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Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Sirikharin R, Taengchaiyaphum S, Sanguanrut P, Chi TD, Mavichak R, Proespraiwong P, Nuangsaeng B, Thitamadee S, Flegel TW, Sritunyalucksana K - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Examples of histopathological sections of hepatopancreatic (HP) tissue from moribund shrimp treated by reverse gavage with 60% AS fractions from V. parahaemolyticus isolates.(A) A longitudinal section of HP tissue from pre-challenged shrimp showing normal histology with the lumens enclosed by epithelial cell layers comprised of non-vacuolated deeply basophilic (purple stained), embryonic cells (E-cells) at the distal end of the tubule that progress in the proximal direction into a mixture of B-cells with large, single vacuoles, R-cells with multiple vacuoles and F-cells that are non-vacuolated and deeply basophilic. (B) A section of normal HP tissue from the PBS negative control shrimp showing normal tubules mostly in longitudinal section except for a few tubules at the outer (distal) portion of the HP where they are cut in cross-section. The tubule lumens are surrounded by epithelial cells similar to those in (A). (C) Tangential section of HP tissue from shrimp treated with non-AHPND S02 preparation and showing normal HP and showing the same cell types as in (A) and (B). (D) Section HP tubules (mostly in cross-section) from shrimp treated with 5HP preparation and showing AHPND pathology characterized by absence of normal epithelia containing B-cells, R-cells and F-cells as seen in (A) to (C) and instead by massive sloughing of epithelial cells into tubule lumens in the absence of bacteria. The inset shows a magnification of the sloughed epithelial cells in a tubule lumen. (E) Section of HP tubules (cross-section) from shrimp treated with CN preparation and showing AHPND pathology similar to that in (D) but more severe in that all of the tubule lumens are completely filled with sloughed cells except for two tubules cut in cross-section through the E-cell region.
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pone.0126987.g001: Examples of histopathological sections of hepatopancreatic (HP) tissue from moribund shrimp treated by reverse gavage with 60% AS fractions from V. parahaemolyticus isolates.(A) A longitudinal section of HP tissue from pre-challenged shrimp showing normal histology with the lumens enclosed by epithelial cell layers comprised of non-vacuolated deeply basophilic (purple stained), embryonic cells (E-cells) at the distal end of the tubule that progress in the proximal direction into a mixture of B-cells with large, single vacuoles, R-cells with multiple vacuoles and F-cells that are non-vacuolated and deeply basophilic. (B) A section of normal HP tissue from the PBS negative control shrimp showing normal tubules mostly in longitudinal section except for a few tubules at the outer (distal) portion of the HP where they are cut in cross-section. The tubule lumens are surrounded by epithelial cells similar to those in (A). (C) Tangential section of HP tissue from shrimp treated with non-AHPND S02 preparation and showing normal HP and showing the same cell types as in (A) and (B). (D) Section HP tubules (mostly in cross-section) from shrimp treated with 5HP preparation and showing AHPND pathology characterized by absence of normal epithelia containing B-cells, R-cells and F-cells as seen in (A) to (C) and instead by massive sloughing of epithelial cells into tubule lumens in the absence of bacteria. The inset shows a magnification of the sloughed epithelial cells in a tubule lumen. (E) Section of HP tubules (cross-section) from shrimp treated with CN preparation and showing AHPND pathology similar to that in (D) but more severe in that all of the tubule lumens are completely filled with sloughed cells except for two tubules cut in cross-section through the E-cell region.

Mentions: A preliminary study confirmed that shrimp treated by reverse gavage with 12-hour, cell-free, crude culture broth of VPAHPND bacteria exhibited AHPND pathology, as previously reported [1]. We thus hypothesized that if the toxic VPAHPND substances were proteins, they could be fractionally precipitated by stepwise addition of ammonium sulfate (AS) to the cell-free culture broth. The 40%, 60% and 80% AS-precipitate fractions from the VPAHPND isolates 5HP and CN and the non-AHPND VPS02 were subjected to rehydration and dialysis to remove excess AS. Preliminary electrophoresis tests (not shown) revealed that no suspected toxin bands were present in the 40% AS fraction so it was not used in bioassays. When fractions from 60% and 80% were used in reverse gavage bioassays (1 μg total protein each) (Table 1), mortality with the 60% AS fraction for 5HP and CN, respectively, was 40% and 70% while there was no shrimp mortality in the shrimp group treated with PBS or with the 60% AS fraction from VPS02. The moribund shrimp showed typical signs of AHPND pathology (i.e., massive sloughing of HP tubule epithelial cells) (Fig 1). Mortality also occurred with the 80% AS fractions from 5HP and CN, but histological examination revealed no AHPND pathology in shrimp treated with them (not shown). The latter results indicated that other toxins were present in the broth and that these might exacerbate shrimp mortality while not causing HP cell sloughing. Since there was also high mortality without AHPND pathology in the 80% AS fraction from VPS02, it is possible that some protein(s) present at low levels in the broth were concentrated to a lethal level in the 80% AS precipitate. Differences in protein bands in the 80% AS fractions from the 3 isolates were not studied further because our focus was on the AHPND toxins.


Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Sirikharin R, Taengchaiyaphum S, Sanguanrut P, Chi TD, Mavichak R, Proespraiwong P, Nuangsaeng B, Thitamadee S, Flegel TW, Sritunyalucksana K - PLoS ONE (2015)

Examples of histopathological sections of hepatopancreatic (HP) tissue from moribund shrimp treated by reverse gavage with 60% AS fractions from V. parahaemolyticus isolates.(A) A longitudinal section of HP tissue from pre-challenged shrimp showing normal histology with the lumens enclosed by epithelial cell layers comprised of non-vacuolated deeply basophilic (purple stained), embryonic cells (E-cells) at the distal end of the tubule that progress in the proximal direction into a mixture of B-cells with large, single vacuoles, R-cells with multiple vacuoles and F-cells that are non-vacuolated and deeply basophilic. (B) A section of normal HP tissue from the PBS negative control shrimp showing normal tubules mostly in longitudinal section except for a few tubules at the outer (distal) portion of the HP where they are cut in cross-section. The tubule lumens are surrounded by epithelial cells similar to those in (A). (C) Tangential section of HP tissue from shrimp treated with non-AHPND S02 preparation and showing normal HP and showing the same cell types as in (A) and (B). (D) Section HP tubules (mostly in cross-section) from shrimp treated with 5HP preparation and showing AHPND pathology characterized by absence of normal epithelia containing B-cells, R-cells and F-cells as seen in (A) to (C) and instead by massive sloughing of epithelial cells into tubule lumens in the absence of bacteria. The inset shows a magnification of the sloughed epithelial cells in a tubule lumen. (E) Section of HP tubules (cross-section) from shrimp treated with CN preparation and showing AHPND pathology similar to that in (D) but more severe in that all of the tubule lumens are completely filled with sloughed cells except for two tubules cut in cross-section through the E-cell region.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4446338&req=5

pone.0126987.g001: Examples of histopathological sections of hepatopancreatic (HP) tissue from moribund shrimp treated by reverse gavage with 60% AS fractions from V. parahaemolyticus isolates.(A) A longitudinal section of HP tissue from pre-challenged shrimp showing normal histology with the lumens enclosed by epithelial cell layers comprised of non-vacuolated deeply basophilic (purple stained), embryonic cells (E-cells) at the distal end of the tubule that progress in the proximal direction into a mixture of B-cells with large, single vacuoles, R-cells with multiple vacuoles and F-cells that are non-vacuolated and deeply basophilic. (B) A section of normal HP tissue from the PBS negative control shrimp showing normal tubules mostly in longitudinal section except for a few tubules at the outer (distal) portion of the HP where they are cut in cross-section. The tubule lumens are surrounded by epithelial cells similar to those in (A). (C) Tangential section of HP tissue from shrimp treated with non-AHPND S02 preparation and showing normal HP and showing the same cell types as in (A) and (B). (D) Section HP tubules (mostly in cross-section) from shrimp treated with 5HP preparation and showing AHPND pathology characterized by absence of normal epithelia containing B-cells, R-cells and F-cells as seen in (A) to (C) and instead by massive sloughing of epithelial cells into tubule lumens in the absence of bacteria. The inset shows a magnification of the sloughed epithelial cells in a tubule lumen. (E) Section of HP tubules (cross-section) from shrimp treated with CN preparation and showing AHPND pathology similar to that in (D) but more severe in that all of the tubule lumens are completely filled with sloughed cells except for two tubules cut in cross-section through the E-cell region.
Mentions: A preliminary study confirmed that shrimp treated by reverse gavage with 12-hour, cell-free, crude culture broth of VPAHPND bacteria exhibited AHPND pathology, as previously reported [1]. We thus hypothesized that if the toxic VPAHPND substances were proteins, they could be fractionally precipitated by stepwise addition of ammonium sulfate (AS) to the cell-free culture broth. The 40%, 60% and 80% AS-precipitate fractions from the VPAHPND isolates 5HP and CN and the non-AHPND VPS02 were subjected to rehydration and dialysis to remove excess AS. Preliminary electrophoresis tests (not shown) revealed that no suspected toxin bands were present in the 40% AS fraction so it was not used in bioassays. When fractions from 60% and 80% were used in reverse gavage bioassays (1 μg total protein each) (Table 1), mortality with the 60% AS fraction for 5HP and CN, respectively, was 40% and 70% while there was no shrimp mortality in the shrimp group treated with PBS or with the 60% AS fraction from VPS02. The moribund shrimp showed typical signs of AHPND pathology (i.e., massive sloughing of HP tubule epithelial cells) (Fig 1). Mortality also occurred with the 80% AS fractions from 5HP and CN, but histological examination revealed no AHPND pathology in shrimp treated with them (not shown). The latter results indicated that other toxins were present in the broth and that these might exacerbate shrimp mortality while not causing HP cell sloughing. Since there was also high mortality without AHPND pathology in the 80% AS fraction from VPS02, it is possible that some protein(s) present at low levels in the broth were concentrated to a lethal level in the 80% AS precipitate. Differences in protein bands in the 80% AS fractions from the 3 isolates were not studied further because our focus was on the AHPND toxins.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus