title: Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.
abstract: Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.
authors.affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Ratchanok
authors.initials: R
authors.lastname: Sirikharin
authors.affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Suparat
authors.initials: S
authors.lastname: Taengchaiyaphum
authors.affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Piyachat
authors.initials: P
authors.lastname: Sanguanrut
authors.affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Thanh Duong
authors.initials: TD
authors.lastname: Chi
authors.affiliation: Aquatic Animal Health Research Center, Charoen Pokphand Co. Ltd., Rama 2 Rd., Km 41.5, T. Bangtorat, A. Muang Samutsakorn, Samutsakorn 74000, Thailand.
authors.forename: Rapeepat
authors.initials: R
authors.lastname: Mavichak
authors.affiliation: Aquatic Animal Health Research Center, Charoen Pokphand Co. Ltd., Rama 2 Rd., Km 41.5, T. Bangtorat, A. Muang Samutsakorn, Samutsakorn 74000, Thailand.
authors.forename: Porranee
authors.initials: P
authors.lastname: Proespraiwong
authors.affiliation: Broodstock Multiplication Center (BMC), Faculty of Marine Technology, Burapha University Chanthaburi Campus, Chanthaburi, 22170, Thailand.
authors.forename: Bunlung
authors.initials: B
authors.lastname: Nuangsaeng
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Siripong
authors.initials: S
authors.lastname: Thitamadee
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Flegel
authors.affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Kallaya
authors.initials: K
authors.lastname: Sritunyalucksana
mesh_terms: D000820:Animal Diseases; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D043143:Hepatopancreas / Q000382:microbiology / Q000473:pathology; D033561:Penaeidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014735:Vibrio Infections / Q000382:microbiology / Q000662:veterinary*; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*
chemical_list: D001427:Bacterial Toxins