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

• Published in: Aquaculture Vol. 497; pp. 494 - 502
• Main Authors: Wangman, Pradit, Longyant, Siwaporn, Taengchaiyaphum, Suparat, Senapin, Saengchan, Sithigorngul, Paisarn, Chaivisuthangkura, Parin
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-12-2018
• Subjects: Acute hepatopancreatic necrosis disease (AHPND); Early mortality syndrome (EMS); Monoclonal antibody (MAb); PirA and PirB toxins; Vibiro parahaemolyticus; Vibrio campbellii; Acute hepatopancreatic necrosis disease (AHPND); Vibrio campbellii; Monoclonal antibody (MAb); Early mortality syndrome (EMS); Vibiro parahaemolyticus; PirA and PirB toxins
• ISSN: 0044-8486; 1873-5622
• DOI: 10.1016/j.aquaculture.2018.08.025

Toxins PirvpA and PirvpB 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 PirvpA and PirvpB 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 PirvpA/B toxins of previously described VPAHPND isolates. PCR analysis using VPAHPND detection methods that target the PirvpA and PirvpB 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 PirvpA and PirvpB 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 clade. 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.
•Three archived isolates of V. campbellii were positive for PirvpA and PirvpB toxins by using specific monoclonal antibodies.•Those three virulent isolates were collected in Thailand 5–10 years prior to reported Thai AHPND outbreaks.•The PirvpA and PirvpB toxin genes from V. campbellii shared 99% identity with that of previously described VPAHPND isolates.