Characterization and pathogenicity of acute hepatopancreatic necrosis disease natural mutants, pirAB_vp (‐) V. parahaemolyticus, and pirAB_vp (+) V. campbellii strains

Two Vibrio parahaemolyticus virulence genes, pirA_vp and pirB_vp, are known to encode a binary photorhabdus insect-related (Pir) toxin that causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. These genes are flanked with repeats of a mobile element (insertion sequence) in a large plasmid. This insertion sequence is closely (92%) related to the known insertion sequence ISVal1. The pirAB_vp genes and the flanking ISVal1 forms a 5535-bp composite transposon, designated Tn6264. There are pirAB_vp gene deletions in some strains of V. parahaemolyticus. During 2013–2016, we found 2 types of pirAB_vp deletion mutants from AHPND-affected farms. The type I mutants included 3 strains with deletions (4.4-kb or 6.0-kb) of entire pirAB_vp genes and the downstream ISVal1, and these mutants were named pirAB_vp(‐). The type II mutants included 3 strains with smaller deletions (1.5-kb or 1.6-kb) including a pirA_vp gene and a partial pirB_vp gene, and were named pirA_vp(‐). In laboratory bioassays, these were not pathogenic to shrimp confirming that both pirA_vp and pirB_vp are required for AHPND pathogenicity. During 2016, we also isolated 4 V. campbellii strains carrying pirAB_vp genes from diseased shrimp. These V. campbellii stains were found, through laboratory bioassays and histological evaluation, to cause AHPND.