Dietary sanguinarine enhances disease resistance to Aeromonas dhakensis in largemouth (Micropterus salmoides).

Herbal medicines offer a natural and safe alternative to antibiotics, functioning as immunostimulants to enhance immune responses, growth performance, and disease resistance in aquaculture species. This study investigated the efficacy of dietary sanguinarine (SA), a quaternary benzophenanthridine alkaloid, in augmenting immunity and protecting largemouth bass (Micropterus salmoides) against Aeromonas dhakensis infection. In vitro assays determined the potent antibacterial activity of SA against A. dhakensis, with a minimum inhibitory concentration (MIC) of 5 μg/mL and a minimum bactericidal concentration (MBC) of 10 μg/mL Time-kill curve analysis further confirmed SA's concentration- and time-dependent bactericidal effects. For the in vivo study, fish were fed diets supplemented with SA at three concentrations: 10 mg/kg (Diet A, low), 30 mg/kg (Diet B, medium), and 50 mg/kg (Diet C, high) for 7 days prior to intraperitoneal challenge with A. dhakensis (1 × 10⁷ CFU/mL). Serum analysis revealed significant enhancements (P < 0.05) in innate immune parameters in SA-fed groups compared with the control group (0 mg/kg SA). Specifically, fish receiving Diets B and C exhibited significantly elevated glutathione (GSH) levels and superoxide dismutase (SOD) activity. Serum acid phosphatase (ACP) and alkaline phosphatase (AKP) activities were also significantly increased, particularly in the Diet C group. Quantitative RT-PCR analysis of spleen tissue demonstrated that dietary SA significantly upregulated the expression of key immune-related genes (TGF-β, IL-10, MYD88, TNF-α, TLR2, IgM) in a dose-dependent manner. Furthermore, dietary SA supplementation significantly reduced bacterial load within the liver tissue post-challenge. Crucially, challenge tests demonstrated a pronounced dose-dependent increase in survival: survival rates were 55 %, 75 %, and 90 % in the Diet A, Diet B, and Diet C groups as compared with the control group (45 %). Collectively, these findings demonstrate that SA is an effective dietary immunostimulant for largemouth bass. It significantly enhances both innate and adaptive immune responses, improves antioxidant capacity, and confers substantial dose-dependent protection against A. dhakensis infection.