The porcine response to infection with Salmonella is the result of differential expression of host-specific genes. To characterize these alterations in gene expression, functional genomic analyses were performed on swine tissues following experimental inoculation of the pigs with Salmonella enterica serovars Choleraesuis and Typhimurium. Suppression subtractive hybridization and quantitative real-time RT-PCR revealed that the transcriptional profiles of the porcine response to the swine-adapted strain (Choleraesuis) and the non-host-adapted strain (Typhimurium) exhibit unique differences

Bearson, Shawn M. D.

Kim, Jae Woo

Lunney, Joan K.

Royaee, Atabak R.

Tuggle, Chris K.

Uthe, Jolita J.

Zhao, Shu-Hong

English

The porcine response to infection with Salmonella is the result of differential expression of host-specific genes. To characterize these alterations in gene expression, functional genomic analyses were performed on swine tissues following experimental inoculation of the pigs with Salmonella enterica serovars Choleraesuis and Typhimurium. Suppression subtractive hybridization and quantitative real-time RT-PCR revealed that the transcriptional profiles of the porcine response to the swine-adapted strain (Choleraesuis) and the non-host-adapted strain (Typhimurium) exhibit unique differences.</p

Bearson, Shawn

Uthe, Jolita

Royaee, Atabak

Lunney, Joan

Tuggle, Chris

Digital Repository @ Iowa State University (ISU)

