Bioencapsulation and disease suppression studies of beneficial bacteria against Edwardsiella tarda infection in climbing perch larvae (Anabas testudineus, Bloch)

This study aimed to develop the best bioencapsulation condition for potential probiotic bacteria to control Edwardsiella tarda infection in climbing perch, Anabas testudineus larvae. Preliminary metagenomic analyses unveiled the pathogenic bacteria (e.g. Edwardsiella tarda, Pseudomonas fluorescens and Serratia marcescens) and beneficial bacteria (e.g. Bacillus subtilis) were the natural microflora in the climbing perch gastrointestinal (GI) tract. Compared to other pathogens, E. tarda was largely residing in the wild- and farmed-typed A. testudineus GI tract. Multiple in vitro screenings such as antagonistic, hemolytic, antibiotic and bacteriosin-like substance (BLIS) assays were performed in the study to determine the best probiotic against E. tarda, through isolating and evaluating the microflora isolated from intestines of Oreochromis niloticus, Clarias batrachus, Ophicephalus striatus, Trichogaster pectoralis, Anabas testudineus and Pangasius pangasius. Data suggested that L. lactis subsp. lactis (CF4MRS) at > 106 CFU mL-1 could be effectively used to control E. tarda in laboratory condition. This bacterial isolate also showed a strong antimicrobial activity against other fish pathogens such as P. fluorescens, P. aeruginosa, K. pneumonia, E. coli, A. hydrophila, and S. marcescens. HPLC analysis showed that this antimicrobial activity was not attributed to any compounds but the organic acids i.e. lactic acid. When L. lactis subsp. lactis CF4MRS (108 CFU mL-1) was bioencapsulated in Artemia franciscana, it could be effectively used to protect the live feed against E. tarda up to 50% of survival rate. Colonization efficiency of the probiotic was also highly demonstrated in A. franciscana when gfp-transformed L. lactis was used in the bioencapsulation process. In addition, this probiotic also improve the production of amylase, protease, lipase and cellulase enzymes in A. franciscana. For the best bacterial encapsulation result, sodium alginate was suggested in the A. franciscana bioencapsulation instead of starch, gum Arabic and gelatin, as it showed the highest total bacterial count in Artemia (2.44 × 107 CFU mL-1). To determine the effects of the probiotic on climbing perch larvae, A. testudineus larvae were produced by comparing different hormones (LHRHa and SGnRHa). Results suggested induced breeding using LHRHa was the most effective way to produce the fish larvae. In the in vivo pathogenic assays, fish larvae were divided into various groups: Artemia nauplii enriched with 108 CFU mL-1 L. lactis (LA); Nauplii enriched 0.5 g L-1 sodium alginate (SA); Nauplii enriched 0.5 g L-1 sodium alginate containing 108 CFU mL-1 L. lactis (LSA); and nauplii only as the control (A). Fish larvae fed with LSA showed highest larval survival rate (50%) compared to LA (41.7%), SA (31.7%) and A (20%). Lysozyme activity was also highest in LSA (13.89 units mg-1 protein), followed by LA (10.00 units mg-1 protein), SA (8.33 units mg-1 protein) and A (1.33 units mg-1 protein). The result indicated a strong improvement of immune response in fish larvae when LSA was included in larval diet. In a nutshell, combination of L. lactis CF4MRS (108 CFU mL-1 for 8 h) and alginate in Artemia bioencapsulation could improve survival rate of E. tarda-infected climbing perch larvae through up-regulating their innate immune response