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United States Department of Agriculture

Agricultural Research Service

Title: Using Suppression Subtractive Hybridization to Analyze Porcine Differential Gene Expression Following Challenge with Salmonella Enterica Serovar Choleraesuis

Authors
item Uthe, J - IOWA STATE UNIVERSITY
item Stabel, Thomas
item BEARSON, SHAWN
item Zhao, S - IOWA STATE UNIVERSITY
item Tuggle, C - IOWA STATE UNIVERSITY

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 27, 2004
Publication Date: May 23, 2004
Citation: Uthe, J.J., Stabel, T.J., Bearson, S.M., Zhao, S.H., Tuggle, C.K. 2004. Using suppression subtractive hybridization to analyze porcine differential gene expression following challenge with Salmonella enterica serovar Choleraesuis [abstract]. American Society for Microbiology. p. 208.

Technical Abstract: An important aspect of salmonellosis that continues to elude our understanding is the mechanism(s) of Salmonella enterica serovar-host specificity. We hypothesize that Salmonella enterica serovar Choleraesuis (S. choleraesuis) in swine affects host gene expression in a species-specific manner. This initial study was designed to characterize the differential gene expression in pigs after S. choleraesuis infection using a suppression subtractive hybridization assay. Twenty-four Salmonella-free male and female piglets were raised to 7 weeks of age in isolation facilities at the National Animal Disease Center, Ames, IA. Pigs were divided in two groups: non-infected (n = 9) and infected (n = 15). Three non-infected control pigs were necropsied 3 d prior to infection. On day 0, pigs in the infected group were intranasally challenged with 1 x 10**9 CFU S. choleraesuis Chi3246. At 8 h, 24 h, 48 h, 7 d, and 21 d post infection, one control pig (two control pigs on day 21) and three infected pigs were necropsied. Tissue samples from spleen, lung, liver and mesenteric lymph-node were aseptically collected and immediately frozen in liquid nitrogen for future mRNA isolation. Analysis of differential gene expression has been completed on mesenteric lymph-node mRNA at 24 h post infection. Forward and reverse suppression subtractive hybridization was performed using pooled mRNA samples from 3 infected and 3 non-infected pigs. From each library, 288 subtracted clones were differentially screened. Sequence was determined for 33 forward-subtracted cDNA clones and 44 reverse-subtracted clones and then examined by BLAST analysis. Sequenced clones showed 89% homology with known genes. Real-time RT-PCR was used to independently confirm differential expression of sequences deemed relevant to the host response to Salmonella infection. Expression of interferon-gamma-inducible protein 10 (IP-10) was significantly increased in porcine mesenteric lymph node 24 h post infection with S. choleraesuis. IP-10 has been shown to be important in the development of the innate immune response to bacterial infection. Understanding host-specificity is an important step in the prophylactic treatment of salmonellosis in swine.

Last Modified: 9/10/2014
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