Abstract
Massive mortalities caused by bacterial infections in intensive aquaculture result in serious economic losses. In this study, a novel antimicrobial peptide gcIFN-20H was efficiently expressed in Pichia pastoris (GS115) and loaded on carboxylmethyl chitosan (CMCS) to prepare CMCS-20H nanoparticles. Through physical characterization assays (TEM, DLS, BCA, and Raman) and biological activity tests (antimicrobial activity and cytotoxicity), CMCS-20H nanopeptide was verified to be spherical nanoparticles with sustained release, antimicrobial activity, and negligible toxicity. CMCS-20H nanoparticles are more resistant to intestinal degradation than unloaded gcIFN-20H by indirect immunofluorescence assay. Oral administration was then carried out for 42 days. Complement C3 content, lysozyme, and total superoxide dismutase activities are highest in CMCS-20H group by serum biochemistry index assays. After challenge with Aeromonas hydrophila, the survival rate in CMCS-20H group is highest (46%), which is 64% higher than the control group (28%). Meanwhile, the tissue bacterial loads (intestine, spleen, head kidney, trunk kidney, hepatopancreas, muscle, and blood) in the CMCS-20H group are significantly lower than other groups. By PAS staining analysis, the number of intestinal villi goblet cells and the thickness of mucin in the CMCS-20H group obviously increased. CMCS-20H effectively enhances mRNA expressions of some important immune genes (IL-1β, IL-6, TNF-α, IL-2, IFN-γ2, and IgM). The minimal tissue lesions (Intestine, spleen, and trunk kidney) were seen in the CMCS-20H group by histopathological examination. 16S rRNA sequencing showed that oral CMCS-20H maintains the intestinal microbiome homeostasis in bacterial infection. The results indicate that the novel nanopeptide CMCS-20H as the immunopotentiator can remarkably boost fish immunity and precautionary effect by oral administration and address the theoretical mechanisms and insights into the promising application prospect in aquaculture.