Integrated Genomics to Enhance Host Resistance to Bacterial Colonization in Poultry

1
Integrated Genomics to Enhance Host Resistance to
Bacterial Colonization in Poultry
Chinese Poultry Conference July 2009 Harbin,
CHINA

• Susan J. Lamont

Department of Animal Science Iowa State
University, Ames, Iowa USA
2
Iowa State University -- in wintertime
Financial Support to Lamont Lab Animal Health
Formula Funds BARD Research and Postdoctoral
Grants Cargill Research Excellence
Fellowship Iowa Agriculture Experiment
Station Midwest Poultry Research Program Poultry
Breeding and Genetics Companies USDA-CSREES
National Research Initiative USDA National Needs
Fellowships US Poultry Genome Coordinators
3
Why study host response to Salmonella?

• Millions of Salmonella food-poisoning cases
  annually in US, with 500-1000 deaths in
  susceptible individuals
• Many trace-backs to improperly handled poultry
  products (Proper food handling prevents the
  problem!)

• Horizontal transmission (fecal shedding) and
  vertical transmission from hens to table
  and fertile eggs (chicks)
• Consumer concern about antibiotic use in food
  animals
• Goal identify and use naturally occurring
  biodiversity to enhance host resistance to
  Salmonella colonization of live birds

4
Integrated research to elucidate host bird
response to Salmonella enteritidis

• Biological resources
• Advanced intercrosses
• Cell lines
• SNPs
• Candidate genes
• Genome-wide QTL
• Gene expression
• Q-PCR
• Microarrays
• siRNA gene knock-downs
• Immunohistochemistry
• Proteomics

5
Anticipated Impacts

• Practical tools to use the existing
  biodiversity in poultry populations for genetic
  selection to improve host resistance to bacterial
  colonization
• Enhanced fundamental understanding of host
  resistance mechanisms to bacteria, at the tissue,
  cellular and molecular levels
• Improved poultry health, production efficiency,
  and pre-harvest food safety

6
Broiler
Reduce linkage disequilibrium by producing
advanced intercross lines
7
Reduction of linkage disequilibrium in AIL F2
versus F8 Haploview Chromosome 3

• Why is linkage disequilibrium (LD) important?
• To precisely locate genetic control of traits

8
Reduction of linkage disequilibrium in AIL F2
versus F8 Haploview Chromosome 3
9
Experimental Design Salmonella InfectionF8
Advanced Intercrosses
Candidate gene studies
SE challenge
104 CFU of SE
Day 1
Day 7 or 8
Organ Harvest
SE Count
Frozen Tissue
Total RNA Isolation
10
Gallinacins (Chicken beta-defensins)Biological
Candidate Gene Family

• Anti-microbial peptides
• Found in species from plants to insects to
  mammals
• Kill broad range of bacteria
• Bind to microbial membranes and form membrane
  channels
• Cluster of genes on GGA3

Ganz, T. 2003
11
Gallinacin SNPs and Salmonella response
12
Gallinacin SNPs and Salmonella response
X
X
Hasenstein Lamont. 2007. Avian Dis. 51561-567
X
Two recombination hotspots in the Gallinacin
gene cluster
13
Gallinacin SNPs and Salmonella response
X
X
Bacterial load in cecum of Broiler x Leghorn
AIL
Significant (P lt 0.05) in 4 of 10 gene tests P lt
0.008 for haplotype effect of Gal 11,12, 13
Gallinacin gene SNPs associated with bacterial
load in cecum
14
Experimental Design Salmonella InfectionF8
Advanced Intercrosses
Candidate gene studies
SE challenge
High-density SNP genotyping(Illumina)
104 CFU of SE
Day 1
3072 SNP panel designed by U.S. Poultry Genome
Co-Coordinator, Hans Cheng et al.
Day 7 or 8
Organ Harvest
SE Count
Frozen Tissue
Total RNA Isolation
15
Lots and lots of chicken SNPs
Nature 432 717-722, 2004
Slide modified from Hans Cheng
16
Genome-wide QTL scan for bacterial load in spleen
and cecum (GGA3)
Beta-defensins
Spleen bacterial burden
Red line FDR of 0.25
Cecum content bacterial burden
Hasenstein et al. 2008 Dev. Biolog. 132 213-218
17
Immune and apoptosis genes identified in
genome-wide QTL study

• TIRAP TLR4 controlled response to endotoxins
• IBTK activated by LPS and required for TNF
  production
• STK39 response factor to hypotonic stress,
  serves as an immediate response to cellular
  stress
• TNS1 involved in signal transduction,
  negatively regulates activation of NF?B
• IGF1R mediates proliferation and apoptosis
• MAPK9 transmembrane tyrosine kinase, implicated
  in cellular apoptosis

18
Immune function genes and signaling pathways in
Salmonella infection
Bold squares genes identified in current
genome-wide SNP study with SE and AIL
chickens Dashed squares genes previously
identified with SNP associated with SE response
in chickens
19
Experimental Design Salmonella InfectionF8
Advanced Intercrosses
Candidate gene studies
SE challenge
High-density SNP genotyping(Illumina)
104 CFU of SE

