Construction Of Salmonella Enterica Serovar Enteritidis C50041Crp, SpiC Mutants And Their Immunobiological Features

Food-borne diseases caused by Salmonella enterica could bring an important public health problem worldwide. Because poultry is one of the main host of Salmonella, it would be very significant to strengthen the prevention and control of Salmonella in poultry, which not only can ensure the poultry production, but also can protect human from diseases caused by Salmonella.Following the progress of gene engineering techniques, we could selectively knock out interesting virulence genes to make attenuated live Salmonella vaccine candidates, which were attracting considerable attention, because these attenuated live vaccines could provide effective immunity and prolonged exposure of antigens to the immune system and result in the production of long-lasting memory cells, having obvious advantage in controlling Salmonella infections.In this study, three mutants were constructed by homologous recombination, based on virulent strain C50041of Salmonella enterica serovar Enteritidis. The immunobiological features of the gene-deleted mutants were tested for their possibility as live attenuated vaccines.1. Construction and identification of S. Enteritidis C50041spiC, crp mutantsFor constructing crp-deleted strain, two DNA fragments which were the upstream925bp and the downstream936bp of crp gene were amplified using the C50041genome DNA as PCR template. The chloramphenicol-resistant gene was inserted in the two fragments to construct pMD-Δcrp/Cm. Then amplified the fragment which consisted of the chloramphenicol resistance gene(CmR) flanked by two homology arms of crp gene. The PCR products were introduced into wild type C50041with pKD46plasmid by electroporation, with the help of λRed-mediated recombination system, the target gene was replace by homology extensions connected with CmR. The chloramphenicol-resistant strains were selected by chloramphenicol antibiotic in the agar, CmR gene was eliminated by using a helper plasmid of pCP20which encoded the Flp recombinase.The recombinant suicide plasmid pGMB151-ΔspiC/Km was used to delete the spiC gene of C50041and C50041Δcrp. After the mutants C50041Δcrp, C50041ΔspiC and C50041ΔcrpΔspiC/Km were constructed, their phenotype, growth property and virulence characteristics were determined. The results showed that the growth characteristic of ΔspiC mutant was consistent with those of its parent strain C50041, but the growth of Δcrp mutant was lower because the mutant was not able to metabolize many kinds of carbohydrate. The LD50of3mutants had a significant drop about1,000folds comparing with their parent strain for1-days-old chickens by being inoculated intramuscularly.2. Preliminary analysis on immunobiological features of the C50041mutantsThe macrophages cell line RAW264.7was used as the model in vitro to investigate the characterization of mutants. The results showed that the invasion efficiency of the three mutant strains was significantly declined comparing with C50041strain (p<0.01), the invasion efficiency of the C50041Δcrp mutant and C50041ΔcrpAspiC/Km mutant was obviously lower than the C50041ΔspiC mutant(p<0.01), the C50041ΔcrpΔspiC mutant was obviously lower than the C50041Δcrp mutant(p<0.05). The results of persistence assays, which1-days-old chickens were inoculated intramuscularly, showed that all of the3mutants were eliminated at10days post inoculation (dpi) in liver. But in spleen, C50041ΔcrpΔspiC/Km mutant was eliminated at13dpi, the C50041Δcrp mutants at16dpi, the C50041ΔspiC mutants at20dpi, as the control, the chicken of wild type C50041group died out7dpi.The humord responses of chickens inoculated the mutants had been examined by indirect ELISA. The levels of serum IgG could be observed at1weeks post inoculation (wpi) and peaked at2wpi. In the group of C50041ΔspiC mutant imunization, the mucosal sIgA antibodies were observed at2wpi, but in the group of C50041Δcrp mutant and C50041ΔcrpΔspiC/Km mutant, the mucosal sIgA antibodies was observed at3wpi. In the protective efficacy assay, chickens were immunized at3days-old with3mutants and then challenged with S. Enteritidis C50041at8dpi. In the feces, the total number of Samonella in the groups of immunization was notablely lower than the group of PBS control(p<0.01). In the liver and spleen, the CFU/g of imunization-groups was obviously lower than the group of PBS control(p<0.01), and in the group of PBS control, the wild type C50041was able to persist longer time.