Evaluation of protective efficacy of live attenuated Salmonella enterica serovar Gallinarum vaccine strains against fowl typhoid in chickens

Abstract

Salmonella enterica serovar Gallinarum is the etiological agent of fowl typhoid, which constitutes a considerable economic problem for poultry growers in developing countries. The vaccination of chickens seems to be the most effective strategy to control the disease in those areas. We constructed S. Gallinarum strains with a deletion of the global regulatory gene fur and evaluated their virulence and protective efficacy in Rhode Island Red chicks and Brown Leghorn layers. The fur deletion mutant was avirulent and, when delivered orally to chicks, elicited excellent protection against lethal S. Gallinarum challenge. It was not as effective when given orally to older birds, although it was highly immunogenic when delivered by intramuscular injection. We also examined the effect of a pmi mutant and a combination of fur deletions with mutations in the pmi and rfaH genes, which affect O-antigen synthesis, and ansB, whose product inhibits host T-cell responses. The S. Gallinarum Δpmi mutant was only partially attenuated, and the ΔansB mutant was fully virulent. The Δfur Δpmi and Δfur ΔansB double mutants were attenuated but not protective when delivered orally to the chicks. However, a Δpmi Δfur strain was highly immunogenic when administered intramuscularly. All together, our results show that the fur gene is essential for the virulence of S. Gallinarum, and the fur mutant is effective as a live recombinant vaccine against fowl typhoid.

FIG 1

Phenotype characterization of S. Gallinarum Δfur mutants. (A) Fur production in S. Gallinarum wild-type (287/91), Δfur-453::cam (χ11575), and Δfur-712 vaccine strains (χ11798, χ11820, χ11821, and χ11823). Whole-cell lysates were obtained from overnight cultures, electrophoresed on a 12% SDS-PAGE gel, transferred onto nitrocellulose, and probed with anti-Fur serum (top). The blot was also probed with anti-GroEL antibodies (bottom) to serve as a loading control. (B) IROMP production in S. Gallinarum vaccine strains. OMPs were obtained by Sarkosyl extraction from overnight cultures, electrophoresed on a 10% SDS-PAGE gel, and stained with Coomassie blue. wt, wild type. (C) Effect of acid shock on viability of S. Gallinarum fur mutants. Strains 287/91 (wt), χ 11741 (Δpmi-2426), χ11797 (Δfur-712), and χ11798 (Δpmi-2426 Δfur-712) were grown in LB to early logarithmic phase, washed in E medium (pH 7.0), and then challenged with E medium (pH 3.0). Survival was monitored by plating samples on LB agar. The data shown are the mean and standard error of the mean (SEM) values from four independent experiments. A statistical analysis was carried out using two-way ANOVA, followed by Tukey's multiple-comparison test. All possible pairs of data within each time point except 287/91 versus χ11741 were significantly different (P < 0.01).

FIG 2

Colonization of spleen and liver in birds that survived the challenge with wild-type S. Gallinarum. Survivors from experiment (expt.) 3 (Table 4) were euthanized 19 days postinfection, and spleens and livers were collected to recover viable S. Gallinarum from each tissue. The organs were homogenized, diluted, and plated on LB agar. Negative samples were additionally enriched using RV broth and plated on SS agar.