Recipient of 2014 ADSA-EAAP Travel Award sponsored by Novus International & ADSA

Transcription

1 Marlene Paibomesai - University of Guelph Advisor: Dr. Bonnie Mallard Sept 1 st, 2015 Recipient of 2014 ADSA-EAAP Travel Award sponsored by Novus International & ADSA

2 Never start a presentation with a quiz!!

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6 above genetics gene and environment interaction Acts like a switch - turning genes on and off Genomics = the blueprints Epigenetics = the engineers Zaidi S K et al. Mol. Cell. Biol. 2010;30:

7 Zaidi S K et al. Mol. Cell. Biol. 2010;30:

8 GENETICS SNPs EBV Histone Modifications DNA Methylation EPIGENETICS Phenotype Pathogens Toxins Hormones Management ENVIRONMENT

9 F1 Generation F2 Generation Environmental Factor

10 The Epigenetic Landscape DNA Methylation turns OFF genes DNA Methylation turns ON genes Hochedlinger K, Plath K Development 2009;136:

11 Antibody Mediated Immune Response Parasite Virus Bacteria Fungi Cell Mediated Immune Response T-cell Ag Ag B-cell AMIR T-HELPER CELLS CMIR IL-4 CYTOKINES IFNγ Ag Infected Cell

12 The Immune System AMIR Extracellular ex E. coli Phenotype CMIR Intracellular ex MAP Epigenetics Immune Response Epigenome Immune Response Genotype Wilkie, B. and Mallard, B Vet Immunol Immunopath 72:

13 Isolated CD4+ T-cells from Average Immune Response Cows (n=5) Th1 Skewed CD4+ T-cells 20 μg/ml anti-il4 10 ng/ml rpil-2 10 ng/ml rpifnγ 2.5μg/mL ConA Th2 Skewed CD4+ T-cells 20 μg/ml anti-ifnγ 10 ng/ml rpil-2 10 ng/ml rpil4 2.5μg/mL ConA rp IL-2 rest for 24 hours rp IL-2 rest for 24 hours Stimulate ConA Harvest: Cell culture supernatant for measuring cytokines (ELISA) RNA for quantifying transcript ( Realtime PCR) DNA for methylation profiling (Bisulfite Conversion Assay)

14 Magnetic bead Isolated T-Helper Cells PCR Product Capturing Extract DNA Pyrosequencing Treat with Bisulfite Site of Methylation

15 Fold Change IFNG IL4-2 Th1 Skewed CD4+ T-cells Th2 Skewed CD4+ T-cells

16 DNA Methylation of IFNG Promoter of Th1-Th2 Skewed Bovine CD4+T-cells(n=5 cows) Basepairs from IFNG TSS DNA Methylation of IL4 Promoter of Th1-Th2 Skewed Bovine CD4+T-cells (n=5 cows) Basepairs from IL-4 TSS Th1 Th2 Th1 Th2

17 The How can we apply this on farm? THE EPIGENOME The Epigenome

18 Pregnancy Parturition Postpartum AMIR CMIR AMIR/CMIR?

19 Peripartum period reflects a period of stress in dairy cows Higher occurrences of disease after calving e.g. Mastitis, ketosis, metritis Evidence of suboptimal immune response (Mallard et.al.,1998) Characteristic shifts in Th1- Th2 responses (Shafer-Weaver et.al.,1999)

20 Cell-Mediated Immune Response (CMIR) Antibody- Mediated Immune Response (AMIR)

21 CMIR Residuals High and low groups are +/-1 standard deviation from mean Hine, et al, LOW 12 HighCMIR/LowAMIR Cows AMIR Residuals HIGH 12 HighAMIR/LowCMIR Cows

22 % Disease Occurence 16 Disease Occurrence by Immune Response Category High Average Low Wagter LC J. Dairy Sci. 83(3):488; 2008 DeLapaz, J. MSc Thesis University of Florida; Thompson-Crispi K JDS 95(7):3888; Thompson-Crispi K Clin Vacc Immuno. 20(1):106.

23 Peripartum Period - 28 days Calving +4 days Prepartum Postpartum +21 days Postpartum 10 H-CMIR/L-AMIR & 11H-AMIR/L-CMIR cows Whole Blood as a source of CD4+ T lymphocytes (Density gradient and MACS) Stimulated with T-cell mitogen (ConA) Cell culture supernatant for measuring cytokines (ELISA) DNA for methylation profiling (Bisulfite Conversion Assay) PROC MIXED in SAS9.2.1.; log (concentration) to normalize the data. Y= μ+group+time+cytokine+group*time*cytokine+parity+ε GROUP= HiAMIR/LoCMIR or HiCMIR/LoAMIR; Time= -28, +4, +28 Days; Cytokine= IFN, IL4, IL17A; Parity=1 st & 2 nd

24 IFN-γ Concentration (pg/ml) # p< a a 1 a -28 Days + 4 Days + 21 Days H-AMIR Cows H-CMIR Cows # 2

25 IL-4 Concentration (pg/ml) a 1 1 a -28 Days + 4 Days + 21 Days b * 1 * p<0.05 H-AMIR Cows H-CMIR Cows

26 More cytokine after stimulation of T-helper cells isolated from H-CMIR = Less methylation for both cytokines in H-CMIR cows

27 Magnetic bead Isolated T-Helper Cells PCR Product Capturing Extract DNA Pyrosequencing Treat with Bisulfite Site of Methylation

28 % of DNA Methylation DNA Methylation of IFNG Promoter from Isolated CD4+ T-cells of H-AMIR (n=11) & H-CMIR (n=10) Dairy Cows * * * * * * Basepairs from IFNG Transcription Start Site H-AMIR UNSTIM H-AMIR STIM H-CMIR UNSTIM H-CMIR STIM

29 % of DNA Methylation * Basepair from IL4Transcription Start Site +195 H-AMIR UNSTIM H-AMIR STIM H-CMIR UNSTIM H-CMIR STIM

30 H-AMIR cows less cytokine production DNA methylation H-CMIR cows more cytokine production DNA methylation

31 GENETICS PHENOTYPE EPIGENETICS ENVIRONMENT

32 Advisor: Dr. Bonnie Mallard Dr. Shayan Sharif Dr. Niel Karrow Summer Students Technicians at Elora Research Center Funding: NSERC Discovery National Milk Federation PhD Paper Competition: Production Division Marlene Paibomesai Dairy Specialist American Dairy Science Association Novus International