| Salmonella Choleraesuis,one type of short bacillus Gram-negative bacterium without capsule and spore,was initially isolated in 1885 by Salmon.It forms medium size,colorless,transparent and smooth colonies on the traditional agar medium.The bacterium is one of the main pathogens causing paratyphoid fever in piglets aged 2～4 months,which characterizes as acute sepsis,refractory diarrhea and chronic necrotizing enteritis.The delayed treatment will lead to a large number of deaths in weaning piglets and cause huge losses to the pig industry.Vaccination is the most effective strategy to prevent this disease.However,the inactivated vaccine has defects such as poor immunogenicity,heavy workload and more intense stress reaction for animal.Due to unclear mechanism of the attenuation,live attenuated vaccine has a risk of virulence returning。Fortunately,the attenuated strains originated from gene deletion mutant overcome these defects.In this study,five gene-deleted Salmonella Choleraesuis mutant strains were successfully constructed,which wereΔcrp,Δcya,Δaro A,Δaro B single deletion strains andΔaro AΔaro B double deletion strain.Their biological characteristics and pathogenicity were evaluated.The immunological characteristics immune protection efficacy of theΔaro AΔaro B double deletion strain was also measured.The main content includes:1.Construction of gene deletion strains:The crp,cya,aro A and aro B genes of Salmonella Choleraesuis were knocked out byλRed homologous recombination in this study.TheΔcrp、Δcya、Δaro A、Δaro B andΔaro AΔaro B gene deletion strains were successfully constructed and verified by PCR amplification and sequencing comparison.2.The result of biological characteristics:the growth rate of the 5 mutant strains was slightly slower than WT strain on the logarithmic growth phase.And the motility of 5 mutants were significantly lower than WT strain.There were no significant differences in biochemical characteristics between the gene-deleted strains and the parental strain,except theΔcrp andΔcya single strains that cannot metabolize mannitol and sorbitol.TheΔcrp strain had no significant changes in sensitivity to other drugs except streptomycin and rifampicin.The antibiotics susceptibility of other mutants had no significant difference.All of the mutants could be stably inherited for at least 20 generations in vitro.3.Pathogenicity assay ofΔcrp,Δcya,Δaro A,Δaro B andΔaro AΔaro B of Salmonella Choleraesuis:In this study,100 times LD50doses of the WT strain was used as the challenged dose to inoculate BALB/c mice.Their clinical symptoms ofΔcrp andΔcya infected groups appeared later than that in the WT group,and both groups resulted a 20%survival rate of Meanwhile,the clinical symptom of theΔcya infected group was delayed by 2 days compared with theΔcrp challenged group.Luckily,theΔaro A,Δaro B andΔaro AΔaro B offered a 100%protection to challenged mice,showing no clinical symptoms.All livers and spleens in WT-,Δcrp-andΔcya-infected groups became swollen,while the necrotic nodules were observed in livers of WT andΔcrp infected groups.However,theΔaro A,Δaro B andΔaro AΔaro B infected groups showed no obvious symptoms.Ultimately,the immunological properties of theΔaro AΔaro B strains were preliminarily evaluated.4.The evaluation of immunological characteristics:The results of virulence test showed that the virulence ofΔaro AΔaro B strain was at least 1.02×107lower than that of WT strain.The results of immune protection trail demonstrated that theΔaro AΔaro B strain could fully protect the mice against the challenge by 629.9 LD50parental strain and partially（40%）protected the mice from extreme high does(6 299 LD50)challenge with the parental strain.In conclusion,the Salmonella CholeraesuisΔaro AΔaro B strain could be stably inherited,the virulence was significant attenuated.This strain was safe and with good immune protective effect,which laid a solid foundation for further prepare the recombinant strains to express exogenous antigen. |
