Co-administration of recombinant Pichia pastoris-CXCL20a and immunostimulatory polysaccharides enhances resistance to Aeromonas hydrophila in grass carp (Ctenopharyngodon idella).

Aeromonas hydrophila poses a serious threat to aquaculture, necessitating the development of effective and sustainable control strategies. To this end, we evaluated the protective efficacy of recombinant Pichia pastoris-CXCL20a (Pp-C20a), administered alone or in combination with polysaccharides-β-glucan, chitosan (CS), and Astragalus polysaccharide (APS)-against A. hydrophila infection in grass carp. Results demonstrated that Pp-C20a alone improved survival (32%) compared with the control (20%) and empty vector control (22%) groups. Notably, co-administration with polysaccharides further enhanced protection, with the Pp-C20a + β-glucan group showing the highest survival (46%), followed by Pp-C20a + APS (40%) and Pp-C20a + CS (38%). Furthermore, serum immune assays showed that combination treatments increased total superoxide dismutase (SOD) activity, lysozyme activity, and complement C3 content, with the β-glucan group exhibiting the strongest enhancement. Moreover, RT-qPCR analysis revealed that dietary combinations upregulated immune-related genes (il-1β, tnf-α, ifn-γ2, igm) in major immune tissues (head kidney, spleen, and intestine). In addition, bacterial load analysis revealed that combination treatments reduced bacterial burdens in the head kidney, spleen, and hepatopancreas. Histopathological examination further demonstrated that co-administration alleviated tissue damage in the spleen and intestine. In conclusion, Pp-C20a confers effective protection against A. hydrophila, which is further enhanced by co-administration with polysaccharides, particularly β-glucan. This study provides a promising, sustainable, and antibiotic-free strategy for bacterial disease management in aquaculture.