Transcriptomic defense mechanism of Japanese Eel (Anguilla japonica) against Edwardsiella anguillarum infection after bath immunization with Bacillus subtilis spores displaying OmpA of E. anguillarum

this study investigated the effecacy and transcriptomic mechanisms of bath immunization using Bacillus subtilis spore-displayed outer membrane protein A (OmpA) against the infection of Edwardsiella anguillarum , a pathogen causing hemorrhagic septicemia in Japanese eels ( Anguilla japonica ). Eels were immunized with B. subtilis spores displaying OmpA (WA group) or inactivated whole-cell E. anguillarum (EA group), alongside two control groups receiving wild-type B. subtilis spores (WC group) or PBS (Con group). Following 28-day (d) immunization, all groups were challenged with E. anguillarum , and mortality was monitored for 14 d. Histopathology and gene expression were assessed 48 hours (h) post-infection. The WA and EA groups showed significantly lower mortality rates (64.3 % and 57.1 % relative survival, respectively) compared to the Con group. Histopathological examination revealed attenuated hepatic and renal in immunized groups, relative to pathology observed in control groups. Transcriptomic profiling identified 1973 differentially expressed genes (DEGs) in WA-bath infected group (WA_inf) and 866 in EA-bath infected group (EA_inf) relative to PBS-bath (Con_inf) and WC-bath (WC_inf) infected groups. Gene Ontology (GO) and KEGG annotation of DEGs highlighted 5 GO terms associated with "catalytic activity", "binding", "cell part", "membrane part", "biological regulation", and "cellular process", and 4 KEGG pathways related to "Cancer", "Infectious disease", "Immune system", and "Signal transduction". Enrichment analysis emphasized 3 GO terms—transporter activity, heme binding, and oxidoreductase activity—and 10 immune-related KEGG pathways, especially those involving phagosomes, in the WA_inf vs WC_inf comparison. Chord diagram and protein-protein interaction network analyses identified 17 immune-related DEGs and four hub proteins critical for anti- E. anguillarum immunity. These results underscore the potential of bath immunization to boost eel survival against E. anguillarum infection and elucidates the molecular mechanisms mediating the immune response. • The RPS reached 64.3 % after the E. anguillarum infection in the eels immunized through a bath with OmpA-displaying spores. • Eels bathed with OmpA-displaying spores exhibited only mild pathological alterations after the E. anguillarum infection. • There were 17 key DEGs involved in immune regulation against E. anguillarum post-bath immunization. • Four DEG-encoded proteins played a pivotal role in the immune response to E. anguillarum post-bath immunization.