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

Development of a Bovine Macrophage Specific cDNA Library to Investigate Tropical Theileriosis. Kirsty Jensen. Tropical Theileriosis. Causative agent: Theileria annulata Vector: Hyalomma ticks Prevalence: 200 million cattle at risk Economic impact: $384.3 million in India/year.

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

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

  2. Tropical Theileriosis Causative agent: Theileria annulata Vector: Hyalomma 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 indicusBos 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

  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. RNA RT-PCR Denaturation & Reannealing Denaturation & Reannealing Column Purification Column Purification 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 Represent 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 (P<0.05, fold change >2). • Over 100 genes have been identified that exhibit breed-specific differential expression during infection (P<0.05, fold change >2). • Over 20 of these genes exhibit breed-specific differential expression in resting cells (P<0.05, fold change >2).

  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

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