Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp
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 solub...
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| creator | Sirikharin, Ratchanok Taengchaiyaphum, Suparat Sanguanrut, Piyachat Chi, Thanh Duong Mavichak, Rapeepat Proespraiwong, Porranee Nuangsaeng, Bunlung Thitamadee, Siripong Flegel, Timothy W Sritunyalucksana, Kallaya |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0126987 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0126987</identifier><identifier>PMID: 26017673</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Ammonium ; Ammonium sulfate ; Animal Diseases ; Animals ; Bacteria ; Bacterial Toxins - genetics ; Bacterial Toxins - metabolism ; Bacterial Toxins - toxicity ; Bioassays ; Biocompatibility ; Biomedical materials ; Biotechnology ; Causes of ; Cell culture ; Cloning ; Cultures ; Diagnosis ; Dialysis ; Electrophoresis, Polyacrylamide Gel ; Epidemics ; Epithelial cells ; Escherichia coli - genetics ; Gangrene ; Gel electrophoresis ; Gene Expression Regulation, Bacterial ; Genetic aspects ; Genetic engineering ; Genomes ; Government agencies ; Health aspects ; Hepatopancreas ; Hepatopancreas - microbiology ; Hepatopancreas - pathology ; In vivo methods and tests ; Insects ; Laboratories ; Litopenaeus vannamei ; Mass spectrometry ; Mass spectroscopy ; Medical research ; Methods ; Molecular biology ; Mortality ; Necrosis ; Pathology ; Penaeidae - microbiology ; Photorhabdus ; Physiological aspects ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Proteins ; Recombinant ; Science ; Similarity ; Sodium lauryl sulfate ; Stomach ; Sulfate ; Sulfates ; ToxA gene ; Toxins ; Vibrio ; Vibrio Infections - microbiology ; Vibrio Infections - veterinary ; Vibrio parahaemolyticus ; Vibrio parahaemolyticus - genetics ; Vibrio parahaemolyticus - isolation & purification ; Vibrio parahaemolyticus - pathogenicity ; Water-borne diseases ; Waterborne diseases</subject><ispartof>PloS one, 2015-05, Vol.10 (5), p.e0126987-e0126987</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Sirikharin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Sirikharin et al 2015 Sirikharin et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-b8d231532e5146379b94c9fb66f5177b53ae5431ca614926251c4e19e04674f43</citedby><cites>FETCH-LOGICAL-c692t-b8d231532e5146379b94c9fb66f5177b53ae5431ca614926251c4e19e04674f43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446338/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446338/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,729,782,786,866,887,2104,2930,23873,27931,27932,53798,53800</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26017673$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Jiravanichpaisal, Pikul</contributor><creatorcontrib>Sirikharin, Ratchanok</creatorcontrib><creatorcontrib>Taengchaiyaphum, Suparat</creatorcontrib><creatorcontrib>Sanguanrut, Piyachat</creatorcontrib><creatorcontrib>Chi, Thanh Duong</creatorcontrib><creatorcontrib>Mavichak, Rapeepat</creatorcontrib><creatorcontrib>Proespraiwong, Porranee</creatorcontrib><creatorcontrib>Nuangsaeng, Bunlung</creatorcontrib><creatorcontrib>Thitamadee, Siripong</creatorcontrib><creatorcontrib>Flegel, Timothy W</creatorcontrib><creatorcontrib>Sritunyalucksana, Kallaya</creatorcontrib><title>Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Ammonium</subject><subject>Ammonium sulfate</subject><subject>Animal Diseases</subject><subject>Animals</subject><subject>Bacteria</subject><subject>Bacterial Toxins - genetics</subject><subject>Bacterial Toxins - metabolism</subject><subject>Bacterial Toxins - toxicity</subject><subject>Bioassays</subject><subject>Biocompatibility</subject><subject>Biomedical materials</subject><subject>Biotechnology</subject><subject>Causes of</subject><subject>Cell