68ORALPRESENTATIONSSafePork 2005THE TRANSCRIPTIONAL RESPONSE TO SALMONELLAINFECTION IN SWINEShawn M.D. Bearson*1, Jolita J. Uthe1,2, Atabak R. Royaee3, Joan K. Lunney3, Shu-HongZhao2, Jae Woo Kim2, Chris K. Tuggle2*1USDA, ARS National Animal Disease Center, 2300 Dayton Rd, Ames, IA, 50010, USA, Ph:  515-663-7455, Email: sbearson@nadc.ars.usda.gov; 2Iowa State University, Ames, IA, USA; 3USDA, ARS, Beltsville Agricultural ResearchCenter, Beltsville, MD, USAAbstract The porcine response to infection with Salmonella is the result of differential expressionof host-specific genes. To characterize these alterations in gene expression, functional genomicanalyses were performed on swine tissues following experimental inoculation of the pigs withSalmonella enterica serovars Choleraesuis and Typhimurium. Suppression subtractive hybridizationand quantitative real-time RT-PCR revealed that the transcriptional profiles of the porcine responseto the swine-adapted strain (Choleraesuis) and the non-host-adapted strain (Typhimurium) exhibitunique differences. Our research demonstrates not only a difference in gene expression in ani-mals infected with two different strains of Salmonella (host-specificity), but also alterations in thetranscriptome over a time course of infection from the acute state (48 hr) to the chronic or carrierstage (21 days).  Introduction The ability of higher animals to quickly and specifically alter their gene expression inresponse to pathogenic bacteria is an important mechanism to fight infection (Rosenberger et al.,2000; Strieter et al., 2002). Studies have documented differences in the immune responsivenessamong animal breeds (Warner et al., 1987; Edfors-Lilja et al. 1998; Edfors-Lilja et al., 2000) andsignificant genetic control of disease resistance has been reported in pigs (Sellwood, 1979;Rothschild et al., 1984; Lunney and Butler, 1998; Meijerink et al., 2000; Lunney, 2003). Thus,understanding and improving the genetic control of disease resistance could benefit the swineindustry by identifying useful tools for livestock breeding and production. Furthermore, since thetranscriptional response mounted by the host’s immune system is highly pathogen-specific, profil-ing of the host immune gene expression is a recent revolutionary approach for diagnosing infec-tious diseases (Bodrossy and Sessitsch, 2004; Bryant et al, 2004; Campbell et al, 2004; Dawsonet al., 2005).  As Salmonella is a foodborne pathogen that compromises animal health and food safety via itsability to cause intestinal and systemic disease, the objective of this research was to use a molec-ular approach to identify differentially expressed swine genes in response to Salmonella infection.The goals were to 1. investigate the host-specific response of Salmonella-infected pigs using two strains of Salmonella, a swine-adapted strain (Choleraesuis) and a generalist strain (Typhimurium) and 2. characterize the transcriptional profile of disease over a time course of infection from the acute to the chronic stage. Understanding the immunological response of the pig and identifying the host signal(s) that con-fer resistance to Salmonella may provide targets for new drug discovery and diagnostic tests,improve intervention strategies, and/or pinpoint modifications in production practices to addresspathogen load.Materials and Methods Conventionally raised piglets from Salmonella spp.-free sows wereweaned at 10 days (d) of age, shipped to the NADC in Ames, IA and raised in isolation facilities. Toconfirm that all piglets were free of Salmonella prior to the experiment, bacteriological culturingwas performed twice on rectal swabs. At 7 weeks of age, the pigs were divided into non-infectedand infected groups with the infected groups receiving an intranasal challenge of 1 x 109 CFU ofeither S. Choleraesuis x3246 or S. Typhimurium x4232. Rectal temperatures and clinical signs ofinfection (lethargy, loss of appetite and diarrhea) were recorded for each animal daily. At 8 hours(h), 24 h, 48 h, 7 d and 21 d post-infection (p.i.), three infected pigs were necropsied. Tissue sam-ples from the mesenteric lymph nodes and lungs were aseptically collected and immediatelyfrozen in liquid nitrogen for future mRNA isolation. Samples of the ileocecal lymph node wereused for quantitative bacteriology.Total RNA was prepared by homogenization of the frozen tissues in liquid nitrogen, extracted ORALPRESENTATIONS69SafePork 2005using TRIzol reagent (Invitrogen), and purified using the RNeasy Midi kit and a RNase-free DNaseset (Qiagen). Messenger RNA was isolated from total RNA using an Oligotex mRNA mini kit(Qiagen). The concentration and purity of the total RNA and mRNA was determined using theAgilent 2100 Bioanalyzer and the Genequant pro spectrophotometer (Amersham Biosciences).  Suppression subtractive hybridization was performed using the PCR Select cDNA Subtractionkit (Clontech). Forward and reverse subtractions were carried out using pooled mesenteric lymphnode mRNA samples from three S. Choleraesuis and three S. Typhimurium infected pigs at 24 hpost infection. Secondary (nested) PCR-amplified forward and reverse subtracted cDNA popula-tions were cloned into the pBAD vector (pBAD TOPO TA Expression Kit, Invitrogen) and trans-formed into E. coli DH5a. Following overnight growth on selective media, random colonies werepicked into 96-well plates for storage at -70°C.Differential screening was performed on the subtracted clones to confirm their unique geneexpression. The cDNA inserts were amplified by PCR using the following conditions: 95°C for 1min followed by 30 cycles at 94°C for 30 sec, 68°C for 30 sec, 72°C for 1 min and a final cycle at72°C for 5 min. PCR products were analyzed on 2% agarose gels to identify insert-containingclones. To prepare for Southern hybridization, 2 identical nylon membranes were made using theBio-Dot apparatus (BioRad) by blotting 5 ml of denatured, PCR amplified cDNA inserts. For probepreparation, the forward and reverse subtracted secondary PCR products were digested with RsaI to remove the adaptors then purified using QIAquick PCR purification kit (Qiagen). Probes (l00ng of denatured cDNA) were generated by alkaline phosphatase labeling with Gene ImagesAlkPhos Direct Labeling and Detection System (Amersham Biosciences). The two identical mem-branes were hybridized overnight at 55°C, one with the forward and the other with the reversesubtracted probes. The hybridization signals were visualized and quantified using a Multi-ImagerLight Cabinet (Alpha Inotech Corporation). cDNA clones were considered differentially expressedwhen blots probed with the subtracted tester repeatedly demonstrated a signal intensity >1.5-folddifferent than blots probed with the subtracted driver. Plasmids containing differentially expressedcDNA were extracted and sequenced by dideoxy chain termination using an ABI 3700 DNAAnalyzer (Applied Biosystems Inc.) at the Iowa State University DNA sequencing and synthesisfacility. DNA