Nanoconjugation of bicistronic DNA vaccine against Edwardsiella tarda using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in Labeo rohita vaccinated by different delivery routes

•Nanoconjugation of DNA vaccine against E. tarda using chitosan nanoparticles.•Rohu fingerlings were immunized through oral, immersion and injection routes.•CNPs-pGPD + IFN vaccinated fish exhibited high RPS post E. tarda challenged.•CNPs-pGPD + IFN immunized group showed higher specific and innate immune response.•Innate immune genes were significantly upregulated in immunized fish. DNA-based immunization has proven to be an effective prophylactic measure to control aquatic animal diseases. In order to improve the efficiency of vaccine against fish pathogen, novel delivery mechanism needs to be adopted. In the present study we nanoconjugated the previously constructed DNA vaccine (pGPD + IFN) with chitosan nanoparticles (CNPs) by complex coacervation process. After construction of the vaccine, an in vivo vaccination trial was conducted in which 2 groups of rohu (L. rohita) fingerlings were vaccinated with CNPs-pGPD + IFN, one group by oral route (incorporated in feed for 14 days) and the other by immersion route (primary and booster immunised), whereas, a third group was intramuscularly (I/M) injected (initial and booster immunised) with naked pGPD + IFN and subsequently challenged with E. tarda (8.7 × 104 CFU/fish) at 35-day post initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response, expression kinetics of immune-related genes and pathological manifestation. Evaluation of RPS analysis revealed that CNPs-pGPD + IFN groups recorded highest RPS (81.82% and 72.73% in oral and immersion vaccinated fish group respectively) while the naked pGPD + IFN injected group showed 63.62% RPS when compared with 55% cumulative mortality of control group. In addition, NBT, myeloperoxidase activity, serum lysozyme activity and specific antibody titre in case of CNPs-pGPD + IFN groups showed higher activities during all the time points. Furthermore, CNPs-pGPD + IFN groups showed significant (p