culture</subject><subject>Cloning</subject><subject>Cultures</subject><subject>Diagnosis</subject><subject>Dialysis</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Epidemics</subject><subject>Epithelial cells</subject><subject>Escherichia coli - genetics</subject><subject>Gangrene</subject><subject>Gel electrophoresis</subject><subject>Gene Expression Regulation, Bacterial</subject><subject>Genetic aspects</subject><subject>Genetic engineering</subject><subject>Genomes</subject><subject>Government agencies</subject><subject>Health aspects</subject><subject>Hepatopancreas</subject><subject>Hepatopancreas - 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microbiology</subject><subject>Vibrio Infections - veterinary</subject><subject>Vibrio parahaemolyticus</subject><subject>Vibrio parahaemolyticus - genetics</subject><subject>Vibrio parahaemolyticus - isolation & purification</subject><subject>Vibrio parahaemolyticus - pathogenicity</subject><subject>Water-borne diseases</subject><subject>Waterborne diseases</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk9tu1DAQhiMEoqXwBggsIaFWYpf4EGdzg7Rsga5UtVVbemtNvJONSzYOtoNanojHxHto1UW9QLlwNP7mn4NnkuQ1TYeU5_Tjte1dC82wsy0OU8pkMcqfJLu04GwgWcqfPvjfSV54f52mGR9J-TzZYTKlucz5bvJnUoMDHdCZ3xCMbQm0M3I2OSeHGFCvLKcV-WxacLcfyJlxg2PzA8mlvTGtJ5WzC3JlSmcs6aJQDbiwzW0wuvdk6m0DAT0JNQQygd4jGes-IDnCDoLtoNUOY1RNTlA7640nh8YjRG5_fHR2cnhATEsuamcW3cvkWQWNx1ebcy_5_vXL5eRocHz6bToZHw-0LFgYlKMZ4zTjDDMqJM-LshC6qEopq4zmeZlxwExwqkFSUTDJMqoF0gJTIXNRCb6XvF3rdo31atNkr6gc8SwfiSi9l0zXxMzCtepicrE1yoJRK4N1cwUuFtWgopTySvJyVqalKEHH7LAsKE1BImM0j1qfNtH6coEzjW1w0GyJbt-0plZz-0sJEavjoyiwvxFw9mePPqiF8RqbBlq0_SpvIXiWcRnRd_-gj1e3oeYQCzBtZWNcvRRVY8GZSDO20ho-QsVvhguj40RWJtq3HA62HCIT8CbM41B4Nb04_3_29Gqbff-ArRGaUMex65eD67dBsQaXg-YdVvdNpqlaLtRdN9RyodRmoaLbm4cPdO90t0H8L664Gts</recordid><startdate>20150527</startdate><enddate>20150527</enddate><creator>Sirikharin, Ratchanok</creator><creator>Taengchaiyaphum, Suparat</creator><creator>Sanguanrut, Piyachat</creator><creator>Chi, Thanh Duong</creator><creator>Mavichak, Rapeepat</creator><creator>Proespraiwong, Porranee</creator><creator>Nuangsaeng, Bunlung</creator><creator>Thitamadee, Siripong</creator><creator>Flegel, Timothy W</creator><creator>Sritunyalucksana, Kallaya</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20150527</creationdate><title>Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp</title><author>Sirikharin, Ratchanok ; Taengchaiyaphum, Suparat ; Sanguanrut, Piyachat ; Chi, Thanh Duong ; Mavichak, Rapeepat ; Proespraiwong, Porranee ; Nuangsaeng, Bunlung ; Thitamadee, Siripong ; Flegel, Timothy W ; Sritunyalucksana, Kallaya</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-b8d231532e5146379b94c9fb66f5177b53ae5431ca614926251c4e19e04674f43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Ammonium</topic><topic>Ammonium sulfate</topic><topic>Animal Diseases</topic><topic>Animals</topic><topic>Bacteria</topic><topic>Bacterial Toxins - genetics</topic><topic>Bacterial Toxins - metabolism</topic><topic>Bacterial Toxins - toxicity</topic><topic>Bioassays</topic><topic>Biocompatibility</topic><topic>Biomedical materials</topic><topic>Biotechnology</topic><topic>Causes of</topic><topic>Cell culture</topic><topic>Cloning</topic><topic>Cultures</topic><topic>Diagnosis</topic><topic>Dialysis</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Epidemics</topic><topic>Epithelial cells</topic><topic>Escherichia coli - genetics</topic><topic>Gangrene</topic><topic>Gel electrophoresis</topic><topic>Gene Expression Regulation, Bacterial</topic><topic>Genetic aspects</topic><topic>Genetic engineering</topic><topic>Genomes</topic><topic>Government agencies</topic><topic>Health aspects</topic><topic>Hepatopancreas</topic><topic>Hepatopancreas - microbiology</topic><topic>Hepatopancreas - pathology</topic><topic>In vivo methods and tests</topic><topic>Insects</topic><topic>Laboratories</topic><topic>Litopenaeus vannamei</topic><topic>Mass spectrometry</topic><topic>Mass