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

Zhu W, Yang G, Zhang Y, Yuan J, An L - J. Biomed. Biotechnol. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Animal Resistance Biology of Shandong, College of Life Sciences, Shandong Normal University, Jinan 250014, China.

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.

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Related in: MedlinePlus

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).
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fig2: 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).

Mentions: Since F. columnare G4cpN22 was high sensitive to chloramphenicol, but not to ampicillin, a specific lysis vector with the cat gene had to be constructed (Figure 1). The lysis plasmid pBV E cat was successfully electroporated into F. columnare G4cpN22 which growth characteristics were similar to F. columnare G4. Generation of F. columnare ghosts (FCGs) was performed by shifting the incubation temperature to 42°C to inactivate the repressor protein and activate lysis gene E. Onset of the lysis occurred 1 h after temperature elevation because the numbers of viable cells began to decrease at 1 h after induction, and the lysis process was completed 10 h after induction (Figure 2). The results of three replicate experiments showed the efficiency of FCGs induction was 99.99% at the end of the lysis process. The lyophilized FCGs were analyzed for survivors by inoculating 100 mL of growth media with a tenfold immunization dose at 26°C, but no bacterial growth was detected. The lyophilized FCG preparation was used for vaccination studies in fish.


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

Zhu W, Yang G, Zhang Y, Yuan J, An L - J. Biomed. Biotechnol. (2012)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376489&req=5

fig2: 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).
Mentions: Since F. columnare G4cpN22 was high sensitive to chloramphenicol, but not to ampicillin, a specific lysis vector with the cat gene had to be constructed (Figure 1). The lysis plasmid pBV E cat was successfully electroporated into F. columnare G4cpN22 which growth characteristics were similar to F. columnare G4. Generation of F. columnare ghosts (FCGs) was performed by shifting the incubation temperature to 42°C to inactivate the repressor protein and activate lysis gene E. Onset of the lysis occurred 1 h after temperature elevation because the numbers of viable cells began to decrease at 1 h after induction, and the lysis process was completed 10 h after induction (Figure 2). The results of three replicate experiments showed the efficiency of FCGs induction was 99.99% at the end of the lysis process. The lyophilized FCGs were analyzed for survivors by inoculating 100 mL of growth media with a tenfold immunization dose at 26°C, but no bacterial growth was detected. The lyophilized FCG preparation was used for vaccination studies in fish.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Animal Resistance Biology of Shandong, College of Life Sciences, Shandong Normal University, Jinan 250014, China.

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.

Show MeSH
Related in: MedlinePlus