The synergistic toxic effects of ammonia nitrogen and IHNV co-stress on the immune and intestinal health of rainbow trout.

Ammonia nitrogen can cause physiological damage in fish, but its combined effect with viral infection remains unclear. This study investigated how ammonia stress influences rainbow trout susceptibility to Infectious Hematopoietic Necrosis Virus (IHNV). Four experimental groups were designed: control (A: 0.86 mg/L ammonia), ammonia-stressed (B: 4 mg/L for 10 days), IHNV-infected (C: 0.86 mg/L ammonia), and combined stress (D: ammonia pre-exposure then IHNV). Liver transcriptome sequencing and multi-tissue RT-qPCR analysis revealed that individual stressors (B, C) up-regulated pro-inflammatory cytokines (IL-1β, TNF-α) in the head kidney and spleen. Conversely, combined stress (D) down-regulated IL-1β in liver, intestine, and spleen, indicating immune dysregulation. Interferon pathway genes (IFN-α, IRF7, Mx) were broadly suppressed, particularly in liver, impairing innate antiviral immunity. Apoptosis analysis showed combined stress reduced Bcl-2 expression and increased Bax/Bcl-2 ratio across tissues, systematically activating apoptosis. Adaptive immunity genes (IgM, CD4, TGF-β) showed tissue-specific alterations, predominantly suppressed in the intestine. KEGG enrichment confirmed synergistic pathogenicity in the intestine, where ammonia pre-activation exacerbates post-viral metabolic disturbances, inhibiting cellular functions and activating ferroptosis and apoptosis. These findings indicate ammonia stress compromises immune function, highlighting the need for monitoring ammonia levels during epidemics to reduce IHNV susceptibility. This provides a theoretical basis for ecological prevention of IHNV in rainbow trout farming.