• Gene expression studies
• Single genes
• Microarray
• siRNA

Day 1
3072 SNP panel designed by U.S. Poultry Genome
Co-Coordinator, Hans Cheng et al.
Day 7 or 8
Organ Harvest
SE Count
Frozen Tissue
Total RNA Isolation
20
Microarrays
A global approach to gene expression
analysis .
What is happening at the pathway level?
21
Experimental design for Affymetrix array
Pheno-group
Microarray
Harvest time
H-SE (3)
6
AIL F8
Day 7 (8)
Br ? Leg (16)
L-SE (3)
6
Day 8 (8)
NO-SE (2)
4
H-SE (3)
6
Day 7 (8)
Br ? Fay (16)
L-SE (3)
6
Day 8 (8)
NO-SE (2)
4
H high bacteria load, L Low bacteria load,
NO-SE not inoculated Red number sample size
22
Affymetrix Chicken GeneChip

• 33K transcript oligo array, with 28K genes
  represented
• Cecal tissue, one wk post-infection with
  Salmonella
• Individual hybridizations

• 146 transcripts consistently differentially
  expressed with SE inoculation (challenge vs
  no-challenge), regardless of genetic line cross
  or tissue harvest day
• Applied bioinformatic analyses

23
Affymetrix Chicken GeneChip

• 33K transcript oligo array, with 28K genes
  represented
• Cecal tissue, one wk post-infection with
  Salmonella
• Individual hybridizations

• 146 transcripts consistently differentially
  expressed after SE inoculation (challenge vs
  no-challenge), regardless of genetic line cross
  or tissue harvest day
• Applied bioinformatic analyses

Gene examples CD3, CD4, CD8
IL2RG, IL13RA2, IL20RA IL16, IL18, K203
LITAF, SOCS1 TLR1, TLR15
24
Bioinformatics shows the important genes fitted
into networks and pathways for response to
Salmonella infection Example
shown NF-kappa-B signaling
25
What is happening at the cellular level?
Whole tissue Cell populations
functions gene expression

• Proliferation
• Differentiation
• Migration
• Death

26
Immunohistochemistry in the cecum one week after
SE infection

• Labeled frozen sections
• Macrophages
• Lymphocytes (B T cells)
• Apoptosis
• Annexin (early)
• ssDNA (late)

27
Immunohistochemistry in the cecum one week after
SE infection

• Labeled frozen sections
• Macrophages
• Lymphocytes (B T cells)
• Apoptosis
• Annexin (early)
• ssDNA (late)

Uninfected
SE Infected
Macrophage staining
ssDNA staining
28
Cecal cell composition differencesone week after
SE infection
P lt 0.05
P lt 0.05
Cell Population/Functional Type
Stealthy SE Increased macrophage number, but
decreased late apoptotic cells
29
Mechanisms at the single-gene level?

• Study selected genes in intensive system
• Apply RNA interference (RNAi) technology to
  examine gene function in chicken macrophages
• Question Does siRNA-mediated knock-down of iNOS
  gene in chicken macrophages alter nitric oxide
  (NO) production and gene expression?

30
RNAi technology
Iorns, E., et al. 2007. Nat. Rev. Drug Discov.
6556-568.
31
Key role of iNOS in antimicrobial response
LPS IFNG
Lowenstein and Padaldo. 2004. J Cell
Sci1172865-2867.
32
Experimental design Chicken macrophages and
siRNA
HD11 chicken macrophage
line Targeted iNOS gene with
siRNA and IFN? Gene expression (iNOS)
NO production
33
Reduced iNOS mRNA expression in iNOS
siRNA-treated HD11 macrophages
Each siRNA reduces iNOS expression cocktail
reduces more
34
Reduced NO production in iNOS siRNA-treated HD11
macrophages
35
Mechanisms at the single-cell level?

• Study cell lines in intensive in vitro system
• Apply microarray technology to examine global
  gene expression in chicken macrophages
• Question Does endotoxin from Salmonella alter
  chicken macrophage gene expression and what
  pathways are affected?

36
Experimental Design endotoxin stimulation of
chicken HD11 macrophages
Chicken Macrophage Cell line HD11
Affymetrix GeneChip chicken genome array
Statistical analysis
Bioinformatic analysis, Ingenuity Pathway
Analysis software
37
Consistent signature of NFKBIA, IL1B, IL8
38
NFKB key node
39
Summary Structural Genomic Studies

• Successful implementation of
• AILs in chickens to reduce LD
• Analyzed beta-defensin gene cluster
• Utilize a high-density SNP array
• for genome-wide QTL scan
• Identified 21 SNPs for host
• genetic control of SE colonization
• in or near 19 candidate genes
• New candidate genes identified for targeted
  investigation
• Many genes in known pathways of immune response
• Little overlap (2 of 19 genes) in genetic control
  of bacterial burden in different sites in host
  tissue-specific control

40
Summary Functional Genomic Studies

• Pathways associated with host
• response to Salmonella by testing
• variation in gene structure or
• gene expression or both
• Cytokines and chemokines
• TLRs
• Apoptosis
• iNOS
• NFKB
• Infected cecum showed increase in macrophages and
  decrease in late apoptotic cells stealthy
  Salmonella
• RNAi applied to verify gene function in important
  mechanism of resistance to bacterial colonization

41
The good news

• Despite long-term and stringent genetic selection
  for growth or egg production, domestic poultry
  continue to improve in the traits under genetic
  selection
• Genetic mutation
• Interaction of genes in complex networks
• Immunity and quality traits are almost untapped
  resources, with much opportunity for genetic
  improvement

National Geographic, April 2005
42
E Sandford
C Ciraci
S Redmond
L Pernis
Thank you to the lab and collaborators!
2008
J Cheeseman
B Abasht
X Ye
R Fernando
J Hasenstein
M Kaiser
C Andreasen
J Dekkers
A Hassen
2005
D Nettleton
P Kaiser
H Cheng
C Tuggle
D Palic
H Lillehoj
L Nolan
L Nolan
P Liu