Development of a Bovine Macrophage Specific cDNA Library to Investigate Tropical Theileriosis

1
Development of a Bovine Macrophage Specific cDNA
Library to Investigate Tropical Theileriosis

• Kirsty Jensen

2
Tropical Theileriosis
Causative agent Theileria annulata Vector Hyalo
mma ticks Prevalence 200 million cattle at
risk Economic impact 384.3 million in
India/year
3
Cytospin of T. annulata Infected Macrophages
4
Cattle Breeds Exhibiting Differential Response to
T. annulata Infection.
Sahiwal Holstein-Friesian Bos indicus Bos
taurus
5
Tropical Theileriosis

• The breed-specific divergence in disease
  progression coincides with the schizont stage of
  the parasite.
• Previous work has implicated the infected
  macrophage as being central to-
• the progression of the disease.
• the breed-specific differences.

6
Aims

• To investigate the host-pathogen interactions
  between bovine macrophages and T. annulata.
• To elucidate the mechanisms involved in the
  breed-specific resistance of cattle to tropical
  theileriosis.

7
Bovine Microarrays
Microarray Format Source of Material
BOTL cDNA Bovine total leukocyte cDNA library immune gene amplicons
Pyxis CattleArray 7600 cDNA Bovine spleen placental cDNA libraries
NBFGC cDNA MARC BARC cDNA libraries
BMAM cDNA MARC BARC cDNA libraries Mammary gland specific
Endometrial/placental microarray cDNA Bovine placental endometrial cDNA libraries
Bovine immune-endocrine microarray cDNA Bovine immune endocrine gene amplicons
Bovine MHC class I microarray oligos BoLA DRB3 DQA sequences
RIGUA oligos Bovine immune inflammatory gene amplicons
Affymetrix oligos Various cDNA libraries
ARK-Genomics cDNA Various cDNA libraries
8
cDNA Microarray

• Source of clones
• Publicly available resources
• Collaboration
• In house library construction

9
cDNA Microarray

• Construction of cDNA library
• Tissue
• Tissue status
• Normalization v Subtraction

10
Normalization Subtraction
11
Bovine Macrophage cDNA Library

• Normalized
• Bos taurus Bos indicus
• Bovine monocytes

12
Monocytes infected in vitro with T. annulata 1,
4, 12, 24, 48 96 hour post infection
Resting monocytes
Activated monocytes PMA Ionomycin 1, 2, 4, 8,
16 24 hour post activation
Bovine macrophage cDNA library
Activated monocytes LPS IFN-? 1, 2, 4, 8, 16
24 hour post activation
Established ex vivo T. annulata infected cell
lines
13
Sequence Analysis

• 10,000 clones were sequenced in collaboration
  with the Sanger Institute
• Initial analysis
• Assessment of library quality
• Quality of sequences
• Sequence length
• Cluster analysis within the library

14
Sequence Analysis

• Cluster analysis with EMBL bovine EST dataset.
• Cluster analysis with EMBL Theileria annulata EST
  dataset.
• BLAST analysis with EMBL EST NR datasets
• BLAST analysis with human ENSEMBL dataset.

15
Composition of the Bovine Macrophage Library
Other 2.8
Parasite 5.4
Human 5.6
Unique 45.0
Bovine 41.2
16
RT-PCR Analysis of Unique Sequences
Monocytes
Activated Infected Ovarian Parasite
- - -
- RT enzyme
GAPDH
Tams1
U2
U8
17
GO Ontologies Biological Processes
Cancer related
Immune response
Response to stress
Cell adhesion
Cell communication
Cell surface linked signal transduction
Intracellular signaling cascade
Cell organization biogenesis
Cell growth and/or maintenance
Cell cycle
Cell proliferation
Cell death
Metabolism
Transport
Others
18
Composition of the bovine macrophage microarray
Source Description Number Bovine Macrophage
Library ENSEMBL BLASTN hits 2372 EST hits
- bovine 1076 human 139 other 43 Represe
nt unique clusters 234 ESTscan
hits 204 Singletons 632 MARC1-4/BARC5 312
Amplicons 25 Total 5037
19
Microarray Experiments

• Experiment 1.
• Investigation of the gene expression dynamics of
  Holstein Sahiwal derived macrophages at 2 72
  hours post T. annulata infection.
• 1) Host-pathogen interactions.
• 2) Breed-specific differences.
• Experiment 2.
• Investigation of the gene expression dynamics of
  Holstein Sahiwal derived macrophages at 2 16
  hours post LPS IFN-? activation.

20
The BoMP Microarray
21
Infection Study

• Over 500 genes have been identified that exhibit
  differential expression during infection (Plt0.05,
  fold change gt2).
• Over 100 genes have been identified that exhibit
  breed-specific differential expression during
  infection (Plt0.05, fold change gt2).
• Over 20 of these genes exhibit breed-specific
  differential expression in resting cells (Plt0.05,
  fold change gt2).

22
Breed-Specific Differential Expression in
Unactivated Monocytes.
4
Fib
2
0
- 2
CD9
- 4
C1r
NF-kB AP 373
AOX1
- 6
- 8
- 10
Holstein
Sahiwal
- 12
23
Validation of Microarray Results
Holstein Sahiwal
H1 H2 H3 H4 H5 H6 S1 S2 S3
S4 N
28s
AOX1
NF-kB AP 373
C1r
Fib
CD9
24
Validation of Microarray Results
30
Fibronectin
CD9
25
20
mRNA fold change
15
10
5
0
H1
H2
H3
H4
H5
S1
S2
S3
S4
S5
Holstein Sahiwal
25
Acknowledgements
Roslin Institute Liz Glass Mary Clapperton Susan
Craigmile Edith Paxton Giles Makins John
Williams Wilson Lee David Speed ARK-Genomics
Facility Richard Talbot Alison Downing Frazer
Murray
University of Edinburgh Paul Wright ENMV,
Tunisia Mohammad Darghouth Wellcome Trust
Sanger Centre Ruth Taylor Jane Rogers BBSRC