Optimized Detoxification of a Live Attenuated Vaccine Strain (SG9R) to Improve Vaccine Strategy against Fowl Typhoid

Abstract

The live attenuated vaccine strain, SG9R, has been used against fowl typhoid worldwide, but it can revert to the pathogenic smooth strain owing to single nucleotide changes such as nonsense mutations in the rfaJ gene. As SG9R possesses an intact Salmonella plasmid with virulence genes, it exhibits dormant pathogenicity and can cause fowl typhoid in young chicks and stressed or immunocompromised brown egg-laying hens. To tackle these issues, we knocked out the rfaJ gene of SG9R (named Safe-9R) to eliminate the reversion risk and generated detoxified strains of Safe-9R by knocking out lpxL, lpxM, pagP, and phoP/phoQ genes to attenuate the virulence. Among the knockout strains, live ΔlpxL- (Dtx-9RL) and ΔlpxM-9R (Dtx-9RM) strains induced remarkably less expression of inflammatory cytokines in chicken macrophage cells, and oil emulsion (OE) Dtx-9RL did not cause body weight loss in chicks. Live Dtx-9RM exhibited efficacy against field strain challenge in one week without any bacterial re-isolation, while the un-detoxified strains showed the development of severe liver lesions and re-isolation of challenged strains. Thus, SG9R was optimally detoxified by knockout of lpxL and lpxM, and Dtx-9RL and Dtx-9RM might be applicable as OE and live vaccines, respectively, to prevent fowl typhoid irrespective of the age of chickens.

Keywords: SG9R; Salmonella enterica serovar Gallinarum biovar Gallinarum; detoxification; lipid A; pro-inflammatory cytokines; vaccines.

Figure 1

Deletion of rfaJ in Safe-9R and detoxified strains. (A) Deletion of rfaJ in Safe-9R and Safe-9R-derived knock-out strains. Lanes: 1, SG9R; 2, Safe-9R; 3, ΔphoP/Q; 4, ΔlpxL; 5, ΔlpxM; 6, ΔpagP. (B) Deletion of lipid A biosynthesis-related genes of Safe-9R-derived knock-out strains. The amplicon of Safe-9R for each gene was placed at the first lane of each rectangle to compare the amplicon of knock-out mutant strain.

Figure 2

Comparison of stimulatory effects of knock-out mutant strains on transcriptions of pro-inflammatory cytokines and related genes in HD11 cells. Comparison of relative transcription levels of IL-1β, IL-18, iNOS and TLR4 genes in HD11 after infection of knock-out mutant strains was performed by using the 2–∆∆Ct method. Statistical significance indicated as follows: ns not significant, * significantly different compared with Neg (p < 0.05).

Figure 3

Humoral immune responses of the OE Safe-9R vaccines. Each group received OE Safe-9R at 1 week of age, and serum samples were collected at 2 weeks post-vaccination (wpv) and 7 wpv, respectively. The immune response was analyzed by ELISA made by Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) immunogenic outermembrane proteins (OMP), OmpA and OmpX, and total OMP extracts. * Indicates a significant difference compared with Neg (p < 0.05).

Figure 4

In vivo verification of detoxification by chick body weight model. Inactivated vaccines were inoculated in (A) 1 week-old and (B) 2 week-old chicks and the differences in body weight were examined. The significance was compared to the negative control. Statistical significance indicated as follows: ns not significant, * significantly different compared with Neg (p < 0.05).

Figure 5

Humoral immunogenicity of oil emulsion vaccines of the detoxified strains. Vaccines were inoculated at 1 week of age, and antibody titers were determined after 2 weeks. * Indicates a significant difference compared with Neg (p < 0.05).

Figure 6

Humoral and mucosal immunogenicity of live vaccines with the detoxified strains after challenge. Detoxified vaccines were inoculated in 1 week-old chicks and the virulent field strain was challenged after 1 week of vaccination. Blood and bile samples were collected after 2 weeks of the challenge and analyzed with the ELISA. Statistical significance indicated as follows: ns not significant, * significantly different compared with Neg (p < 0.05).

Figure 7

Proportion of CD8+ T cells in peripheral blood mononuclear cells (PBMCs) determined by fluorescence activated cell sorting. Detoxified vaccines were inoculated in 1 day-old chicks and the whole blood samples were collected in heparin-containing tubes. The samples were pooled by group and PBMCs were isolated. The percentage of (A) CD8+ T cells and (B) CD4+ T cells were analyzed. * Indicates a significant difference compared with Neg (p < 0.05).