Optimization of immune priming interval using Imuno-FS Plus enhances the efficacy of oral formalin-killed Vibrio parahaemolyticus vaccine in Pacific white shrimp (Litopenaeus vannamei)

Acute hepatopancreatic necrosis disease (AHPND), caused by toxigenic Vibrio parahaemolyticus, remains a major constraint in Pacific white shrimp ( Litopenaeus vannamei ) aquaculture. This study evaluated whether the interval between adjuvant-induced immune priming and oral vaccination could be optimized to enhance the efficacy of a formalin-killed V. parahaemolyticus vaccine. Shrimp were fed a diet supplemented with Imuno-FS Plus powder (0.75 g kg⁻¹ feed) for seven days to prime the immune system, followed by an interval of 0, 2, 4, or 6 days before oral vaccination (10⁸ CFU shrimp⁻¹) for four days, and were subsequently challenged with virulent V. parahaemolyticus . Among the tested schemes, a four-day interval elicited the strongest immune activation, significantly increasing total hemocyte count, phenoloxidase activity, respiratory burst, and phagocytic activity (P < 0.05), and upregulating expression of the immune-related genes down syndrome cell adhesion molecule (DSCAM), prophenoloxidase (ProPO), Litopenaeus vannamei big penaedin (LvBigPEN), peroxinectin, (PE) and fibrinogen-related protein (FREP). Hepatopancreatic bacterial colonization declined to undetectable levels by day 7 post-challenge in shrimp given the four- and six-day intervals, both of which achieved survival statistically comparable to unchallenged controls, as confirmed by generalized linear modeling of the raw survival data. Relative percent survival was highest in the four-day interval group (72.9%), accompanied by reduced histopathological damage. These findings indicate that synchronizing the timing of immune priming and vaccination — rather than simply combining the two components — is a key determinant of oral vaccine efficacy against AHPND in Pacific white shrimp.