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Infection and Immunity, November 2008, p. 5310-5321, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00960-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Salmonella enterica Serovar Dublin-Specific Sequences by Subtractive Hybridization and Analysis of Their Role in Intestinal Colonization and Systemic Translocation in Cattle{triangledown} ,{dagger}

Gillian D. Pullinger, Francis Dziva, Bryan Charleston, Timothy S. Wallis,{ddagger} and Mark P. Stevens*

Division of Microbiology, Institute for Animal Health, Compton, Berkshire RG20 7NN, United Kingdom

Received 31 July 2008/ Returned for modification 16 August 2008/ Accepted 4 September 2008

Salmonella enterica serovar Dublin is a host-restricted serovar associated with typhoidal disease in cattle. In contrast, the fowl-associated serovar S. enterica serovar Gallinarum is avirulent in calves, yet it invades ileal mucosa and induces enteritis at levels comparable to those induced by S. enterica serovar Dublin. Suppression subtractive hybridization was employed to identify S. enterica serovar Dublin strain SD3246 genes absent from S. enterica serovar Gallinarum strain SG9. Forty-one S. enterica serovar Dublin fragments were cloned and sequenced. Among these, 24 mobile-element-associated genes were identified, and 12 clones exhibited similarity with sequences of known or predicted function in other serovars. Three S. enterica serovar Dublin-specific regions were homologous to regions from the genome of Enterobacter sp. strain 638. Sequencing of fragments adjacent to these three sequences revealed the presence of a 21-kb genomic island, designated S. enterica serovar Dublin island 1 (SDI-1). PCR analysis and Southern blotting showed that SDI-1 is highly conserved within S. enterica serovar Dublin isolates but rarely found in other serovars. To probe the role of genes identified by subtractive hybridization in vivo, 24 signature-tagged S. enterica serovar Dublin SD3246 mutants lacking loci not present in Salmonella serovar Gallinarum SG9 were created and screened by oral challenge of cattle. Though attenuation of tagged SG9 and SD3246 Salmonella pathogenicity island-1 (SPI-1) and SPI-2 mutant strains was detected, no obvious defects of these 24 mutants were detected. Subsequently, a {Delta}SDI-1 mutant was found to exhibit weak but significant attenuation compared with the parent strain in coinfection of calves. SDI-1 mutation did not impair invasion, intramacrophage survival, or virulence in mice, implying that SDI-1 does not influence fitness per se and may act in a host-specific manner.

* Corresponding author. Mailing address: Division of Microbiology, Institute for Animal Health, Compton, Berkshire RG20 7NN, United Kingdom. Phone: 44 (0)1635 577915. Fax: 44 (0)1635 577237. E-mail: mark-p.stevens{at}bbsrc.ac.uk

{triangledown} Published ahead of print on 15 September 2008.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: A. J. Bäumler

{ddagger} Present address: Ridgeway Biologicals Ltd., Institute for Animal Health, Compton, Berkshire RG20 7NN, United Kingdom.

Infection and Immunity, November 2008, p. 5310-5321, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00960-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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