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Figure 3 Evaluation of proposed probiotic bacteria in zebrafish. (A) Evaluation of bacterial biosafety. Ten fish per group were injected intraperitoneally with 1 × 106, 1 × 107 and 1 × 108 CFU per fish for both A. veronii and P. entomophila, separately. Fish injected with PBS buffer were used as controls. Bacterial toxicity was evaluated by recording signs and symptoms of infection and mortality of the injected fish daily for 7 days. (B) Experimental design for protective response against M. marinum. The effect of immunization with zebrafish gut candidate probiotic bacteria was evaluated with α-Gal and PBS used as positive and negative controls, respectively. Thirty LRZ were randomly allocated to Group A, commercial diet with probiotic A. veronii; Group B, commercial diet with probiotic P. entomophila; Group C, commercial diet with PBS; and Group D, commercial diet with α-Gal immersion. Fish were weighted at the weeks 1–5 and 10 at the end of the experiment. Gut and sera were collected at weeks 3 (T1), 4 (T2) and 5 (T3) and at the end of the experiment (week 10; T4) and processed for gut and serum collection for analysis of antibody levels by ELISA, mycobacteria levels by RT-qPCR, expression of selected immune response gene markers by RT-qPCR, oxidative stress biomarkers and gut microbiome.