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.

Quorum Sensing Attenuates Virulence in Sodalis praecaptivus

“…They were subsequently found in other pathogenic bacteria including Vibrio parahaemolyticus , in which they play an important role in shrimp pathogenesis (Sirikharin et al, 2015). It was shown that the crystal structure of the V. parahaemolyticus Pir toxin complex resembles those of the Bacillus thuringiensis Cry toxins, suggesting that they may have a conserved mode of action that ultimately causes destabilization of the host cell cytoskeleton, leading to cell death (Lee et al, 2015).…”

Necrosis aguda del hepatopáncreas: una revisión de la enfermedad en Penaeus vannamei

“…En el continente americano el AHPND se diagnosticó en el año 2013, generando altas mortalidades y disminución de producción en México, específicamente en los estados de Sonora, Nayarit y Sinaloa Nunan et al, 2014;Pantoja y Lightner, 2014;Sánchez et al, 2014;Sirikharin et al, 2015). Por último, se identificó en Centroamérica en el 2015 (Han et al, 2015b), donde no se especificó el país, pero posteriormente, se informó su presencia en los cultivos en Belice (Shrimp News International, 2015).…”

Recombinant PirA‐like toxin protects shrimp against challenge with Vibrio parahaemolyticus, the aetiological agent of acute hepatopancreatic necrosis disease

“…; Sirikharin et al . ). Remarkably, the oral administration of recombinant PirA‐like toxin alone did not cause histopathological signs or mortality (Lee et al .…”

“…Figure shows the patterns observed in the SDS‐PAGE analysis, where a strong band of 16 KDa is observed, which is very close to the theoretical and native PirA‐like toxin molecular weight (Sirikharin et al . ). Evaluation by SDS‐PAGE allowed to determine that PirA‐like toxin predominantly remains in the soluble fraction (data not shown).…”

“…; Sirikharin et al . ). Remarkably, the quantity of combined PirA‐like and PirB‐like proteins required by the oral route to cause 100% mortality and AHPND histopathological signs was 10 μg g −1 shrimp of each protein, effects that were not observed at doses of 2 or 5 μg g −1 shrimp (Sirikharin et al .…”