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.
Highlights
Bacterial infections cause massive mortalities in intensive aquaculture, leading to serious economic losses [1, 2]
carboxylmethyl chitosan (CMCS)-20H nanoparticles form through the adsorption of negatively charged CMCS and positively charged gcIFN-20H
Raman spectroscopy analysis indicated that numerous specific peaks were observed in the CMCS group, and no peak was observed in the gcIFN-20H group
Summary
Bacterial infections cause massive mortalities in intensive aquaculture, leading to serious economic losses [1, 2]. The bacterial diseases of aquaculture animals are often prevented and treated by chemotherapeutants. This method may result in many problems, such as the production of drugresistant bacteria, decrease of fish immune functions, and drug residues which could further affect human health [3, 4]. Bacterial infections can be prevented by vaccination [5, 6]. Commercial vaccines only prevent specific pathogens, and numerous vaccines increase the costs [7, 8]. Facing the severe bacterial diseases in aquaculture, it is urgent to develop ideal antimicrobial agents
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