Immunization of pigs to prevent disease in humans: construction and protective efficacy of a Salmonella enterica serovar Typhimurium live negative-marker vaccine

Abstract

Zoonotic infections caused by Salmonella enterica serovar Typhimurium pose a constant threat to consumer health, with the pig being a particularly major source of multidrug-resistant isolates. Vaccination, as a promising approach to reduce colonization and shedding, has been scarcely used, as it interferes with current control programs relying on serology as a means of herd classification. In order to overcome this problem, we set out to develop a negative-marker vaccine allowing the differentiation of infected from vaccinated animals (DIVA). Applying an immunoproteomic approach with two-dimensional gel electrophoresis, Western blot, and quadrupole time-of-flight tandem mass spectrometry, we identified the OmpD protein as a suitable negative marker. Using allelic exchange, we generated an isogenic mutant of the licensed live vaccine strain Salmoporc and showed that virulence of Salmoporc and that of the mutant strain, SalmoporcDeltaompD, were indistinguishable in BALB/c mice. In a pig infection experiment including two oral immunizations with SalmoporcDeltaompD and challenge with a multiresistant S. enterica serovar Typhimurium DT104 clinical isolate, we confirmed the protective efficacy of SalmoporcDeltaompD in pigs, showing a significant reduction of both clinical symptoms and colonization of lymph nodes and intestinal tract. OmpD immunogenic epitopes were determined by peptide spot array analyses. Upon testing of several 9-mer peptides, each including an immunogenic epitope, one peptide (positions F(100) to Y(108)) that facilitated the detection of infected animals independent of their vaccination status (DIVA function) was identified. The approach described overcomes the problems currently limiting the use of bacterial live vaccines and holds considerable potential for future developments in the field.

FIG. 1.

Discriminatory efficacy of OmpD-derived immunogenic epitopes. Biotinylated epitopes 1 to 4 were used as solid-phase antigens on streptavidin-coated ELISA plates and incubated with internal positive and negative control sera, respectively, obtained from animals in the vaccination trial. The bars show the means and standard deviations of three independent ELISA experiments. OD%, relative optical density of the negative control serum compared to that of the positive control serum (defined as 100 OD%) for each coating peptide.

FIG. 2.

Characterization of S. enterica serovar Typhimurium SalmoporcΔompD. Phenotypes were characterized using membrane preparations of Salmoporc (lane 1), SalmoporcΔompD (lane 2), and SalmoporcΔompD complemented in trans with plasmid pSOM810 (lane 3). Protein bands A to C were sequenced using Q-TOF MS/MS and identified as being OmpC (A), OmpD (B), and OmpF (C), respectively. LMW, low molecular weight marker (Amersham Pharmcia Biotech AB, Uppsala, Sweden).

FIG. 3.

Clinical scores of pigs in the vaccination and challenge trial. The total scores of individual animals for days 1 to 7 postinfection and the arithmetic means (horizontal bars) calculated for groups 1 to 3 (six animals each) are given. The asterisks indicate statistical significance (Wilcoxon test), and the P values are given.

FIG. 4.

Serological responses of pigs in a discriminatory ELISA upon immunization and challenge. Animals were immunized on days 1 and 21 and challenged on day 43. Blood was taken on days 0, 36, and 50 (six animals per group) and on days 57 and 65 (three animals). Anti-OmpD antibody titers were determined using an ELISA with OmpD-derived peptide 2 as a solid-phase antigen. The horizontal line marked with “+” (OD% = 100) indicates the position of the internal positive control consisting of the pooled sera from day 50. OD%, relative optical density of the text sera compared to that of the internal positive control serum (defined as 100 OD%).