The effect of recombinant ToxA and ToxB administration on shrimp.

<p>(A) Graph of cumulative mortality up to 48 h from single and mixed toxins at various concentrations. The graph contains results from two experiments, each with a BSA control (10 ug/g shrimp) (SD = 0) and with co-administration of 10 ug/g each of ToxA+ToxB (SD bars). The other treatments were not duplicated (no SD bars). (B) Graph of mortality at 24 h post administration versus mixed toxin concentration, yielding a linear regression line and rough LD_<b>50</b> for the mixed toxins of approximately 6 ug/g of each.</p

Bacterial expression of ToxA and ToxB.

<p>(A) ToxA expressed with a 6-His tag and purified by Ni-NTA affinity chromatography. Lane 1: Bacterial cell lysate from a non-induced bacterial culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted protein from the Ni-NTA column. The deduced molecular weight for ToxA-His was 12.7 kDa. (B) ToxB was expressed as a GST-fusion protein. Lane 1: Bacterial cell lysate from a non-induced culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted fraction from Sepharose 4B beads; Lanes 4&5: Fraction eluted from Sepahrose 4B after thrombin-cut. The estimated molecular weights for GST-ToxB and ToxB were approximately 76 and 50 kDa, respectively.</p

Examples of histopathological sections of hepatopancreatic (HP) tissue from moribund shrimp treated by reverse gavage with 60% AS fractions from <i>V</i>. <i>parahaemolyticus</i> isolates.

<p>(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.</p

Agarose gel of PCR amplicons from VP_AHPND using the AP3 method.

<p>Lane M; DNA marker, Lane N: negative control; Lanes 1–3: Positive amplicons (333 bp) from 3 isolates of VP_<b>AHPND</b> bacteria; Lanes 4–10: No amplicons from 8 non-AHPND bacteria; Lane P: positive control (333 bp).</p

SDS-PAGE analysis of 60% AS fractions from broth of non-AHPND isolate S02 (Lane 2), and VP_AHPND isolates 5HP (Lane 3) and CN (Lane 4).

<p>Lane 1: 60% AS from culture broth without bacteria. There are 2 major bands (ToxA at ~16kDa and ToxB at ~50 kDa) present in lanes for the VP_<b>AHPND</b> isolates 5HP and CN, but absent or at low level in non-AHPND isolate S02.</p

Examples of HP histology of moribund shrimp administered single or combined doses of ToxA and ToxB by reverse gavage.

<p>(A) Tissue sections from a BSA (10/20 μg/g) negative control shrimp (tubule longitudinal sections) and (B) a ToxA (5 μg/g) treated shrimp (tubule cross sections) showing only normal histology with morphology and cell types as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126987#pone.0126987.g001" target="_blank">Fig 1A to 1C</a>. (C) ToxB only (5 μg/g) and (D) ToxA+ToxB (2 μg/g each) showing mostly normal histology, but with some thin HP tubule epithelia (black arrows) when compared to epithelia of normal thickness (grey arrows). (E) ToxA+ToxB (5 g/g each) showing enlarged HP tubules (compared to A-C) with collapsed epithelia (black arrows) but no cell sloughing. (F) ToxA+ToxB (10 μg/g each) showing massive sloughing (black arrows) and dissolution of HP tubule epithelial cells (i.e., severe AHPND histopathology).</p