Effects of dietary humic acid supplementation on survival, immune response, and gut microbiota in Nile tilapia (Oreochromis niloticus) challenged with Lactococcus garvieae

Aquaculture is a cornerstone in protein production with the cultivation of Nile tilapia (Oreochromis niloticus), but it has faced challenges from pathogens such as the emerging bacterium Lactococcus garvieae. Humic acid (HA) was used as a prophylactic measure to evaluate its effect on the intestinal microbiota, immune system, and survival in tilapia challenged with L. garvieae under laboratory conditions. Four concentrations of HA were tested in feed over 83 days. The intestinal microbiota was analyzed in treatment IV (1.0 g/kg of feed) and control through 16S rRNA gene sequencing (V3). Liver and gill tissue were collected for immune system gene expression analysis (COX-2, TGF, IL-1B, and TNF-α) through real-time qPCR methods. On day 71, the fish were inoculated by immersion with L. garvieae (CL₅₀ = 8.5 × 10⁵ CFU/mL). Gene expression in the liver (COX-2 and TNF-α) and gills (COX-2, TGF-β, and IL-1β) increased significantly in treatment IV. Cetobacterium was among the most abundant genera in the tilapia gut. Fusobacteriota (phylum) and the genera Aeromonas, Rhodobacter, Gemobacter, and Ideonella were enriched in HA, whereas the phylum Patescibacteria and genera Pelomonas, Nitrobacter, and Clostridioides decreased significantly. Dietary HA supplementation significantly altered gut microbiota diversity and the interaction network core. Humic acid supplementation significantly improved tilapia survival against L. garvieae by enhancing immune gene expression and modulating gut microbiota—enriching Fusobacteriota and beneficial genera (Rhodobacter, Ideonella) while reducing pathogens (Pelomonas, Clostridioides). These findings support HA as a prophylactic alternative against lactococcosis in Nile tilapia (O. niloticus) farming.