Isolation of Potential Probiotic Bacillus spp. from the Intestine of Nile Tilapia to Construct Recombinant Probiotic Expressing CC Chemokine and Its Effectiveness on Innate Immune Responses in Nile Tilapia

Abstract

This study aimed to investigate the potential probiotic Bacillus spp. from the intestine of Nile tilapia in order to construct a recombinant probiotic for the enhancement of the Nile tilapia immune response. One hundred bacterial isolates from the intestine of Nile tilapia were characterized for species identification using the 16s ribosomal RNA (rRNA). Only Bacillus isolates with exhibited antagonistic activity were investigated for their biological functions, which included protease-producing capacity, bile salts and pH tolerance, antibiotic susceptibility, and pathogenicity tests. According to the best results, Bacillus isolate B29, as closely related to B. subtilis, was selected to construct a recombinant probiotic for the delivery of CC chemokine protein (pBESOn-CC). The existence of recombinant probiotics was confirmed by Western blotting before the feeding trial. In addition, the CC chemokine mRNA level was quantified in the intestine of fish fed probiotics after 30 days of feeding. Total immunoglobulin, lysozyme activity, alternative complement 50 activity (ACH50), and phagocytic activity of fish fed either wild-type or recombinant probiotics were significantly increased, indicating that probiotics could stimulate the Nile tilapia immune system through different processes. Interestingly, the dietary supplementation of recombinant probiotics has a stronger immune response enhancement than the wild-type strain.

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

“…Furthermore, our previous in vitro study con rmed that the potential probiotic B. subtilis, isolated from the intestine of Nile tilapia, exhibited antibacterial activity and effectively antagonized pathogenic S. agalactiae. [34]. Together, these ndings suggest that the improvement of antagonistic activity against S. agalactiae in recombinant B. subtilis may be attributed to the synergistic effect of B. subtilis and vitamin C, modulating innate and adaptive immunity in Nile tilapia.…”

“…Prior to commencing the feeding experiment, the presence of secreted GULO produced from probiotic B. subtilis was con rmed through the western blot method as within the method of our previous study [34].…”

“…At 30 and 90 days of the trial, serum LZM activity [37], serum total Ig concentration [34], and serum ACH50 activity [38] were performed in a slightly modi ed manner. The optical density of each sample was measured using an absorbance microplate reader Epoch BioTek instruments (Agilent Technologies, CA, USA).…”

“…Since the ascorbate biosynthesis pathway, starting with D-glucose-1-phosphate as the initial precursor and progressing until L-gulonate, is conserved in all animal species [33], it may be possible to reestablish this pathway by integrating the GULO gene into probiotic B. subtilis using recombinant probiotic technology. In addition, our previous study revealed the bene cial effects of B. subtilis isolated from the intestine of Nile tilapia, including, antagonistic activity, bile salts and pH tolerance, protease-producing capacity, antibiotic susceptibility, and pathogenicity tests [34]. Therefore, the application of recombinant probiotic B. subtilis expressing GULO may provide a possible alternative option to achieve the combined effect of probiotic B. subtilis and vitamin C supplementation.…”

Importance of Probiotics in Fish Aquaculture: Towards the Identification and Design of Novel Probiotics

“…Recombinant probiotics have also been used to express proteins that stimulate the immune response in fish, such as cytokines [ 145 , 146 ] and chemokines [ 147 ], the intestinal barrier [ 148 ], or enzymes that disrupt chemical communication in pathogens [ 149 ]. In the case of the cytokines, L. lactis has been used to deliver Interferon I and II, conferring protection against IPNV and F. psychrophilum , respectively [ 145 , 146 ].…”