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The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1β (IL-1β), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (˜44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.The recombinant OmpF of A. hydrophila was assessed for its vaccine potential in Labeo rohita.L. rohita immunized with the recombinant OmpF (rOmpF) generated specific immune response.The rOmpF immunization stimulated the expression of IL1- β, C3, TLR22, TNF- α and CXCa chemokines.The rOmpF-immunization improved the survival rate of L. rohita against A. hydrophila challenge.The anti-rOmpF antisera specifically cross-reacted with all Aeromonas strains/isolates tested.