Heat‐killed Pseudoalteromonas piscicida 2515 decreased bacterial dose and improved immune resistance against Vibrio anguillarum in juvenile olive flounder ( Paralichthys olivaceus )

The Pseudoalteromonas piscicida 2515 is a potent probiotic with broad effect against Vibrio bacteria. Juvenile olive flounder (Paralichthys olivaceus) (9.04 ± 0.89 g) were used to evaluate the safety of P. piscicida 2515 (control, 106, 107, 108 and 109 CFU/g) in terms of haemolysis activity and mortality rate. To investigate the effects of P. piscicida 2515 (live and heat-killed) on the immune response, and intestinal microbiota and morphology in olive flounder (24.72 ± 2.21 g), five treatment groups, including control, L5 (live bacteria, 105 CFU/g), L7 (live bacteria, 107 CFU/g), HK5 (heat-killed bacteria, 105 CFU/g) and HK7 (heat-killed bacteria, 107 CFU/g), were set up over 35 days. The results showed that treatment with 109CFU/g live P. piscicida 2515 could lead significantly high haemolytic activity. Live- and heat-killed- P. piscicida 2515 significantly increased peroxidase (POD) and serum lysozyme (LZS) activities, while heat-killed P. piscicida 2515 increased SOD and TP. Moreover, L7 could increase expressions of IL-1β and TNF-α in kidney, whereas HK5 could raise IL-10 and TNF-α in spleen, and upregulate IL-1β in kidney. P. piscicida 2515 modulated microbiota richness (Ace and Chao1) and composition at the phylum level. In addition, supplementation of P. piscicida 2515 (live- and heat-killed) could significantly raise intestinal goblet cell number and microvilli length (p < 0.05). Particularly, fish in the treatment groups of HK5 and L7 exhibited higher survival rate against V. anguillarum infection. Therefore, P. piscicida 2515 demonstrated probiotics potency at 108 CFU/g or less based on feed supplement, and heat-killed (105 CFU/g) P. piscicida 2515 has decreased probiotic level dose and has improved the non-specific immunity, intestinal function and protected the olive flounder against V. anguillarum.