Joan Lunney

Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes 

Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD) 

Genomics of interaction of Salmonella with 

porcine lymph nodes and enterocytes 

Joan Lunney 

APDL, BARC, ARS, USDA, Beltsville, MD 

Thursday 7th June 2007 

E. coli and Salmonella workshop 

Utrecht, The Netherlands 

Transcriptional Response of Pigs to Salmonella 

infection: Comparison of responses to infection with 

Salmonella enterica serotype Typhimurium as that 

observed in S. Choleraesuis infection 

JJ Uthe 1,2 , SMD Bearson 2 , YF Wang 1 , L Qu 1 , 

D Kuhar 3 , TJ Stabel 2 , SH Zhao 4 , OP Couture 1 , 

D Nettleton 5 , JC Dekkers 1 , CK Tuggle 1 , JK Lunney 3 

1 Dept. of Animal Science, Iowa State University, Ames, IA USA 

2 

National Animal Disease Center, USDA-ARS, Ames, IA USA 

3 

Animal Parasitic Diseases Lab, USDA-ARS, Beltsville, MD USA 

4 

Lab of Molecular Biology and Animal Breeding, Huazhong 

Agricultural Univ., Wuhan, PR China 

5 

Dept. of Statistics, Iowa State University, Ames, IA 

Funding: USDA ARS funds and CSREES grants 

Research Goals 

General: Understand immune and genetic basis of swine infectious 

disease responses to pathogens 

Specific: Compare effects of Salmonella enterica serotypes 

Choleraesuis (SC) and Typhimurium (ST) on gene and protein 

expression in infected pig tissues and in monolayer cultures of 

porcine epithelial cells (IPEC J2 cells) 

Innate 

Immunity 

Targets: 

• Predict host protective responses to bacteria 

• Improve strategies for vaccination to prevent infection 

• Discover new targets for antibacterial agents for disease control 

• Identify differences in host responses between Salmonella 

pathogens 

Long term goal: Prevent bacterial persistence 

E. Thacker. The Pig Journal 54: 55 (2004) www.thepigsite.com/FeaturedArticle/Default.asp?Display=1284 

Differentiation Swine Immune Gene 

Expression: T helper 1 (Th1) versus Th2 

Definition of Th1 (Toxoplasma gondii) and Th2 

(Ascaris suum) immune responses in swine. 

Use of real-time RT-PCR technology to measure targeted gene 

expression at multiple tissues sites 

Link between Innate and Adaptive Immune Systems 

IFNA? 

? 

Dawson HD, Beshah E, Nishi S, Solano-Aguilar G, Morimoto M, 

Zhao A, Madden KB, Ledbetter TK, Dubey JP, Shea-Donohue T, 

Lunney JK, Urban JF Jr. 2005. Infection and Immunity 73: 1116. 

PIN (Porcine Immunology and Nutrition) database 

http://www.ars.usda.gov/Services/docs.htm?docid=6065 

Curated by Harry Dawson, NRFL, BHNRC, BARC 

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL 

This presentation is the property of Joan Lunney of the USDA. 

This presentation should not be reproduced without the author's permission. 1/7



Toll-Like Receptor and NFκB Pathways [Tuggle] 

www.biocarta.com 

Swine Salmonella infections 

Comparative gene expression response studies 

Salmonella enterica serovar Typhimurium [S. Typhimurium, ST] 

pig and human pathogen - broad host range 

clinical disease - usually enterocolitis 

food safety issue 

Salmonella enterica serovar Choleraesuis [S. Choleraesuis, SC] 

pig pathogen - narrow host range 

clinical disease - septicemia, enterocolitis, pneumonia and/or 

hepatitis 

References: 

Zhao et al, Mamm Genome. 17: 777, 2006 

Uthe et al, Vet Micro 114: 60, 2006; Molec Immunol 44: 2900, 2007 

Wang et al, Genomics In Press, 2007 (ST); In preparation (SC). 

Tuggle et al, next talk 

Swine Gene Arrays 

swine long oligo microarrays 

• NRSP8-Qiagen 12,500 probes, 2003 design 

Zhao et al. Genomics. 86: 618, 2005; 

Mamm Genome 17: 777, 2006 

• updated NRSP8-Illumina 20,000 probes, 2006 design 

Testing underway 

Affymetrix arrays 

• 20,000 probes, 2004 

Wang et al. Genomics. 2007 May 11 epub 

Arrays enable broad tissue specific gene expression analyses; 

data affirmed with real time PCR analyses and protein 

assays [when assay and relevant sample are available.] 

Quantitative bacteriology of the ileocecal lymph nodes 

in swine inoculated with Salmonella 

[Most probable number method used for Salmonella cfu; 

A 

cfu/g ICLN 

1000000 

100000 

10000 

1000 

100 

10 

1 

# statistically different at p



Gene expression profiling in Salmonella serovar Choleraesuis 

infected lung using a long oligonucleotide Qiagen-NRSP-8 swine array 

Real-time 

immune gene 

expression in 

S. Choleraesuis 

infected porcine 

lung 

Function classification of increased genes 

at 48 hour infection with p values < 0.001 

Use real time assays to 

target sets of genes 

involved in pathways 

identified with arrays 

and with anti-bacterial 

immune responses. 

