| Tilapia is one of the most important fish species for freshwater breeding as well as one of the species that is highly promoted to culture in southern China. In recent years, the problems of Streptococcus agalactiae infection in tilapia culture have become gradually apparent. Severe streptococcal infection in tilapia caused by S. agalactiae has occurred in China, causing heavy losses. In July 2009, a severe streptococcal infection in tilapia outbroke in intensive tilapia cultures in Hainan Province, China. The pathogen had been isolated and characterized as S. agalactiae in our laboratory. In this research, we cloned the gene encoding a portion of Sip into pVAXl and introduced the recombinant plasmid pVAX1-sip into attenuated S. typhimurium SL7207 by electrotransformation. We evaluated the safety, stability and the distribution in tilapia tissues of the recombinant DNA vaccine delivered by SL7207 as well as the immunity effect to tilapia by oral vaccination.1. Construction of expression plasmids and expression of pVAX1-sip in EPC cellsS. agalactiae DNA was extracted from pure cultures of the bacterial isolate. The sip segment was amplified from the DNA. The purified PCR product of sip gene was linked into pMD19-T and then sequenced. The sip gene fragment was cut by restriction enzyme and ligated into pVAXl. The empty vector pVAX1 and recombinant plasmid pVAX1-sip were transiently transfected into the epithelioma papulosum cyprinid (EPC) cells by using Lipofectamine and the transient expression of pVAX1-sip in EPC cells was detected by an indirect immunofluorescence assay, together with Western blot analysis. The results showed that a 1000 bp gene was amplified by PCR from the DNA of S. agalactiae. The purified amplification product of sip gene was linked into pMD19-T vector and identified by PCR and restriction enzyme analysis. The amplified sequence was 1002 bp in length and encoded 333 amino acids according to sequence analysis (KJ398146.1). The sip gene was inserted into the pVAX1 and identified by PCR and restriction analysis. EPC cells transfected with pVAX1-sip showed a strong cytoplasmic fluorescence due to expression of Sip protein compared to vector control. An apparent molecular weight of approximately 40 kDa was identified by Western blot with reaction to antiserum against S. agalactiae while no expression was detected in cells transfected with pVAXl.2. Transformation into attenuated S. typhimuriunt and biological characterizationThe purified plasmid pVAX1-sip or pVAX1 was electroporated into S. typhimurium SL7207. The positive transformants selected for kanamycin resistance were identified by PCR amplification, restriction endonuclease analysis and sequence analysis with universal 16S rDNA primers. The recombinant Salmonella strains containing plasmid pVAXl-sip or pVAXl were named SL7207-pVAXl-sip and SL7207-pVAX1, respectively. The stability of plasmid in vitro and in vivo was evaluated as well as the biological characteristics of the recombinant S. typhimurium SL7207-pVAXl-sip and SL7207-pVAX1. We also evaluated the stability of plasmid in food pellets with different drying or storage temperatures. The results showed that the growth curve of recombinant strains were almost the same as the original strain SL7207. Meanwhile, the stability of pVAX1-sip into Salmonella was over 90% after 50 generations with antibiotic selection in vitro while remained stable over 80% during 35 generations under antibiotic-free conditions. Recombinant bacteria strains could be isolated from tissues (intestinal tract, livers and spleens) of the immunized tilapia and could be finally eliminated from the animals within a month. The results of the stability of plasmid in food pellets indicated that low-temperature drying and low temperature storage can reduce the loss rate of plasmid pVAXl-sip in recombinant bacteria, suggesting that it is best for the food pellets to be with the current feed in order to ensure the optimal immunity effect.3. The safety study on DNA vaccineThe safety of the DNA vaccine was evaluated. First, the virulence of attenuated S. typhimurium to tilapia was tested. Results indicated that the LD50 of SL7207-pVAX1-sip to tilapia was 1.7×1011 cfu/fish by intragastric administration, an extremely high value, which indicated a quite low virulence. Second, there were no deaths or any side effect in tilapia after immunization with attenuated S. typhimurium SL7207-pVAX1-sip at different dosages (1×108 CFU, 1×109 CFU,5×109 CFU). The histopathological observations of tissus also showed that there were no apparent pathological changes after immunization. Third, we tested the potential for integration into host DNA in tilapia following intramuscular injection instead of intragastric administration. The assay sensitivity was approximately 100 plasmid copies/μg DNA and the examination of 6 tissues failed in revealing any evidence of integration at the sensitivity level. In short, it is relatively safe to use attenuated S. typhimurium as a carrier for DNA vaccine plasmid in tilapia.4. The distribution of DNA plasmid and the expression products in tissues of tilapiaThe distribution of DNA plasmid and the expression products in tissues of tilapia were investigated. The results showed that after immunization the DNA plasmid could be distributed in intestine, liver, spleen and kidney of tilapia detected by PCR. The expression products Sip could be distributed in spleen and liver by immunohistochemistry. The duration of DNA plasmid or the expression products in tissus was correlated positively with the number of immunization times. The results showed that the attenuated Salmonella could carry the sip gene to the target tissue as a DNA vacccine carrier. And the DNA plasmid could be expressed in tilapia cells effectively which laid the foundation for post-immunization trials.5. The immunity effect of the DNA vaccine delivered by attenuated S. typhimuriumTilapias were immnunized by intragastric administration and feeding food pellets respectively with different immunization times. The changes of serum total protein, serum SOD activity, serum lysozyme activity, serum alexine C3 and serum antibacterial activity as non-specific immune parameters were evaluated. Meanwhile, the serum antibody titers, the relative expression of cytokines IL-1β and TNF-a in both kidney and spleen and the protective efficacy were also evaluated. The results showed that vaccination in the two oral inoculation routes can both effectively induce various immune responses of tilapia and make tilapias gain the ability against S. agalactiae infection. The highest relative rates of protection in intragastric administration groups and food pellets groups were 57% and 63%, respectively. Considering the safety and vaccine efficiency, the DNA vaccine SL7207-pVAX1-sip at a dosage of 108 CFU repeated three times at a 1-week interval is the most effective vaccination program by oral administration according to the study. |
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