The Role of Natural Antimicrobials in Reducing the Virulence of Vibrio parahaemolyticus TPD in Shrimp Gut and Hepatopancreas Primary Cells and in a Post-Larvae Challenge Trial.

Some Vibrio parahaemolyticus strains cause translucent post-larvae disease (Vp(TPD)), leading to significant economic losses in shrimp farming. We aimed to identify whether a mixture of natural antimicrobials, AuraAqua (Aq), can protect white-leg shrimp (Penaeus vannamei) against the lethal effects of Vp(TPD) and to understand its biological mode of action. Herein, we demonstrate that Aq, an antimicrobial mixture composed of a blend of organic acids, citrus, and olive extracts, suppressed Vp(TPD) virulence at sub-inhibitory concentrations and conferred robust protection to shrimp. The minimum inhibitory and bactericidal concentrations against the Vp(TPD) isolate were at 0.05% and 0.2%, respectively. At 0.05-0.1%, Aq reduced bacterial growth and downregulated six major virulence genes (vhvp-1, vhvp-2, vhvp-3, pirA(Vp), pirB(Vp), pirAB(Vp)), while leaving metabolic ldh expression unaltered. Parallel in vitro assays revealed diminished adhesion of Vp(TPD) to primary shrimp gut and hepatopancreas epithelial cells and a ≈50% reduction in infection-induced extracellular H(2)O(2), indicating an antioxidant effect. The treatment also triggered a time-dependent surge in extracellular alkaline phosphatase (ALP) activity, consistent with membrane permeabilization. In vivo, a challenge of post-larvae with 10(4) CFU/mL Vp(TPD) resulted in 91% mortality after 45 h; co-treatment with 0.1% and 0.2% Aq reduced mortality to ≈12% and ≈6%, respectively, while 1% Aq achieved ≈98% survival. The clinical protection test confirmed that 0.1% Aq preserved high survival across four pathogen inocula (10(1)-10(4) CFU/mL). Conclusively, Aq destabilized the pathogen and therefore transcriptionally silenced multiple virulence determinants, translating into significant in-pond protection for controlling Vp(TPD) for shrimp aquaculture.