Introduction
A bottleneck to the growth of the aquaculture industry is disease outbreak. The most recent disease outbreak in shrimp farming systems, known as Acute Hepatopancreatic Necrosis Disease (AHPND), occurred in 2009 and resulted in enormous economic losses across the globe (FAO, 2022; Sarker et al., 2021; Mishra et al., 2017). The opportunistic pathogenic bacterium Vibrio parahaemolyticus (VP) produces plasmid encoded (pVA1) binary toxins pirAVP/pirBVP. The toxins cause up to 100% mortality by degenerating the epithelial cells of the hepatopancreases during the early stages of shrimp stocking (Lightner et al., 2012; Lai et al., 2015). Bacillus strains have been used widely in combating diseases in aquaculture. There is also evidence that Bacillus treatment improve brine shrimp survival against V. parahaemolyticus (Nguyen et al., 2021). Interestingly, the protection mechanism is still not clear. In this study, eight distinct Bacillus strains as potential AHPND disease controlling or mitigating agents were examined in vitro for their ability to degrade AHPND toxins and other phenotypes potentially contributing to their probiotic nature.
Materials and Methods:
Eight Bacillus strains have been used for the study. The Bacillus were collected from BCCM/LMG and DSMZ collection centre. The recombinant binary toxins (rPirABVP) were purified using His Spintrap column (Cytiva 28-4013-53, USA). In vitro degradation (over a period of 48h by 107 CFU/ml) of recombinant AHPND toxins were detected using SDS-PAGE followed by Western Blot techniques. Strains were tested for their capacity to degrade quorum sensing molecules (only acyl homoserine lactons; AHL) in an LB background over a period of 24 h. The flow cytometer was used to test their capacity to bind four fluorescently (FITC) labeled lectins (from plant origin). Biolog GENIII microplate (BiOLOG, Hayward) assays were used for metabolic profiling analyses and data were processed by PCA analysis. Extracellular proteins harvested from concentrated Bacillus culture were separated by SDS-PAGE and analysed using the BioNumerics 7 software (setting: 1% optimization of band migration) (Applied Maths, Kortrijk, Belgium).
Results and discussions:
Bacillus strain LMG9300, DSM1668 & DSM8785 exhibited strong AHL-degrading properties. Apart from AHL-degrading capacity, LMG9300 also showed significant rPirB toxin degradation overtime (Fig. 1). Each Bacillus strain produced different secreted proteins and dendrograms of secretome profiles from all Bacillus strains were constructed based on two strategies (namely based on presence/absence of bands or band intensity) (Fig.2). In both cases, 2 main clusters of strains could be observed, harboring the same Bacillus species. Yet, in the band based approach of clustering, LMG9300 was singled out. All Bacillus strains bind predominantly either the lectin WGA or the lectin ConA (Fig. 2). The grouping of the secreted protein profiles correlated to a large extent with the capacity to bind either the WGA or ConA. Metabolic profiles for carbon sources of each strain were examined using Biolog GENIII plates. The metabolic data were processed using heatmap and principal component analysis (PCA). Generally, three clusters were detected by heat map analysis. The heat map analysis for amino acids and peptides was not significantly different from heatmap analysis of carbon sources. Finally, the tested in vitro phenotypes (in vitro AHPND toxins degradation, secretome profile, metabolic profile and lectin binding profile) will be correlated with their in vivo capacity to control AHPND, hopefully facilitating establishing links with their in-vivo functionality.
References:
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