Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease caused by virulent strain of Vibrio parahaemolyticus (VPAHPND) that results in significant economic losses to shrimp aquaculture. The V. parahaemolyticus encodes deadly toxin (VPAHPND toxins) that are responsible for shrimp mortality during AHPND. Therefore, to better understand the toxicity mechanism of VPAHPND toxins, we have used the gnotobiotic Artemia franciscana model to determine the toxicity of recombinant PirAVP and PirBVP toxins in vivo. Subsequently, the study was validated by VPAHPND ECP30, ECP10 and ECP3 concentrated with 30, 10 and 3 kDa amicon filters to establish the toxicity towards brine shrimp larvae. It was found that recombinant PirBVP is more toxic to brine shrimp larvae as compared to PirAVP. Moreover, the survival of brine shrimp larvae challenged with a mixture of PirAVP and PirBVP toxins decreased ~2-fold as compared to PirBVP toxin and ~3-fold as compared to PirAVP toxin, as anticipated, as these 2 toxins seem to form an active complex. The study also confirms that VPAHPND ECP10 and ECP3, comprising PirAVP and PirBVP toxins, exhibits toxic effect to brine shrimp larvae. The ECP30, lacking PirAVP, showed reduced toxicity. The VPAHPND ECP10 and ECP3 (containing PirAVP and PirBVP toxins, along with other proteins) were more toxic than their respective equivalent amounts of pure toxins, suggesting that VPAHPND ECP10 and ECP3 contains additional, but uncharacterized, toxins. Hence, our study provides substantial evidence that toxicity of V. parahaemolyticus AHPND strains is mediated by VPAHPND ECP comprised of PirAVP and PirBVP (mostly) and other ECP or toxins produced by the bacterium. |
