Physiology, pathogenicity and immunogenicity of lon and/or cpxR deleted mutants of Salmonella Gallinarum as vaccine candidates for fowl typhoid

Abstract

To construct a novel live vaccine candidate for fowl typhoid (FT) caused by Salmonella Gallinarum (SG), the lon and cpxR genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Deltalon) and JOL916 (DeltalonDeltacpxR) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (DeltacpxR), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD50 using 1-week-old chickens, were attenuated approximately at 10(1)-, 10(1)-, and >10(3)-fold, respectively. The oral inoculations of 2x10(7) cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT.

Figure 1.

Phenotype examination of Salmonella Gallinarum wild type strain (JOL394), Δlon (JOL914), ΔcpxR (JOL915), and ΔlonΔcpxR (JOL916) mutant strains: A, relative extra-cellular polysaccharide of 24-h culture on LB agar examined by concanavalin-A binding fluorometric assay; B, relative FimA expression of 24-h culture on LB agar by dot-blot analysis. Error bars indicate the SEM. * Significant difference of mutants from the parental strain JOL394 (p < 0.05).

Figure 2.

Antigen-specific immune responses in immunized chickens with Salmonella Gallinarum wild type strain (JOL394), JOL914, JOL915, and JOL916: A, SI of lymphocyte sample from the chickens by peripheral lymphocyte proliferation assay using soluble antigen at 10th day post inoculation; B, plasma IgG concentration (μg/mL) by indirect-ELISA at second week post immunization. Error bars indicate the SEM (n = 5). * Immunized chickens showed the significant immune responses compared to the control group that was inoculated with PBS (p < 0.05).