A Live Salmonella enterica Serovar Enteritidis Vaccine Allows Serological Differentiation between Vaccinated and Infected Animals

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Abstract
Three precisely defined deletion mutants of Salmonella enterica serovar Enteritidis were constructed, a guanine auxotrophic Δ guaB mutant, a nonflagellated Δ fliC mutant, and an auxotrophic and nonflagellated Δ guaB Δ fliC double mutant. All three mutants were less invasive than the wild-type strain in primary chicken cecal epithelial cells and the human epithelial cell line T84 and less efficiently internalized in the chicken macrophage cell line HD11. The Δ fliC mutant was pathogenic in orally infected BALB/c mice, while the Δ guaB mutant was attenuated and conferred protection against a challenge with the pathogenic parent strain. The Δ guaB Δ fliC double mutant was totally asymptomatic and conferred better protection than the Δ guaB mutant. This indicates that the major flagellar protein flagellin is not required for efficient vaccination of BALB/c mice against Salmonella infection. The Δ guaB Δ fliC mutant was also safe for vaccination of 1-day-old chickens. After two immunizations, it induced statistically significant protection against infection of the internal organs of the birds by a virulent S. enterica serovar Enteritidis challenge strain but not against intestinal colonization. These data demonstrate that nonflagellated attenuated Salmonella mutants can be used as marker vaccines.