Generation And Animal Testing Of Edwardsiella Tarda Ghost Vaccine

Edwardsiella tarda (E. tarda) is widely distributed in aquatic environments and is infectious to variety of animals including humans, fish, amphibians, reptiles and birds. This organism's versatility with respect to the broad-range of hosts highlight the importance of developing strategies for the protection of both animals and humans from E. tarda infections.In recent years, chemotherapy has been used effectively in controling fish infections, however, there is significant concern regarding food safety following chemotherapeutic interventions in addition to the danger of selecting for antibiotic-resistant E. tarda isolates which have been reported worldwide. These concerns have prompted the development of novel vaccination strategies for the control of E. tarda infections. Although the development of E. tarda vaccines has been attempted, their efficacy against challenge has been inconsistent. The commercial vaccines presently available consist of heat- or formalin-inactivated E. tarda or subunit formulations, however these strategies can affect the physio-chemical structural properties of surface antigens thereby negatively affecting the development of protective immunity. Subunit vaccines are often less immunogenic, necessitating the use of adjuvants which may have significant negative side effects on the host.Bacterial ghosts are empty cell envelopes that are produced, for example, by the controlled expression of the PhiX174 lysis gene E in Gram-negative bacteria. Expression of lysis gene E leads to the formation of trans-membrane tunnels which consequently lead to the loss of cytoplasmic contents. The resulting bacterial ghosts have been demonstrated to retain functional and antigenic determinants of the envelope. Even highly-sensitive and fragile structures such as pili are well protected following ghost formation. These data suggested that ghosts could be used in place of the traditional live-attenuated vaccine preparations to elicit immunity.In the present study, E. tarda ghosts were generated and used as vaccine candidates. 1 Screening of Edwardsiella tarda Strain for Ghost VaccineThe applicability of morphology, selective culture medium, biochemical tests, PCR and 16S rDNA methods for identification of E. tarda associated in fish and human culture system was studied, and the result suggest that they were all E. tarda. Six pairs of primers were designed according to six virulence gene published nucleotide sequence, and PCR was developed to detect the distribution of above virulence genes in domestic and oversea isolation strains. There was different in the virulence genes distribution between domestic and oversea isolation strains. We tested the haemolysis of E. tarda, and challenged the mouse and zebrafish using E. tarda CD strain, suggesting that CD strain was virulence.2 Generation of Edwardsiella tarda ghosts by PhiX174 lysis gene EBacterial ghosts may be generated by the controlled expression of the PhiX174 lysis gene E in Gramnegative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The objective of this study was to examine the potential utility of the PhiX174 E gene driven by the PR/cI857 regulatory system for the generation of E. tarda ghosts. The E. tarda ghost (ETG) vaccine was successfully prepared using this technology and tested in safe trials.3 Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infectionThe Edwardsiella tarda ghost (ETG) vaccine was tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease.4 Immune Effect of Edwardsilla tarda ghosts on zebrafishIn this work, we describe a method to assay the phagocytic activity of zebrafish leukocytes induced by E. tarda ghosts (ETG) vaccine. Control groups included zebrafish immunized with formalin-killed E. tarda (FKC) or zebrafish treated with phosphate-buffered saline (PBS), respectively. The results showed that the leukocytes phagocytic activity was significantly higher in ETG group than those in the other two groups. Most important, ETG-immunized fish were significantly protected against E. tarda challenge (83.3% survival) compared to 70 and 6.7% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG vaccine could confer protection against infection in a zebrafish model of disease.5 Edwardsiella tarda Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease AA dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of E. tarda ghosts (ETG). The expression of staphylococcal nuclease A in E. tarda resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments. The dual expression system for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. The 26 amino acid N-terminal sequence of SNA fused with theλphage Cro gene, showed successful degradation of bacterial nucleic acids. BG were generated via coexpression of the SNA gene and lysis gene E under the control of eachλPR promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria.