<i>In Vivo</i> Efficacy of Bacteriophage Therapy Against <i>Streptococcus agalactiae</i> in Nile Tilapia ( <i>Oreochromis niloticus</i> ): Delivery Route and a Dose-Dependent Effect of Survival, Immune Responses, and Tissue Recovery

Background: The sustainability of Nile tilapia ( Oreochromis niloticus ) aquaculture is increasingly threatened by bacterial pathogens, such as Streptococcus agalactiae , which cause severe economic losses and high mortality. The overuse of antibiotics in aquaculture has worsened antimicrobial resistance, highlighting the need for alternative therapeutic strategies. This study examines the effectiveness of bacteriophage therapy under experimental conditions as a targeted, potentially environmentally friendly approach to control S. agalactiae infections in Nile tilapia by measuring survival, immune, biochemical, and histological endpoints. Materials and Methods: Three lytic bacteriophages (STRA1, STRA2, STRA3) were isolated and characterized in vitro for host range and lytic activity against S. agalactiae . Experimental application used three administration methods: intramuscular injection of both bacteria and phage (Treat1), intramuscular bacterial infection followed by phage immersion (Treat2), and simultaneous immersion of fish in water containing both bacteria and phages (Treat3). Phage doses of 10 5 , 10 7 , and 10 9 plaque-forming unit (PFU)/mL were tested. Outcomes evaluated included survival rates, immune responses, biochemical markers, histopathological recovery, and cytokine expression. Results: The results showed a dose-dependent decrease in mortality, with Treat1 achieving 100% relative percent survival at 10 9 PFU/mL. Phage therapy significantly enhanced innate markers (nitroblue tetrazolium activity, lysozyme levels) and adaptive markers (total Ig, IgM), as well as glutathione S -transferase activity. Histological assessments revealed improved intestinal villus architecture, reduced kidney pathology, and enhanced tissue regeneration in groups treated with phages. Cytokine and gene expression analysis revealed a dose-dependent increase in interleukin (IL)-1β and IL-8 and in the antiviral MX gene, while TNF-α decreased at high doses. CXC2 (a chemokine) was also induced. Intramuscular administration consistently surpassed immersion methods, especially at intermediate doses, by providing more rapid and effective protection under the tested conditions. Conclusions: This research highlights the potential of bacteriophage therapy as an alternative to antibiotics in aquaculture. The findings emphasize the importance of optimizing phage dosage and delivery techniques to enhance therapeutic effectiveness while reducing environmental impact. Future studies should focus on field validation, phage formulation strategies, and the long-term effects of phage therapy on fish health and microbiomes.