Functional dietary additives enhance host defense and modulate virulence associated-gene transcript abundance in Aeromonas hydrophila-challenged Nile tilapia

Aeromonas hydrophila is a major opportunistic pathogen responsible for disease outbreaks and economic losses in Nile tilapia aquaculture. This study comparatively evaluates diets supplemented with formic acid (FA), black seed oil (BSO), and α-lipoic acid (α-LA), relative to florfenicol (FFL) as an antibiotic reference treatment, on antioxidant defense, immune response, virulence- gene transcript abundance, and resistance to A. hydrophila infection in Nile tilapia. A. hydrophila strain isolated from naturally diseased tilapia was molecularly identified and deposited in GenBank under accession number PV866787. Fish were assigned to five dietary groups and fed a basal diet or diets supplemented with FFL (15 mg/kg body weight), FA (10 mL/kg diet), BSO (20 mL/kg diet), or α-LA (600 mg/kg diet) for a short-term feeding period lasting 15 days and a long-term feeding period lasting 30 days before intraperitoneal challenge with A. hydrophila STN-1 at 1 × 10 5 CFU/mL. Compared with the challenged basal-diet control, dietary FA, BSO, and α-LA significantly increased ( p < 0.05) catalase, superoxide dismutase, reduced glutathione, total antioxidant capacity, total protein, immunoglobulin M, and lysozyme activity, while reducing malondialdehyde levels and improving liver and kidney function indicators ( p < 0.05). At the molecular level, supplemented diets were associated with significantly reduced transcript abundance of virulence-associated genes hemolysin ( hlyA ) and aerolysin ( aerA ) in recovered A. hydrophila , together with reduced post-challenge mortality, particularly after 30 days of feeding. FA showed the strongest in vitro antibacterial activity, whereas α-LA exerted marked antioxidant effects, and BSO improved redox balance and immune responsiveness. Overall, these findings support FA, BSO, and α-LA as promising functional feed additives for enhancing host defense and reducing reliance on antibiotic-based disease control in tilapia aquaculture.