Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent disease that poses significant economic threats to the shrimp aquaculture industry. This disease is caused by toxins secreted by pathogenic strains of Vibrio parahaemolyticus that have acquired a unique 63–70 kb AHPND-associated plasmid (pVA1). The plasmid encodes the binary PirA /BVP toxins that consist of two subunits PirAVP and PirBVP. Our study, with its focus on investigating the ability of Bacillus sp. to degrade these toxins, could potentially lead to the development of a new treatment for AHPND in shrimp aquaculture .
Toxin protein purification was done using MagneHisTM Protein Purification System provided by Promega. A previous study showed that recombinant PirBVP is more toxic to brine shrimp larvae than PirAVP
. The PirBVP subunit is a lectin that seems to participate in bacterial pathogenicity
(Victorio-De Los Santos et al., 2020)
. Our first experiment with crude AHPND toxin also revealed that PirAVP was more stable and apparently unable to degrade by Bacillus strains (data not shown). Based on those facts, in this study, we focused more on investigating the Bacillus strain’s ability to degrade pure PirBVP toxin.
In this study, four different strains of Bacillus sp., namely LMG9300, D10, LT12, and L6, were able to degrade PirB toxin as shown by SDS PAGE . Furthermore, we used those strains for the challenge test toward gnotobiotically cultured brine shrimp larvae Artemia franciscana , either with the addition of PirB toxin or directly challenged with Vibrio parahaemolyticus M0904.
Our in vivo challenge test revealed that adding pure PirAVP and PirBVP together significantly decreased the survival of Artemia. The addition of either a single or mixed culture of Bacillus strains then improved the survival of the Artemia when exposed to both pure PirAVP and PirBVP.