B. subtilis DSM33018 is able to degrade pirB and alleviates AHPND in Artemia
Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND), is a penaeid shrimp disease that causes serious economic losses and significant mortality, up to 100%, in cultured shrimp species.16.07.2020
The dramatically high mortality rates in infected shrimp are caused by disfunction and destruction of the hepatopancreas (Lightner et al. 2014). There is no inflammatory response to the causative Vibrio spp., because AHPND is elicited by a toxin (Han et al. 2015), which is encoded by a plasmid (Yang et al. 2013; Tran et al. 2013). Various Vibrio spp., not only V. parahaemolyticus were demonstrated to carry this pathogenic plasmid.
Comparison of genome sequences revealed that the pathogenic plasmid encodes genes homologous with Photorhabdus insect related (Pir) toxin genes (Kondo et al. 2014). The Pir toxins act as binary proteins, they are encoded by the pirA and pirB genes, and both proteins are necessary for oral toxicity (Blackburn et al. 2006; Ahantarig et al. 2009; Han et al. 2015).
At the Laboratory of Aquaculture and Artemia Reference Center of the University of Ghent it was tested, if B. subtilis DSM 33018 is able to degrade the AHPND-causing pirB toxin in vitro. It could indeed be demonstrated that B. subtilis DSM 33018 is able to degrade the AHPND-causing pirB toxin (Figure 1).
Gram-positive probiotic organisms, such as Bacillus subtilis from Biomin are not involved in horizontal gene transfer processes with Gram-negative organisms such as Vibrio spp. and are thus unlikely to acquire resistance or virulence genes or plasmids from Vibrio species (Moriarty, 1999). B. subtilis DSM 33018 is part of the probiotic product range AquaStar® with scientifically proven positive effects on aquaculture productivity.
It was then tested, if B. subtilis DSM33018 is able to rescue infected animals from AHPND related mortality in gnotobiotic artemia. Bacillus subtilis DSM 33018 (Ba) was tested at 107 cfu/mL and 108 cfu/mL. At the concentration of 107 cfu/mL Bacillus treatment resulted in survival rate of 63% versus 47% in control (Figure 2). Enterococcus (En), Pediococcus (Pe) and Lactobacillus (La) alone were not able to significantly improve the survival rate after toxin challenge. AquaStar® (Mix) at concentration 107 cfu/mL and 108 cfu/mL however rescued the artemia from AHPND related mortality and allowed survival rates of 77% and 70%.
Figure 2. Artemia survival after challenge with purified pir toxins at 26 µg/mL. Bacillus DSM33018 (Ba) was tested at 107 cfu/mL and 108 cfu/mL. At the concentration of 107 cfu/mL it tended to increase survival rate from 47% in control to 63%. Enterococcus (En), Pediococcus (Pe) and Lactobacillus (La) alone tended to improve the survival rate after challenge. AquaStar® (Mix) at concentration 107 cfu/mL and 108 cfu/mL rescued the artemia from AHPND related mortality and allowed survival rates of 77% and 70%. LVS3 is a sterile Aeromonas control, acting as a nutritional support. ‘Non’ is the control challenge without probiotic treatment nor basic nutritional support.
It remains to be determined if B. subtilis alone helps to alleviate AHPND in shrimp culture. Having said that, when Pacific whiteleg shrimp (L. vannamei) were challenged with pirA and pirB toxin producing Vibrio parahaemolyticus (AHPND positive), via an immersion infection model, supplementing AquaStar® Growout (combination of Bacillus sp. plus lactic acid bacteria) appeared to significantly reduce mortalities (Figure 3; Kesselring et al, 2019).