Characterization and protective effects of feed-based bivalent vaccine against streptococcosis and aeromoniasis in red hybrid tilapia (Oreochromis spp.)

Streptococcosis and aeromoniasis are the major bacterial diseases in tilapia farming. Streptococcus agalactiae and Aeromonas hydrophila are the dominant causative agents of streptococcosis and aeromoniasis, respectively. Controlling bacterial diseases by vaccination using feed-based bivalent vaccines has been considered an effective method to prevent multiple diseases, which contributes to economic sustainability. Previously, a novel feed-based bivalent vaccine containing formalin-killed whole organisms of Streptococcus iniae and A. hydrophila for red hybrid tilapia was developed, which provided good immunological responses and high protection against S. iniae and A. hydrophila infections. However, there is still a lack of vaccine characterization regarding stability and mechanism of the oral delivery route. This study aims to characterize and investigate the immunoprotective efficacy of feed-based bivalent vaccine against the causal agents of streptococcosis and aeromoniasis in red hybrid tilapia. The feed-based bivalent vaccine containing killed whole organisms of S. agalactiae and A. hydrophila mixed with 10% palm oil was successfully prepared and characterized regarding stability, nutritional quality, safety and growth performance. The vaccine was delivered orally at 5% of the fish’s body weight for three consecutive days on weeks 0, 2 and 6. Lysozyme and enzyme-linked immunosorbent (ELISA) assays were analyzed to evaluate the immunological responses following vaccination. The vaccination efficacy was assessed according to the relative percentage survival (RPS) and histopathological assessment of the vaccinated fish following challenges with pathogenic bacteria. The vaccinated fish’s hindgut was subjected to transcript response analyses according to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and transcriptome for the expression analysis of immune-related genes and pathways following vaccination, respectively. Results showed that in one g of feed-based bivalent vaccine contained 109 CFU/g of S. agalactiae and A. hydrophila, respectively. Acid tolerance analysis of feed-based bivalent vaccine has caused a 10% reduction in the concentration of killed whole organisms without any morphological changes. The concentrations of killed whole organisms in the feed-based bivalent vaccine remained at approximately 90% when stored at room temperature for 60 days. The vaccine did not influence red hybrid tilapia’s culture water stability, palatability and growth performances. Moreover, vaccinated fish showed high protective efficacy against S. agalactiae (RPS at 80%) and A. hydrophila (RPS at 90%) and partial cross-protective efficacy against S. iniae (RPS at 63%) and Aeromonas veronii (RPS at 60%). The lysozyme activity and IgM antibody level in vaccinated fish’s serum, gut lavage and skin mucus were significantly (p < 0.05) higher than the control fish. The transcript level of IL-1β (5.2-fold change) was found to be highest in the vaccinated fish at week six after the second vaccination booster, followed by MHC-II (4.7-fold change) and CD4 (4.6-fold change). Following the higher immune-related gene expression in the vaccinated fish’s hindgut at week 6, the hindgut sample was subjected to transcriptomic analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed numerous (775) differentially expressed genes (DEG) were enriched in 16 immune-related pathways. This study suggested that the feed-based bivalent vaccine is promising for improving the immunological response against the causal agent of streptococcosis and aeromoniasis.

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