Liposome-encapsulated cinnamaldehyde enhances zebrafish (Danio rerio) immunity and survival when challenged with Vibrio vulnificus and Streptococcus agalactiae

Cinnamaldehyde, which is extracted from cinnamon, is a natural compound with activity against bacteria and a modulatory immune function. However, the antibacterial activity and immunostimulation of cinnamaldehyde in fish has not been well investigated due to the compoundʹs poor water solubility. Thus, liposome-encapsulated cinnamaldehyde (LEC) was used to evaluate the effects of cinnamaldehyde on in vitro antibacterial activity against aquatic pathogens and in vivo immunity and protection parameters against Vibrio vulnificus and Streptococcus agalactiae. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as well as bactericidal agar plate assay results demonstrated the effective bacteriostatic and bactericidal potency of LEC against Aeromonas hydrophila, V. vulnificus, and S. agalactiae, as well as the antibiotic-resistant Vibrio parahaemolyticus and Vibrio alginolyticus. Bacteria challenge test results demonstrated that LEC significantly enhances the survival rate and inhibits bacterial growth in zebrafish infected with A. hydrophila, V. vulnificus, and S. agalactiae. A gene expression study using a real-time PCR showed that LEC immersion-treated zebrafish had increased endogenous interleukin (IL)-1β, IL-6, IL-15, IL-21, tumor necrosis factor (TNF)-α, and interferon (INF)-γ expression in vivo. After the zebrafish were infected with V. vulnificus or S. agalactiae, the LEC immersion treatment suppressed the expression of the inflammatory cytokines IL-1β, IL-6, IL-15, NF-κb, and TNF-α and induced IL-10 and C3b expression. These findings demonstrate that cinnamaldehyde exhibits antimicrobial activity against aquatic pathogens, even antibiotic-resistant bacterial strains and immune-stimulating effects to protect the hostʹs defenses against pathogen infection in bacteria-infected zebrafish. These results suggest that LEC could be used as an antimicrobial agent and immunostimulant to protect bacteria-infected fish in aquaculture.