Generation of biotechnology-derived Flavobacterium columnare ghosts by PhiX174 gene E-mediated inactivation and the potential as vaccine candidates against infection in grass carp

Abstract

Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs) were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus) by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%). These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

Figure 1

Physical map of the lysis plasmid pBV E cat. Gene E, under transcriptional control of the temperature-sensitive repressor; rrBT1T2: ribosome rrnB gene providing translation stop signal terminator sequence; pro: the regulating sequence including promoter; cat: chloramphenicol acetyl transferase gene; ori: replication origin; cIts857: restraining gene of lambda bacteriophage adapted to heat induced expression.

Figure 2

Growth and lysis kinetics of F. columnare G4cpN22 harboring plasmid pBV E cat by temperature induction of gene E expression. At time zero, the growth temperature of three bottles of the cultures was shifted from 26°C to 42°C (filled symbols), whereas the others served as noninduced control at 26°C (open symbols). Growth and lysis were monitored by the measurement of the OD₆₀₀ (open circles and filled circles) and the determination of the number of CFU (open squares and filled squares and discontinuous lines).

Figure 3

Electron microscopy analyses of F. columnare G4cpN22 ghosts. (a) Arrow showed the transmembrane lysis tunnel located mainly at the cell poles via SEM. (b) Loss of cytoplasmic materials of F. columnare G4cpN22 ghosts was shown by TEM. The lysed cells showed uneven and low electron density and retained the basic cell morphology of the bacterial cells, while the unlysed cells showed even and high electron density in an integral cell structure.

Figure 4

Serum agglutination titres of fish vaccinated with FCG and FKC at different time points postprimary immunization. Grass carp were vaccinated with FCG or FKC via intraperitoneal injection with a boost at 4 weeks after primary immunization. Serum agglutination titres represent the highest dilutions that gave rise to positive reaction. The titres of FCG group were significantly higher than FKC group at all the examined time points (P < 0.01).

Figure 5

Serum bactericidal activities of grass carp vaccinated with FCG or FKC. The grass carp were vaccinated with FCG or FKC via intraperitoneal injection with a boost at 4 weeks after primary immunization. Serum bactericidal activities of FCG group were significantly higher than FKC group at the examined time points of postimmunization (P < 0.01).