Zhao et al. 2006. Mammalian Genome. In Press. 

Summary of immune gene expression in 

S. Choleraesuis infected porcine lung 

• qRT-PCR of 61 differentially expressed (DE) genes confirmed 

the microarray results. [23/33 DE genes confirmed] 

• Two transglutaminase family genes (TGM1 and TGM3), 

associated with apoptosis, showed dramatic increases post 

inoculation; affirmed by qRT-PCR for other genes, indicating 

induction of apoptotic pathways 

• Predominant T helper 1 (Th1)-type immune response, with 

interferon gamma (IFNG) significantly increased at 48 hpi 

along with genes induced by IFNs (GBP1, GBP2, C1S, C1R, 

MHC2TA, PSMB8, TAP1, TAP2) in porcine lung infection. 

• Limited changes in innate and Th2 associated genes and 

general immune associated genes. 

Zhao et al. Mammalian Genome. 17: 777, 2006 

www.biocarta.com 

Swine Salmonella infections – 

comparative response studies 

Infections: 

Salmonella enterica serovar Typhimurium (ST) 

S. Typhimurium; pig and human pathogen; clinical 

disease and food safety issue 

S. Choleraesuis (SC) 

pig pathogen; clinical disease 

Animals: 3 pigs/necropsy at 8, 24, 48 hr pi; 7 and 21 dpi 

Tissues collected: lung, mesenteric lymph node (MLN), 

liver, spleen 

Analyses: suppression subtractive hybridization (SSH) 

http://users.path.ox.ac.uk/~ciu/FionaPowrieGroup1.htm 




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Differentially expressed genes identified by SSH in MLN 

from swine experimentally inoculated with S. Choleraesuis 

Uthe et al. Vet Micro 

114: 60, 2006 

MLN Differential gene expression in response 

to S. Choleraesuis and S. Typhimurium 

Suppression subtractive hybridization (SSH) identified 

7 genes as up-regulated in MLN at 24 hpi in S. Choleraesuisinfected 

pigs (ARPC2, CCT7, HSPH1, LCP1, PTMA, SDCBP, 

VCP) 

3 genes in S. Typhimurium-infected pigs 

(CD47/IAP, CXCL10, SCARB2) 

Experimental design: These genes were then targeted for 

comparative analyses of gene expression on MLN RNA 

collected at 8, 24, 48 hr pi; and at 7 and 21 dpi 

Uthe et al., Molec. Immunol. 44: 2900, 2007 

Differential expression of 

the SSH-enriched genes 

from the Salmonellainfected 

swine using realtime 

PCR. 

The results are expressed as 

the fold change in gene 

expression in the 

S. Choleraesuis- (solid) or 

S. Typhimurium- (open) 

infected pigs compared to the 

non-infected controls. 

Statistical differences (* = P



Conclusions: Salmonella immunity studies 

For both pathogens, immune response initiated within 24 hr; 

intensity, nature and persistence differed between pathogens. 

S. Choleraesuis infection stimulated classic innate and 

inflammatory response lasting to 7, even 21, dpi 

S. Typhimurium infection stimulated a mild and transient immune 

response, potentially evading (suppressing?) pig’s immune 

system. This may aid progression into persistent infection, and 

carrier state. 

Immune gene expression correlates well with the clinical signs 

(fever) in infected animals, indicating the expression profiles will 

reveal pathways important to disease resistance and prevention 

Gene expression kinetics may reveal genes responsible for the 

variation in disease progression observed in swine infected with 

S. Typhimurium compared to S. Choleraesuis. 

Uthe et al., Molec. Immunol. 44: 2900, 2007 

Salmonella response studies 

Compare early response [0, 8, 24, 48 hpi] to both 

pathogens, S. Typhimurium (ST) and S. Choleraesuis 

(SC) infections 

Affymetrix swine array [23,256 transcripts from 20,201 

genes] 

16,229 and 16,046 probe sets (~70 %) showed MLN 

expression during the ST infection and the SC 

infection, respectively 

848 and 1,949 genes showed differential expression 

across different times after ST and SC inoculation or 

when compared to non-inoculated controls. 

Wang, Qu, et al Genomics 2007 epub; in preparation 

Gene Ontology (GO) categorization of porcine ST infected MLN transcriptome 

Transcriptional profile of genes 

selected as cell-type markers or 

immune response pathway genes 

shows no evidence that 

transcriptional changes are due to 

cell migration into ST infected MLN. 

The fold change were calculated using 

Genecluster. Statistical differences (P



Early Take Home Lessons from 

Gene Expression Studies 

• Developed improved understanding of immune 

pathways (T helper 1, apoptosis, NFκB) regulating 

anti-Salmonella responses 

• Verified complexity of host specific factors 

• Identified potential genes which encode genomic 

controls 

• Highlighted target responses and pathways for new 

vaccination and drug treatment strategies 

• Test use of cell lines (IPEC J2) for in vitro studies 

Combine 24h/0, 48h/0, 24h/8h, 48h/24h up regulated genes in SC. 

