Generation Of Flavobacterium Columnare Ghosts By Lysis Gene Mediated Inactivation And The Potential As Vaccine Candidates Against Infection In Grass Carp

Flavobacterium columnare, a Gram-negative gliding bacterium, is the causativeagent of columnaris disease, one of the most important bacterial diseases of freshwaterfish species. This bacterium is ubiquitous in aquatic environments, affecting wild andcultured fish as well as ornamental fish in aquaria. The onset of columnaris disease ischaracterized by external infections in the fish body surface, gills or fins. The diseaseoften ends in death, leading to large economic losses in the fish farming industry.In recent years, chemotherapy has been used effeetively in controlling fishinfections, while the extensive use of chemical substances results in emergence ofantibiotic-resistant microbes and negative impacts on environment and human beings.Therefore, vaccination has become an increasingly important prevention strategyagainst infectious agents in farmed fishes. Several attempts have been made to induceprotection against columnaris disease with formalin- or heat-inactivated preparationsof F. columnare, yet no protection or only partial protection was achieved followingimmersion or injection immunization. Currently, a modified live columnaris vaccinehas been developed and commercialized in the United States and it is efficacious forprevention of columnaris disease in channel catfish and largemouth bass fry. However,live vaccines may bear the danger of reversion. Then, a new type of F. columnarevaccine is urgently needed to prevent and control the columnaris disease.Bacterial ghosts have been given increasing attention as a promising newapproach in non-living vaccine technology. Generally, Bacterial ghosts are producedby the controlled expression of bacteriophage PhiX174 lysis gene E, and the E protein leads to the formation of small transmembrane pores through which cytoplasmiccontents are expelled. The resultant bacterial ghosts retain the functional and antigenicdeterminants of the envelope with their living counterparts; therefore they possessgood immunogenicity and bio-adhesive properties and are able to induce effectiveimmunoprotection.For the first, the generation and the usage as vaccine candidates of F. columnareghosts (FCGs) were reported.Flavobacterium columnare G4R3 is a highly-virulent bacterial pathogen causinghigh mortality rates for many freshwater fish species. Biological characteristics ofstrain G4R3, such as morphology, growth, biochemical properties, antimicrobialsusceptibilities and virulence, were analyzed. The strain G4R3 was identified by PCRamplification of 16S rDNA. The strain G4R3 was grown in Shieh medium at 26oC andhighly sensitive to chloramphenicol and lowly sensitive to ampicillin and kanamycin.The challenge test of grass carp (Ctenopharyngodon idellus) showed the virulence ofthe strain G4R3 was very high which provided a foundation for further studies.Fish vaccination with a safe and effective vaccine is a potential approach forprevention and control of fish disease. Here, in order to produce bacterial ghostvaccine, the lysis plasmids such as pBV-geneE, pBV-geneE-cat, pET28a-geneE,pET32a-geneE, pET28a-geneE-cat and pET32a-geneE-cat were constructed. A specificFlavobacterium lysis plasmid pBV-E-Fpro-cat was constructed by cloning PhiX174lysis gene E and the cat gene with the promoter of F. columnare into the prokaryoticexpression vector pBV220. According to the electroplation of wild type F. columnareG4R3, there was no effective lysis plasmid. DNase activaty was analyzed and anendogenous plasmid was found in F. columnare G4R3. After sequencing and curing ofplasmid pFcG4, a derivative strain which can harboring the lysis plasmidpBV-geneE-Fpro-cat was successfully screened. The plasmid was successfully electroporated into the strain F. columnareG4R3M22 after curing of its endogenous plasmid pFcG4. F. columnare G4R3M22ghosts (FCG) were generated for the first time by gene E-mediated lysis. Generation ofF. columnare ghosts (FCG) was performed by shifting the incubation temperature to 42oC to inactivate the repressor protein and activate lysis gene E. Onset of the lysisoccurred 1 h after temperature elevation because the numbers of viable cells began todecrease at 1 h after induction, and the lysis process was completed 10 h afterinduction. The results of three replicate experiments showed the efficiency of FCGsinduction was 99.99% at the end of the lysis process. The lyophilized FCGs wasanalyzed for survivors by inoculating 100 mL of growth media with a tenfoldimmunization dose at 26 oC, but no bacterial growth was detected. Electronmicroscopic analysis of FCG revealed no gross alterations in cellular morphologycompared to unlysed cells except for the lysis pore. Pores ranging from 100 to 300nmin diameter were observed in FCG by scanning electron microscopy. The structuralintegrity and the loss of cytoplasmic materials were observed in FCG by transmissionelectron microscopy. The lyophilized FCG preparation was used for vaccinationstudies in fish.The vaccine potential of FCG was investigated in grass carp (Ctenopharyngodonidellus) by intraperitoneal route. The agglutination reaction against F. columnare G4R3was detected in grass carp immunized ip with bacterial ghosts, formalin-killed cells orPBS. Fish immunized with FCG or FKC showed significantly higher agglutination titerthan control fish in which no agglutination reaction was detected, while fishimmunized with FCG showed significantly higher titers than fish immunized withFKC at all the examined time points. Fish immunized with FCG showed significantlyhigher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relativepercent survival (RPS) of fish in FCG group (70.9%) was significantly higher thanFKC group (41.9%). These results showed FCG could confer immune protectionagainst F. columnare infection. In conclusion, FCG have been shown to inducestronger protective immunity than FKC in grass carp. Given its safety and high level ofimmunoprotective efficacy, FCG may provide an ideal alternative to pathogen-basedvaccines against columnaris in aquaculture.In conclusion, FCG were generated for the first time by gene E mediated lysis andshown to induce stronger protective immunity than FKC in grass carp. Given its safetyand high level of immunoprotective efficacy, FCG may provide an ideal alternative topathogen-based vaccines against columnaris in aquaculture.