PirA & B toxins discovered in archived shrimp pathogenic Vibrio campbellii isolated long before EMS/AHPND outbreaks

Toxins Pir(vp)PA and Pir(vp)PB were first described in 2014 from Vibrio parahaemolyticus (VP) isolates discovered in 2013 as the cause of acute hepatopancreatic necrosis disease (AHPND) that emerged as a new disease of Chinese cultivated shrimp in 2009. It was subsequently reported from Thailand in 2012. By using Pir(vp)PA and Pir(vp)PB specific monoclonal antibodies (MAbs) together with reference VPAHPND isolates to screen archived bacterial cultures, we discovered 3 isolates of Vibrio (previously identified as V. harveyi) including VH 639, VH 1526 and VH Surat that were positive for both PirA and PirB toxins, even though the isolates were collected in Thailand over 10 years prior to reported Thai AHPND outbreaks. Western blot analysis revealed that the PirA/B toxins from these Vibrio isolates matched the molecular masses of the Pir(vp)PA/B toxins of previously described VPAHPND isolates. PCR analysis using VPAHPND detection methods that target the Pir(vp)A and Pir(v)(p)B toxin genes (AP4 and TUMSAT-Vp3) gave amplicons of the expected size from the 3 Vibrio isolates, and sequence analysis of the amplicons revealed 99% identity with the sequences of the Pir(vp)A and Pir(vp)B genes of previously described VPAHPND isolates. Pathogenicity tests performed by reverse gavage and immersion challenge tests demonstrated the pattern of AHPND pathology. Species confirmation of these 3 Vibrio isolates by multilocus sequence analysis (MLSA) of 16S rRNA, rpoD, rctB, and toxR genes revealed that they belong to V. campbellii Glade. The results revealed that V. campbellii isolates capable of producing PirA and PirB toxins were present in Thailand up to 7 years before AHPND was reported from China and up to a decade before it was reported from Thailand. The plasmid or chromosomal location of the PirA and PirB toxin genes in these archived V. campbellii isolates and their phylogenetic relationship to current VPAHPND isolates are being determined.