spectroscopy</topic><topic>Medical research</topic><topic>Methods</topic><topic>Molecular biology</topic><topic>Mortality</topic><topic>Necrosis</topic><topic>Pathology</topic><topic>Penaeidae - microbiology</topic><topic>Photorhabdus</topic><topic>Physiological aspects</topic><topic>Polymerase chain reaction</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Proteins</topic><topic>Recombinant</topic><topic>Science</topic><topic>Similarity</topic><topic>Sodium lauryl sulfate</topic><topic>Stomach</topic><topic>Sulfate</topic><topic>Sulfates</topic><topic>ToxA gene</topic><topic>Toxins</topic><topic>Vibrio</topic><topic>Vibrio Infections - microbiology</topic><topic>Vibrio Infections - veterinary</topic><topic>Vibrio parahaemolyticus</topic><topic>Vibrio parahaemolyticus - genetics</topic><topic>Vibrio parahaemolyticus - isolation & purification</topic><topic>Vibrio parahaemolyticus - pathogenicity</topic><topic>Water-borne diseases</topic><topic>Waterborne diseases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sirikharin, Ratchanok</creatorcontrib><creatorcontrib>Taengchaiyaphum, Suparat</creatorcontrib><creatorcontrib>Sanguanrut, Piyachat</creatorcontrib><creatorcontrib>Chi, Thanh Duong</creatorcontrib><creatorcontrib>Mavichak, Rapeepat</creatorcontrib><creatorcontrib>Proespraiwong, Porranee</creatorcontrib><creatorcontrib>Nuangsaeng, Bunlung</creatorcontrib><creatorcontrib>Thitamadee, Siripong</creatorcontrib><creatorcontrib>Flegel, Timothy W</creatorcontrib><creatorcontrib>Sritunyalucksana, Kallaya</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Access via ProQuest (Open Access)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sirikharin, Ratchanok</au><au>Taengchaiyaphum, Suparat</au><au>Sanguanrut, Piyachat</au><au>Chi, Thanh Duong</au><au>Mavichak, Rapeepat</au><au>Proespraiwong, Porranee</au><au>Nuangsaeng, Bunlung</au><au>Thitamadee, Siripong</au><au>Flegel, Timothy W</au><au>Sritunyalucksana, Kallaya</au><au>Jiravanichpaisal, Pikul</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-05-27</date><risdate>2015</risdate><volume>10</volume><issue>5</issue><spage>e0126987</spage><epage>e0126987</epage><pages>e0126987-e0126987</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26017673</pmid><doi>10.1371/journal.pone.0126987</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2015-05, Vol.10 (5), p.e0126987-e0126987 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1683578415 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS) Journals Open Access; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Ammonium Ammonium sulfate Animal Diseases Animals Bacteria Bacterial Toxins - genetics Bacterial Toxins - metabolism Bacterial Toxins - toxicity Bioassays Biocompatibility Biomedical materials Biotechnology Causes of Cell culture Cloning Cultures Diagnosis Dialysis Electrophoresis, Polyacrylamide Gel Epidemics Epithelial cells Escherichia coli - genetics Gangrene Gel electrophoresis Gene Expression Regulation, Bacterial Genetic aspects Genetic engineering Genomes Government agencies Health aspects Hepatopancreas Hepatopancreas - microbiology Hepatopancreas - pathology In vivo methods and tests Insects Laboratories Litopenaeus vannamei Mass spectrometry Mass spectroscopy Medical research Methods Molecular biology Mortality Necrosis Pathology Penaeidae - microbiology Photorhabdus Physiological aspects Polymerase chain reaction Polymerase Chain Reaction - methods Proteins Recombinant Science Similarity Sodium lauryl sulfate Stomach Sulfate Sulfates ToxA gene Toxins Vibrio Vibrio Infections - microbiology Vibrio Infections - veterinary Vibrio parahaemolyticus Vibrio parahaemolyticus - genetics Vibrio parahaemolyticus - isolation & purification Vibrio parahaemolyticus - pathogenicity Water-borne diseases Waterborne diseases |
| title | Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp |
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