• Pathogen specific patterns [dependence on pathogen 

isolate, local tissue, cellular interactions, time after 

infection] could be enhanced with microbial arrays 

Salmonella-cell culture invasion assay using IPEC-J2 cells 

Question: Can selected cell cultures reveal bacterial infection 

effects? 

Model: Porcine intestinal epithelial cells (IPEC-J2) grown in 

transwell cultures are enterocyte-like with microvilli, tight 

junctions and glycocalyx-bound mucin. Proven to be a relevant 

in vitro model system for porcine intestinal pathogen-host cell 

interactions; can be infected with Salmonella 

(Schierack et al. Histochem Cell Biol. 125: 293, 2006.) 

Samples collected: 

Cells for RNA by adding Trizol, freezing at –80°C 

and shipping to BARC for gene expression assays 

Supernatant from the Transwell insert and bottom of 

the well for protein expression assays. [not yet tested] 

Salmonella invasion analysis: Triton x-100 to Transwell; plate lysate dilutions 

Salmonella-cell culture invasion assay using IPEC-J2 cells 

I. Preparation of polarized IPEC-J2 cells: 

• Polarize IPEC-J2 cells in 6 well plates-permeable supports 

(Transwell polyester membranes with 0.4 μm pore size). 

• Seed IPEC-J2 cells at density 3.5-2.5x105 cells/well. 

• Polarize cells for 7-9 days. 

II. Preparation of bacterial inoculum: 

• Start bacterial cultures from a single colony. Grow overnight. 

III. IPEC-J2 invasion-gene expression experiment: 

• Prewash IPEC-J2 cells; Add inoculum at MOI=300; control 

medium only. 

• At 2 hr after inoculation, add Gentamicin bacteria killing media 

• Incubate cells at 37°C, 5% CO2 for 2 hr, 4 hr and 8 hr 

Effect of S. Choleraesuis (SC) and S. Typhimurium 

(ST) infection on gene expression in monolayer 

cultures of porcine IPEC J2 epithelial cells 

• In vivo earlier, but limited, immune response to 

S. Typhimurium infection 

• In vitro earlier, but higher, up regulation of chemokines, 

innate cytokines and TLRs with S. Typhimurium infection 

• Similar kinetics of IL8 and CCL20, not TNF, RNA upregulation 

in response to SC and ST infections at lower MOI 

(Skjolaas Vet Im Immunopathol. 115:299, 2007, 10 8 ST/well). 

• Broader array of chemokines (CCL2, CCL3) and apoptosis 

(TGM3) markers shown to be up-regulated. 

• NFkB targets upregulated with ST infections. 

Note: gene not protein expression results. Protein expression 

planned; certain reagents not available. 

Unpublished data 






US Veterinary Immune Reagents Network 

Plan to begin to systematically address the immunological reagent 

gap for the veterinary immunology research community 

Goal: express immune proteins (cytokines, chemokines, acute 

phase reactants) and produce specific monoclonal antibodies to 

immune markers [T cell receptors (TCRs), toll-like receptors 

(TLRs), cell surface differentiation (CD) markers] 

Develop panel of markers for immune proteomic studies 

Coordination + mAb Production: Baldwin, Univ. MA 

Protein Expression: LaBresh, Kingfisher Biotech. 

Ig and TCR Expression: Wagner, Cornell Univ. 

Species: Ruminants, concentrating on cattle - Baldwin, Univ. MA 

Swine - Lunney, ARS BARC 

Poultry, primarily chickens – Lillehoj, ARS BARC 

Horses – Horohov, Univ. KY; Wagner, Cornell Univ. 

Aquaculture species: channel catfish – Miller, MSU; trout – Hansen, USGS 

USDA CSREES Toolkit Network grant 2006-2010 

Swine Toolkit 

Priorities 

• TCRα,β reagents 

• TLRs 

• chemokines 

• CD45RO 

• Cytokines, e.g., IFNA – more sensitive reagents 

• IgGs – note separate NPB grant with J Butler, U Iowa 

Goals: 

• Produce large scale quantities of bioactive protein needed with accurate 

measure of bioactivity; 

• Provide full length cDNAs 

• Produce mAb useful for cytokine and chemokine protein quantitation using 

• ELISAs and ELISpot assays as well as alternate formats, e.g., Luminex, 

intracellular and fixed tissue staining 

• Repository 

What do you need? Send me suggestions 

email: jlunney@anri.barc.usda.gov 

Colleagues 

Nishi 

Lunney Dawson 

Thanks! 

Nishi Samon Royaee Lunney Kuhar 

BARC 

Wang Tuggle Couture 

Iowa State Univ. 

Uthe Bearson 

NADC 

email: jlunney@anri.barc.usda.gov 

soon to be: joan.lunney@ars.usda.gov 

websites: www.anri.barc.usda.gov/pbel/sy_lunney.asp 

www.ars.usda.gov/pandp/people/people.htm?personid=3471

Joan Lunney

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