| Summary: | Multiple infections of several bacterial species are often observed under natural farm conditions. The infections would cause a much more significant loss compared to a single infectious agent. Vaccination is an essential strategy to prevent diseases in aquaculture, and oral vaccination has been proposed as a promising technique since it requires no handling of the fish and is easy to perform. This research attempts to develop and evaluate a potential feed-based polyvalent vaccine that can be used to treat multiple infections by <i>Vibrios</i> spp., <i>Streptococcus agalactiae</i>, and <i>Aeromonas hydrophila</i>, simultaneously. The oral polyvalent vaccine was prepared by mixing formalin-killed vaccine of <i>V. harveyi, S. agalactiae</i>, and <i>A. hydrophila</i> strains with commercial feed pellet, and palm oil as an adjuvant was added to improve their antigenicity. Thereafter, a vaccinated feed pellet was tested for feed quality analysis in terms of feed stability in water, proximate nutrient analysis, and palatability, safety, and growth performance using Asian seabass, <i>Lates calcarifer</i> as a fish host model. For immune response analysis, a total of 300 Asian seabass juveniles (15.8 ± 2.6 g) were divided into two groups in triplicate. Fish of group 1 were not vaccinated, while group 2 was vaccinated with the feed-based polyvalent vaccine. Vaccinations were carried out on days 0 and 14 with oral administration of the feed containing the bacterin at 5% body weight. Samples of serum for antibody and lysozyme study and the spleen and gut for gene expression analysis were collected at 7-day intervals for 6 weeks. Its efficacy in protecting fish was evaluated in aquarium challenge. Following vaccination by the polyvalent feed-based vaccine, IgM antibody levels showed a significant (<i>p</i> < 0.05) increase in serum against <i>Vibrio harveyi</i>, <i>Aeromonas hydrophila</i>, and <i>Streptococcus agalactiae</i> and reached the peak at week 3, 5, and 6, respectively. The high-stimulated antibody in the serum remained significantly higher than the control (<i>p</i> < 0.05) at the end of the 6 weeks vaccination trial. Not only that, but the serum lysozyme level was also increased significantly at week 4 (<i>p</i> < 0.05) as compared to the control treatment. The immune-related gene, dendritic cells, C3, Chemokine ligand 4 (CCL4), and major histocompatibility complex class I (MHC I) showed significantly higher expression (<i>p</i> < 0.05) after the fish were vaccinated with the oral vaccine. In the aquarium challenge, the vaccine provided a relative percentage survival of 75 ± 7.1%, 80 ± 0.0%, and 80 ± 0.0% after challenge with <i>V. harveyi</i>, <i>A. hydrophila</i>, and <i>S. agalactiae</i>, respectively. Combining our results demonstrate that the feed-based polyvalent vaccine could elicit significant innate and adaptive immunological responses, and this offers an opportunity for a comprehensive immunization against vibriosis, streptococcosis, and motile aeromonad septicemia in Asian seabass, <i>Lates calcarifer.</i> Nevertheless, this newly developed feed-based polyvalent vaccination can be a promising technique for effective and large-scale fish immunization in the aquaculture industry shortly.
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