Improvement of antagonistic activity against Streptococcus agalactiae using recombinant Bacillus subtilis expressing L-gulonolactone oxidase: its effects on growth performance, immune response, and antioxidant activity in Nile tilapia, Oreochromis niloticus

Abstract

Abstract Due to the lack of the L-gulonolactone oxidase (GULO) enzyme, Nile tilapia is unable to synthesize vitamin C and thus requires an adequate level of exogenous vitamin C in its diet. In our previous study, we isolated the probiotic Bacillus subtilis from the intestine of Nile tilapia. Our findings revealed its antagonistic activity against major pathogenic bacteria in Nile tilapia, as well as its ability to enhance the immune responses of the fish. In addition, B. subtilis is an ideal bacterial factory to produce heterologous proteins. Therefore, this study aimed to construct a recombinant probiotic B. subtilis expressing GULO and investigated its effects as a dietary supplement in Nile tilapia. The fish were divided into four groups: those fed with a basal diet (CON), a basal diet + vitamin C (VC), a basal diet + wild-type B. subtilis (BS), and a basal diet + recombinant B. subtilis (BS + GULO). At day 90 of the feeding trial, significant enhancements in growth performance, immune response, and antioxidant capacity were observed in fish fed with BS + GULO. The HPLC analysis and qRT-PCR revealed a significant increase in serum ascorbic acid and GULO mRNA levels in the intestine of the BS + GULO group, respectively. In the challenge test, a time-course experiment demonstrated a significant increase in the expression of pro-inflammatory genes and immune response against S. agalactiae in the BS + GULO group, indicating an improvement in antagonistic activity compared to the wild-type B. subtilis.