The Development Of Attenuated Salmonella Gallinarum 1009ΔspiCΔcrp As An Efficient Live Vaccine Candidate

At present, fowl typhoid (FT) is a severe bacterial infectious disease. It was caused by Salmonella Gallinarum (S. Gallinarum) and results in substantial economic losses in many areas with severe anorexia, weight loss, lesions, depression, diarrhea in chickens. Live vaccines can offer more effective protection than killed vaccines because of their induction of highly cellular immune responses. Currently, the live S. Gallinarum vaccine strain 9R has been avialable, but it has some disadvantages, such as residual virulence, low growth rate and insufficient protection.In this study, a AspiC mutant of Salmonella Gallinarum strain 1009 was constructed by the allelic exchange introduced by the transformatin of a recombinant suicide plasmid, the crp gene was then deleted by using the λ Red-based recombination system, to give rise to a double mutant 1009 ΔspiCAcrp which was confirmed by PCR and sequence analysis. Analysis of the biological characteristics of the mutant showed that the strain was stable with both genes deleted. However, its biochemical properties were a little different from those of the parent strain, and the growth velocity of the 1009ΔspicAcrp was slower than the 1009 strain. The chicken lethal test showed that the LD50 of the 1009 Δspic Acrp strain was 107 times higher than that of 1009, implying the downregulation of the mutant.In order to evaluated the safety and protective efficacy of the mutant 1009 AspiCAcrp, three-day-old chickens were intramuscularly immunized with 1009ΔspiCΔcrp for the first time and were subsequently boosted at 7 days later with the same doses and route (vaccinated group), while the control group was immunized with PBS (control group). Specific humoral and cellular immune responses were significantly induced and 1009AspiCAcrp can colonize and persistent in liver and spleen of chickens more than 14 days after immunization. Vaccinated group showed no differences in body weight or clinical symptoms compared with control group. The immunized chickens were then challenged with highly-virulent S. Gallinarum strain Sg9 at 21 dpi, efficient protection and some slight temporary clinical symptoms were observed. All the immunized chickens were survived, while 80% mortality was detected in the control group. Taken together, these results demonstrate the safety and protective efficacy of 1009ΔspiCΔcrp as a live attenuated vaccine candidate.On the basis of perfect efficacy of intramuscular immunity, the safety and protective efficacy of 1009ΔspicAcrp via orally immunized was further evaluated. Three-day-old chickens were orally immunized with 1009AspiCΔcrp in 1 × 108CFU and were subsequently boosted at 7 days later with the same doses and route. The results showed there was no differences in body weight or clinical symptoms between immunized and control group, but the secretion of IgG was singnificantly increased, and the expression level of IL-4、IL-6 and IL-1β were up-regulated. After challenged with S. Gallinarum strain Sg9 at 21 dpi, the survival ratio of immunized chickens was 100% while in unimmunized chickens was 30%. Taken together, these results demonstrate the safety and protective efficacy of 1009ΔspiCΔcrp and potentially to be developed as a live attenuated fowl typhoid (FT) vaccine candidate.