In vivo assessment for oral delivery of Bacillus subtilis harboring a viral protein (VP28) against white spot syndrome virus in Litopenaeus vannamei

Abstract

Bacillus subtilis spores, being consumed as probiotics, have been explored as live carriers for expression and oral delivery of antigen proteins. In our initial experiment, by oral delivery of B. subtilis spores harboring VP28 (rVP28-bs) to Litopenaeus vannamei, the extremely high survival (Relative Percent Survival: 83.3%) upon challenge with white spot syndrome virus (WSSV) can be observed. After ‘vaccination’ with rVP28-bs, the hemocytic phagocytosis and immune-related gene expression levels in hemocytes were analyzed. The percentage of haemocytes phagocytosing WSSV was significantly higher ( p < 0.001) in shrimp previously fed with rVP28-bs (48.2 ± 6.3) than the controls (11.0 ± 3.5 and 8.1 ± 2.5). However, there were no significant differences ( p > 0.05) in all the experimental groups for the percentage phagocytosis of TSV (an unrelated virus of shrimp). This suggests that the heightened phagocytic activity, and thus the high-level survival of shrimp after rVP28-bs ‘vaccination’ are selective or specific towards WSSV. Moreover, immune-related genes (proPO, PE and LGBP) were significantly ( p < 0.05) upregulated in both rVP28-bs and B. subtilis feeding groups compared to the control, though no significant differences ( p > 0.05) were observed between rVP28-bs and B. subtilis groups. It was indicated that the phagocytosis enhanced by rVP28-bs was the essential one to protect shrimp from virus infection, while the rVP28-bs-stimulated humoral response only plays an assistant role in antiviral defense of shrimp. Besides, in vivo fate and dissemination assays of rVP28-bs spores showed that, as robust life forms, spores can survive transit across the gut tract, germinate to express rVP28 and exert probiotic action, and disseminate in the haemolymph to present VP28 to the shrimp defense system before being excreted. These results may raise the application prospective of this recombinant B. subtilis spores against WSSV infection in shrimp farms.