Selection of Vibrio parahaemolyticus isolates as potential vaccine candidates in Litopenaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VpAHPND) remains a major constraint in the culture of Pacific white shrimp ( Litopenaeus vannamei ). This study aimed to identify and evaluate VpAHPND isolates as potential vaccine candidates based on pathogenicity, immunogenicity, and protective efficacy. Bacterial isolates collected from several regions in Indonesia and confirmed as AHPND-causing strains were characterized using biochemical and phylogenetic analyses. The isolates were evaluated through two experiments. The first experiment assessed pathogenicity by injecting shrimp with bacterial isolates and monitoring mortality, clinical signs, and immune responses. The second experiment evaluated immunogenicity and protective efficacy by measuring post-vaccination immune responses and survival following challenge with VpAHPND. Results showed that four isolates (TSK, BYW, IND, and STD) shared 99.9% biochemical similarity with VpAHPND and were phylogenetically related to global VpAHPND strains. Pathogenicity tests indicated that BYW and IND exhibited higher virulence, reflected by increased mortality, shorter mean time to death, and more severe clinical symptoms. These isolates carried pirA and pirB toxin genes, triggered stronger immune responses, and showed higher bacterial colonization. The presence of these toxin genes was associated with characteristic AHPND lesions in histopathological tissue sections, including epithelial sloughing and luminal debris accumulation. Furthermore, immunogenicity and protection assays indicated that BYW and IND significantly improved immune responses and survival compared to TSK and STD. Homologous challenges resulted in higher survival than heterologous ones, indicating isolate-specific protection. Overall, this study indicates that the BYW isolate represents the most promising experimental inactivated vaccine candidate among the isolates evaluated.