Effects of Bacteriophage on Antibacterial Properties, Nonspecific Immune Responses, and Gut Microbiota in Litopenaeus vannamei Post Vibrio parahaemolyticus Infection

Abstract

Effective immune regulation and balanced gut microbiota play important roles in preventing pathogen infections in Litopenaeus vannamei farming. Bacteriophages are a promising candidate in pathogen control for their specific antibacterial properties. While previous studies focused on the direct antibacterial effects of phages, their effects on nonspecific immune responses and gut microbiota after infection remains to be less explored. In this study, a lytic Vibrio parahaemolyticus phage was isolated from wastewater with a broad host range (66.7% lytic efficiency), low multiplicity of infection (MOI; 0.1), and high environmental tolerance (pH: 3–11; temperature: 4–60°C). Whole genome analysis revealed a 93,814 bp double-stranded linear DNA molecule with 45.1% GC. Both the in vitro cocultivation (24 h) and in vivo shrimp cultivation trails (7 days) demonstrated that phage could effectively reduce the quantities of Vibrio (>99%). The in vivo phage fed shrimp exhibited elevated levels of nonspecific immune-related enzymes like alkaline phosphatase (AKP), catalase (CAT), superoxide dismutase (SOD), phenoloxidase (PO), and lysozyme (LZM) and upregulated immune-related gene expression including those of antimicrobial peptides, pattern recognition receptors (PRRs), and pattern recognition proteins. Additionally, phage treatment improved the diversity of the gut microbiota (Shannon-10 index) after Vibrio infection, indicating restored microbial balance in shrimp. These results suggest that phage therapy promotes nonspecific immune responses and repair intestinal dysbacteriosis in shrimp after Vibrio infection, elucidating a promising strategy to treat pathogenic Vibrio in shrimp aquaculture.