Due to grass carp haemorrhagic disease caused by grass carp reovirus (GCRV), aquaculture industry suffers huge economic loss. Oral Lactobacillus casei vaccine, with its excellent safety and effectiveness, is a promising strategy to prevent infectious diseases. While, the oral vaccine encapsulated with alginate-chitosan (SA/CS) can further improve its immune function. In this study, recombinant L. casei (rLc) expressing multiple fragments of VP56 was constructed, which was a key outer capsid protein of GCRV. By mixing rLcs with fodder and fed grass carp, the corresponding anti-VP56 fragment sera was produced, and VP56310-500 was screened as optimal fragment of GCRV-II by adopting enzyme linked immunosorbent assay (ELISA). Furthermore, SA/CS was used as the encapsulating material of L. casei live vaccine expressing VP56310-500, thereby preparing oral microcapsule live vaccine SCL-56310-500. With good resistance to bile (0-0.4%), NaCl (0-0.8%), simulated intestinal fluid, and simulated gastric fluid (pH = 1.5-4.5). Also, it had the ability to maintain high viability after colonizing intestine of grass carp in 7 days. In addition, Flagellin C (FlaC) was adopted as an immune adjuvant, and rLc was con-structed to express VP56310-500-FlaC (56310-500C), thereby enhancing immune effect of oral live vaccine. After oral administration of encapsulated L. casei vaccine, its protective effect on GCRV-II infection was evaluated. As indicated by the results, SCL-56310-500C effectively up-regulated the activity of serum biochemical enzymes (including TSOD, lysozyme, and complement C3), and induced mRNA expression of important immune genes (including IL-1 beta, IFN1, MHC-II alpha, IgM, and IgT in immune tissues). Simultaneously, SCL-56310-500C greatly sup-pressed GCRV replication in head kidney, spleen, and intestine. Correspondingly, grass carp vaccinated orally with SCL-56310-500C have distinctly higher survival rate (58% versus 24% in control) against GCRV infection. These results illustrated that an oral probiotic vaccine SCL-56310-500C effective in GCRV infection prevention was developed. Thus, this study offered an inspiring strategy for the prevention of fish viral diseases.
