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Infection and Immunity, May 2007, p. 2461-2468, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01357-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Connie Adriaensen,¹ Henri De Greve,^1,2 Jean Q. Tian,³ Stéphane De Craeye,^1, Eline Gubbels,¹ Venessa Eeckhaut,⁴ Filip Van Immerseel,⁴ Richard Ducatelle,⁴ Mahesh Kumar,³ and Jean-Pierre Hernalsteens^1*

Viral Genetics Laboratory, Faculty of Sciences, Vrije Universiteit Brussel,¹ Department of Molecular and Cellular Interactions, Flanders Institute for Biotechnology (VIB), Pleinlaan 2, B-1050 Brussels, Belgium,² Fort Dodge Animal Health, 800 Fifth Street NW, Fort Dodge, Iowa 50501,³ Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium⁴

Received 23 August 2006/ Returned for modification 9 October 2006/ Accepted 22 January 2007

Three precisely defined deletion mutants of Salmonella enterica serovar Enteritidis were constructed, a guanine auxotrophic guaB mutant, a nonflagellated fliC mutant, and an auxotrophic and nonflagellated guaB 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 fliC mutant was pathogenic in orally infected BALB/c mice, while the guaB mutant was attenuated and conferred protection against a challenge with the pathogenic parent strain. The guaB fliC double mutant was totally asymptomatic and conferred better protection than the guaB mutant. This indicates that the major flagellar protein flagellin is not required for efficient vaccination of BALB/c mice against Salmonella infection. The guaB 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.

Published ahead of print on 29 January 2007.

Editor: F. C. Fang

Present address: Scientific Institute of Public Health, Pasteur Institute, B-1180 Brussels, Belgium.

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