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Not AvailableDNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eu- karyotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an addi- tional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression of GAPDH and IFN-γ genes in the transfected cell were confirmed by Western blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita) fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1 × 105 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 and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recorded highest RPS of 63.16% while the pGPD vaccinated group showed 47.37% when compared with 63.33% cumulative mortality of control group. The results regarding respiratory burst activity, myeloperoxidase activity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore, the current study displayed pGPD+IFN group having significant (p < 0.05) upregulation of iNOS gene transcript at 24–48 h post-immunization (both initial and booster dose) as well as after challenge. Thus, from this study, we can conclude that the bicistronic vaccine can be an effective immunization strategy in orchestrating a coordinative immune response against E. tarda in L. rohita.Not Availabl