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, 10(6), 10(7), 10(8) and 10(9) 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, 10(5) CFU/g), L7 (live bacteria, 10(7) CFU/g), HK5 (heat-killed bacteria, 10(5) CFU/g) and HK7 (heat-killed bacteria, 10(7) CFU/g), were set up over 35 days. The results showed that treatment with 10(9)CFU/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-1 beta and TNF-alpha in kidney, whereas HK5 could raise IL-10 and TNF-alpha in spleen, and upregulate IL-1 beta 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 10(8) CFU/g or less based on feed supplement, and heat-killed (10(5) 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.