, likely reflecting differences in host-parasite coevolution and antigenic compatibility. These findings demonstrate that inactivated whole-ciliate vaccines coordinately activate innate and adaptive immunity and induce cross-protective responses, highlighting their potential as candidates for broad-spectrum antiparasitic vaccine development in aquaculture.