Innovative approach for vaccinating Nile tilapia,Oreochromis niloticusagainstStreptococcus agalactiaeusing an ozone nanobubble pre-treatment, VAC in BAG and VAC in FEED

Abstract The treatment of Nile tilapia with ozone nanobubbles (ONb) prior to vaccination with an immersible heat-killed Streptococcus agalactiae ( Sa ) vaccine has been reported to modulate and enhance both innate and specific immunity. The efficacy of this novel vaccination strategy is explored further in field trials. This strategy involved a short-term treatment of ONb to activate the fish’s immunity prior to immersion vaccination during their transportation in oxygenated plastic bags (VAC in BAG), followed by two oral boosters during the grow-out stage mixing vaccine in feed (VAC in FEED). The field trial was conducted over 112 days in open cages, comprising four groups: normal aeration control (AC), normal aeration + vaccine (AV), ONb control (NC), and ONb + vaccine (NV). The efficacy of the vaccine was evaluated by measuring specific antibodies for S. agalactiae , monitoring expressions of IgM and IgT transcripts in the gills and head kidney every two weeks, and a laboratory pathogen challenge. Results found that fish in the NV group had significant increases in anti- S. agalactiae antibodies after the primary dose, whereas fish in the AV group required an oral booster dose to produce significant anti- S. agalactiae antibodies. In the vaccinated groups (AV and NV), only IgM was observed to be upregulated at 14 days post-immersion (dpi), while this gene was upregulated in both gills and head kidney in the NC group. No statistically significant upregulation of IgT was recorded in any group at any time point. Despite a decline in the levels of specific antibodies among the vaccinated groups at the time of challenge (88 dpi), the NV and AV groups demonstrated a relative percent survival (RPS) of 50% and 46.7%, respectively, following a relatively high injection dose of S. agalactiae injection (0.1 mL of 10 8 CFU/mL). In summary, this ONb, VAC in BAG and VAC in FEED vaccination strategy represents a promising alternative to the undesirable handling and costly injection approach used within the Nile tilapia industry.