<title>Abstract</title> Aquaculture industry frequently encounters disease outbreaks, high mortalities as well as emergence of new pathogens due to its intensification. <italic>Streptococcus agalactiae</italic> (Lancefield’s group B Streptococcus) is an important pathogen extensively causing infectious diseases in tilapia resulting in huge economic loss and mortality rates. To date, vaccination has been approved to be successful in defending infectious diseases prevailing among farmed fish species. This study aimed to develop an <italic>S. agalactiae</italic> inactivated vaccine (SAIV) using molecular adjuvants- flagellin and tilapia interferon gamma (IFN-γ), and to assess the generated immune response and protective efficacy of the adjuvant incorporated vaccine against the <italic>S. agalactiae</italic> infection in Nile tilapia. The fish were vaccinated with SAIV together with either flagellin or IFN-γ and both together by intraperitoneal injection. The vaccinated fish were challenged with a virulent strain of <italic>S. agalactiae</italic> on day 36 and monitored three weeks for cumulative mortality. The results showed that the vaccine offered significant protection with relative percentage survival (RPS) of 59.37%, 71.87% and 81.25% observed for bacterin vaccine adjuvanted with flagellin, IFN-γ and both, respectively, with an RPS of 15.62% for the unadjuvanted bacterin control group after challenge with <italic>S. agalactiae</italic>. The vaccine induced specific IgM antibodies against <italic>S. agalactiae</italic> in the vaccinated groups and the antibody response was significantly increased following booster vaccination in the fishes administered with vaccine adjuvanted with flagellin, IFN-γ and both. Furthermore, after vaccination MHC-II and IgM gene expression was found significantly upregulated in head kidney and spleen, in line with elevated specific IgM titer. Innate immune parameters including catalase, lysozyme, superoxide dismutase, myeloperoxidase and bactericidal activities were significantly increased in fishes immunized when compared to the unvaccinated controls (<italic>P</italic> &lt; 0.05). Histopathological examinations with tissue sections of head kidney, spleen, liver, kidney, gills and brain were performed from fish vaccinated and non-vaccinated showed mild infiltrations. In conclusion, flagellin and IFN-γ have shown potential for use as molecular adjuvants to enhance the efficacy of fish vaccines against <italic>S. agalactiae</italic> infections.

Analysis of the efficacy of two molecular adjuvants,flagellin and IFN-γ,on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)