Safety, Protective Immunity, and DIVA Capability of a Rough Mutant Salmonella Pullorum Vaccine Candidate in Broilers

Abstract

Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) is highly adapted to chickens causing an acute systemic disease that results in high mortality. Vaccination represents one approach for promoting animal health, food safety and reducing environmental persistence in Salmonella control. An important consideration is that Salmonella vaccination in poultry should not interfere with the salmonellosis monitoring program. This is the basis of the DIVA (Differentiation of Infected and Vaccinated Animals) program. In order to achieve this goal, waaL mutant was developed on a spiC mutant that was developed previously. The safety, efficacy, and DIVA features of this vaccine candidate (Salmonella Pullorum ΔspiCΔwaaL) were evaluated in broilers. Our results show that the truncated LPS in the vaccine strain has a differentiating use as both a bacteriological marker (rough phenotype) and also as a serological marker facilitating the differentiation between infected and vaccinated chickens. The rough mutant showed adequate safety being avirulent in the host chicks and showed increased sensitivity to environmental stresses. Single intramuscular immunization of day-old broiler chicks with the mutant confers ideal protection against lethal wild type challenge by significantly stimulating both humoral and cellular immune responses as well as reducing the colonization of the challenge strain. Significantly lower mean pathology scores were observed in the vaccination group compared to the control group. Additionally, the mutant strain generated cross-protection against challenge with the wild type Salmonella Gallinarum thereby improving survival and with the wild type Salmonella Enteritidis thereby reducing colonization. These results suggest that the double-mutant strain may be a safe, effective, and cross-protective vaccine against Salmonella infection in chicks while conforming to the requirements of the DIVA program.

Keywords: DIVA; Salmonella Pullorum; cross-protection; lipopolysaccharides; live attenuated vaccine.

FIGURE 1

Phenotypic characterization of LPS in mutant and wild type strains. (A) SDS-PAGE of LPS analyzed by silver staining. (B) Western blotting analysis of LPS using an anti-O9 monoclonal antibody. (C) Mutant strain has a rough appearance using the acriflavine agglutination test. (D) Schematic representation of the Salmonella Pullorum LPS molecule (lipid A, core polysaccharides and O-antigen).

FIGURE 2

Colonization and persistence of the vaccine strain in liver and spleen after inoculation of day-old chicks with 10⁷ CFU bacterial cells. Values are represented as log₁₀ CFU/g sample. Negative samples are shown as 0 CFU/g; all samples from the control group were negative.

FIGURE 3

Protective ability of the rough mutant against the virulent Salmonella enterica serovar Gallinarum biovar Pullorum and Gallinarum challenge. At fourteen dpi, chicks from each group were challenged intramuscularly with 1 × 10⁹ CFU wild-type Salmonella Pullorum S06004 (A) or virulent Salmonella Gallinarum SG9 (B). Protection was expressed by percentage survival after challenge (^∗∗∗P < 0.001).

FIGURE 4

Levels of humoral and cellular immune responses in vaccinated versus control chicks. Serum IgG antibody responses against the live vaccine strain measured by indirect-ELISA. Asterisks indicate a significant difference between the immunized and PBS control groups (^∗P < 0.05; ^∗∗P < 0.01). Stimulation index of lymphocyte stimulation responses using soluble antigens in immunized chickens at 3 weeks post vaccination. Asterisks indicate a significant difference compared with the PBS control group (^∗P < 0.05).

FIGURE 5

Capability of DIVA based on a serum plate agglutination test. Serum collected on days 7, 14, 21, and 28 were tested for reactivity to the Salmonella Pullorum standard antigen. Chicks in the S06004ΔspiC control group showed a strong agglutination reaction (+/++) on days 21 and 28 post inoculation.

FIGURE 6

Mutant strain survival rates under different environmental stress. The susceptibility of the Salmonella Pullorum mutant strain and its parental strain to hydrogen peroxide (100 μM for 5 min) and 12.5 mM NaOH for 5 min. Data are the means and standard deviations of three experiments (^∗∗∗P < 0.001).