| Nowadays, the pullorum disease (PD) caused by Salmonella enterica serovar Pullorum is an important disease of considerable economic impact in the poultry industry in China. Antimicrobials were taken to control this disease, but leading to the problem of multiple drug resistance of bacteria and drug residues. Besides, without restrict strategies for practical eradication of PD, the prevalence of PD is relatively high in China, but in developed countries it is nearly eliminated. Recent years, vaccines have been widely employed in many countries and vaccination fulfils the requirement for preventing Salmonella disease. As S. Pullorum is host-restricted for poultry, and zoonotic transmission to man rarely occurs, there is few research on PD vaccines, except killed vaccines which can stimulate strong immune responses, but offer a relatively low degree of protection.The progress of gene engineering techniques allows us to selectively knock out concrete virulence genes to make attenuated live Salmonella vaccine candidates which have been received considerable attention because of the features that strains containing defined deletion mutations cannot revert back, their solid immunity and ability to carry heterologous antigens and capacity to induce mucosal, cellular, and humoral immune responses. Besides, these attenuated vaccines provide prolonged exposure of antigens to the immune system, resulting in the production of long-lasting memory cells. So the conclusion was confirmed that live Salmonella vaccines are superior to killed vaccines and subunit vaccines in controlling Salmonella infections.In an attempt to develop an alternative vaccine, S. Pullorum mutant defective in spiC were constructed by homologous recombination in this study using virulence strain S06004. The immunobiological properties of the spiC gene deletion mutant were tested for their potential as live attenuated vaccines.1. Construction and identification of spiC gene deletion mutant from the S. Pullorum S06004For constructing spiC deletion strain, two DNA fragments which represented the upstream 797bp and the downstream497bp of spiC gene were amplified using the S06004genome DNA as PCR template. These two fragments were combined with kanamycin-resistant gene inserted and cloned into suicide vector pGMB151. The recombinant suicide plasmid was transformed into S06004strain by E.coli.χ7213. The spiC deleted strains with km resistance were selected by two step protocols and the km resistance gene was then eliminated by using a helper plasmid, pCP20, encoding the FLP recombinase. Using this system, spiC gene in chromosome of S. Pullorum was deleted and further confirmed by PCR and sequencing analysis. The phenotype, growth property and virulence characteristics of the mutant strain were carried out. The results showed that the chracteristics of△spiC mutant were consistent with those of its parented strain S06004, and its virulence had a significant drop about1.54×102folds compared with its parented strain for3-days-old chickens inoculated intramuscularly.All results showed that the S06004△spiC mutant of S. Pullorum was successfully constructed and significantly attenuated. This study would pave a way for developing S. Pullorum S06004△spiC as a live attenuated vaccine strain.2. Preliminary analysis on immunobiological features of the attenuated Salmonella pullorum S06004△spiC mutant strainIt was used that the avian macrophages cell line HD-11as the model in vitro to investigate the characterization of S06004△spiC mutant. The results showed the invasion efficiency and the replication of S06004△spiC mutant within HD-11cells were significantly declined compared with S06004strain. In vivo assays3-days-old chickens were inoculated orally or intramuscularly with S. Pullorum. The results showed that the mutants inoculated orally were eliminated at7days post infection (dpi) in liver and9-11dpi in spleen and when inoculated intramuscularly, mutants were cleared by3-4weeks post infection (wpi) in vivo, which were more quickly than wildtype S06004strain.In the protective efficacy assay chickens were immunized at3days-old and then challenged with S.Pullorum S06004or S.Gallinarum Sg910days post inoculation. It was showed that the mutant strain provided significant protection against S06004challenge and also supplied the cross-protection against Sg9challenge, and there was no significant effect on chickens’ body weight. The immune responses induced by S06004△spiC had been examined by indirect ELISA. The levels of serum IgG could be observed at1wpi and peaked at3wpi. The mucosal sIgA antibodies were observed at2wpi and peaked at3-4wpi. Cytokine levels in spleens from immunized animals were quantitatively analyzed by Real-Time qPCR analysis. We found that the pro-inflammatory cytokine IL-1β expression level was consistent with the bacteria’s number in spleen, and the IFN-γ which mediated T-cell responses appeared to be involved in immune clearance, which indicated that Thl-type cellular response could be induced as well as the antibody response after immunization with S06004⊿spiC strains.In summary, the S06004dspiC mutant strain was successfully constructed and had the promising to be used as a live vaccine candidate for S. Pullorum disease. |
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