Oral vaccination for Ctenopharyngodon idella against Flavobacterium columnare using Bacillus subtilis expressing lip gene

Abstract:

Bacillus subtilis is used as a probiotic bacterium in aquaculture and has been explored as a live carrier for the expression and oral delivery of antigen proteins. Flavobacterium columnare, a gram-negative rod-shaped bacterium, is the causative agent of columnaris disease in fish. It is ubiquitous in aquatic environments and causes disease in a variety of freshwater fish worldwide, which leads to severe economic loss. In a previous study, we discovered a protective antigen gene, lip, against F. columnare. In this study, the lip gene was inserted into a shuttle expression vector pBE2R, and then expressed as a recombinant fusion protein in B. subtilis WB800. The expressed protein of 32-kDa, which was estimated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blotting, was consistent with the molecular weight deduced from amino acid sequence. Grass carp (Ctenopharyngodon idella) was immunized through oral administration of B. subtilis WB800 (pBE2R-lip). The effects of protection and immune responses were evaluated. Enzyme-linked immunosorbent assay results showed that lip-specific antibodies could be detected in the sera of immunized fish, and the antibody levels were highest in the fourth week, with an average of 1:117. The outcome of immunization with B. subtilis WB800 (pBE2R-lip) resulted in a relative percent survival of 52.4% against F. columnare. After being immunized via oral administration, the quantity of B. subtilis in the hindgut of grass carp was measured using spread plate count method. Results showed that B. subtilis could not colonize the gut of grass carp. Our study demonstrated that oral administration of B. subtilis expressing lip gene could produce an effective immune response and offer good resistance against F. columnare.