homology searches were conducted using the Basic Local Alignment Search Tool(BLAST) from the National Center of Biotechnology Information (NCBI) (Altschul et al., 1997). Allselected plasmids were re-blotted in triplicate on nylon membranes and re-hybridized to forwardand reverse subtracted secondary PCR products using the above described technique. Southernhybridization data was analyzed using the unpaired t test from GraphPad InStat software(GraphPad Software Inc).Quantitative RT-PCR was performed to verify the differential expression of the clones of inter-est as well as measure specific immune marker gene expression (Dawson et al, 2005; Royaee etal, 2004). Using ABI PRISM 7700 and 7900 Sequence Detection Systems (Applied Biosystems),transcripts were amplified in triplicate and detected using either the QuantiTect SYBR Green RT-PCR kit (Qiagen) with amplification conditions of 50°C for 30 min, 95°C for 15 min, 35 cycles at94°C for 15 sec, 55°C to 60°C for 30 sec (the annealing temperature varied depending on thegene specific primers), and 72°C for 5 sec or published procedures using the Stratagene Brilliantkit and 100 ng cDNA at 50°C for two min; 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°Cfor 1 min; then 4°C (Royaee et al., 2004; Dawson et al., 2005). Relative quantification of targetgene expression was evaluated using cycle threshold (Ct) values. Gene expression data was nor-malized to the amount of RNA/cDNA amplified (Bustin, 2002). Fisher’s LSD post-hoc test wasapplied to assess differences between groups of pigs at different time points post infection.  P<= 0.05 was considered statistically significant for all analyses.Results As Salmonella spp have a large number of specific hosts, including birds, reptiles andmammals, Salmonella enterica serovars Choleraesuis (swine-adapted) and Typhimurium (general-ist) were chosen in investigate Salmonella-host specificity in swine. Suppression subtractivehybridization was performed using pooled mRNA samples from the mesenteric lymph nodes ofthree S. Choleraesuis-infected pigs and three S. Typhimurium-infected pigs at 24 h p.i. Followingdifferential screening by DNA blot hybridization, the DNA sequence was determined for 39 cDNAclones identified as induced in the S. Choleraesuis-infected pigs (compared to the S. Typhimurium-infected pigs) and 44 cDNA clones induced in the S. Typhimurium-infected pigs. Real-time RT-PCR70ORALPRESENTATIONSSafePork 2005on select immune response genes (CXCL10, HSP105, SDCBP) validated the SSH results.  Severalof the genes identified in the SSH study were mapped to specific chromosomes and physical link-age positions using Radiation Hybrid mapping. Two of the genes, SDCBP and CXCL10, mappedwithin known quantitative trait loci (QTL) peaks for mitogen-induced proliferation and lymphocytenumber, respectively (Edfors-Lilja et al. 1998;Edfors-Lilja et al., 2000). The expression of a panel of immune genes was examined by real-time RT-PCR to furtherdefine the porcine response to Salmonella infection. S. Choleraesuis induced a more intense andextended up-regulation of porcine immune gene expression (IFNG, IL1B, IL6, TLR4) whereas S.Typhimurium triggered both a transient up-regulation (IFNG, SOCS1, STAT1) as well as a significantdown-regulation in gene expression of many host immune factors (IL1B, IL4, IL6, TLR4, CSF2).    Discussion The clinical response elicited in swine infected with S. Choleraesuis is more severethan the clinical response to S. Typhimurium (Reed et al., 1986); furthermore, S. Choleraesuis usu-ally does not progress into a carrier state in the host as observed with S. Typhimurium (Gray etal., 1996; Watson et al., 2000). The aim of our research was to investigate the molecular dynamicsresponsible for this difference in Salmonella-host specificity and characterize the porcine responseduring the various stages of Salmonella infection using functional genomic analyses. SuppressionSubtractive Hybridization identified and real-time RT-PCR confirmed the differential expression ofgenes with a role in a variety of host cellular functions in response to Salmonella infection, includ-ing genes involved in innate immunity. Further genetic investigation revealed that two of the dif-ferentially expressed genes mapped to QTL sites for controlling mitogen-induced proliferation ofwhole blood cells (SDCBP) (Edfors-Lilja et al. 1998) and the number of circulating lymphocytes fol-lowing mixing and transport of pigs (CXCL10) (Edfors-Lilja et al., 2000). CXCL10 expression hasbeen shown to increase lymphocyte proliferation in the spleen in vitro (Whiting et al., 2004).Therefore, this expression information will be useful in developing markers and screening for DNApolymorphisms associated disease response, potentially improving pig genetics.As several immune-related genes were identified in the SSH analysis, specific immune geneswere selected for transcriptional profiling using real-time RT-PCR.  Unique differences wereobserved between the porcine gene expression responses to the two Salmonella strains: in gen-eral, a strong Th1 response (IFNG, INDO, STAT1) and expression of genes involved in inflamma-tion and innate immunity (IL1B, IL6, TLR4) were observed in pigs inoculated with S. Choleraesuis.The pigs inoculated with S. Typhimurium exhibited a repression in Th2, innate and inflammatoryresponse gene expression (IL1B, IL4, IL6, TLR4, CSF2) and only a transitory induction in the Th1response (IFNG, SOCS1, STAT1).Conclusions The porcine transcriptional responses during infection with S. Choleraesuis (swine-adapted) and S. Typhimurium (non-host-adapted) revealed unique host-specific differences.Although a response by the pigs to infection with both Salmonella strains was initiated by 24hours p.i., the nature and persistence of that response was different between the two pathogens:S. Choleraesuis infection induced classic inflammatory gene expression, while S. Typhimuriuminduced a mild and transient transcriptional response of immune-related genes.  These investiga-tions suggest that S. Typhimurium may down-regulate the porcine immune response, therebypotentially evading aspects of the host’s immune system and progressing into a carrier state inthe animal. ReferencesAltschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.  Nucleic Acids Res 25:3389-3402Bodrossy L., Sessitsch A. 2004. Oligonucleotide microarrays in microbial diagnostics. Curr Opin Microbiol 7:245-54Bryant P.A., Venter D., Robins-Browne R., Curtis N. 2004. Chips with everything: DNA microarrays in infectious diseases.Lancet Infect Dis 4:100-11Bustin, S.A. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J MolEndocrinol 29:23-39Campbell C.J., Ghazal P. 2004. 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The transcriptional response to Salmonella infection in swine

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The transcriptional response to Salmonella infection in swine

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