Transcriptional Gene Signatures in Tursiops truncatus: An Aid in Differential Diagnostics?
IAAAM 2009
Chia-Shan Lee1; Tatjana Sitt1; Lizabeth Bowen2; Myra T. Blanchard1; Brett R. Smith1; Jeffrey L. Stott1
1Laboratory for Marine Mammal Immunology, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA; 2Aquatic Ecosystems Analysis Laboratory, Center for Watershed Sciences, John Muir Institute of the Environment, University of California, Davis, CA, USA

Abstract

Pursuit of new techniques and approaches for identifying host response(s) to pathogens or other insults is perpetual. Such an expansion of diagnostics would be beneficial to managing cetacean health, as clinical signs of disease are often not apparent until the insult becomes life-threatening. Diagnostically speaking, measurements of pathogen-associated influences on the host have been largely limited to complete blood counts and clinical chemistries. Quantification of gene transcription (messenger RNA) in the peripheral blood leukocytes can provide comprehensive insight into the real-time activity of the host immune system.2,6 Quantitative gene expression assays have been developed and validated in cetaceans,5 and applied to measuring antigen-specific T lymphocyte memory in bottlenose dolphins (Tursiops truncatus) vaccinated for Erysipelas4 and studying potential effects of environmental contaminants in free-ranging T. truncatus3 and harbor porpoises (Phocoena phocoena).1 The current study is designed to study transcriptional patterns of cytokine and other immunologically relevant genes in the peripheral blood leukocytes (PBLs) derived from apparently healthy and diseased T. truncatus, with intent to identify transcriptional signatures with diagnostic potential. Eleven inducible genes including cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), interleukin-17 (IL-17), CD69, IL-2 receptor alpha (IL-2Rα), interferon-alpha (IFN-α), interferon-gamma (IFN-γ), IL-4, IL-10, influenza virus resistance allele Mx+ gene (MX-1), and Fas-associated protein with death domain (FADD) were selected for analysis. This complement of genes represents a broad measure of leukocyte activities including phagocyte activity (COX-2), pro-inflammatory (TNF-α & IL-17) and anti-inflammatory (IL-10) responses, early events of leukocyte (CD69) and lymphocyte (IL-2Rα) activation, T-helper type 1 cell (IFN-γ) or T-helper type 2 cell (IL-4) differentiation, leukocyte apoptosis (FADD), and potential response to viral infection (IFN-α & MX-1). The relative number of mRNA transcripts for each gene was determined using a housekeeping gene (ribosomal subunit S9) as reference. Twenty-five apparently healthy dolphins (ranged from 0.6 to 37.4 years of age) were bled twice at a 4-week interval over a period of two years to establish reference ranges of gene transcripts. Blood was collected in PAXGene vacutainer tubes for immediate fixation of RNA. Total RNA was extracted, cDNA generated and target mRNA expression levels measured by quantitative real-time polymerase chain reaction (Q-PCR) using T. truncatus-specific primers. Messenger RNA transcript levels of the genes of interest were relatively uniform between dual samples from individual animals and between animals (Figure 1). A few outliers for COX-2, IL-4, IL-10, IL-17 and CD69 were observed, suggesting these bio-markers were identifying perturbations in health not identified by standard haematology and clinical chemistry panels. Sequential blood samples from select clinical cases are being evaluated for perturbations in gene expression. All cases showed perturbation at T=0 with most transcription signatures exhibiting unique features. While a large number of clinical cases need to be examined, preliminary data suggests that differential gene expression profiles may be of diagnostic value and assist in development of treatments and temporal assessment of treatment efficacy.

Click on the chart to see a larger view.

Figure 1.

Acknowledgements

This work is supported by a grant of the Office of Naval Research. The authors wish to thank the Sea World Adventure Parks for all their support and providing the animals used in this study.

References

1.  Beineke A., U. Siebert, G. Muller, and Baumgartner. 2007. Increased blood interleukin-10 mRNA levels in diseased free-ranging harbor porpoises (Phocoena phocoena). Vet Imm Immunopath 115: 100-106.

2.  Heid C.A., J. Stevens, K.J. Livak, and P.M. Williams. 1996. Real time quantitative PCR. Genome Res 6: 986-994.

3.  Mancia A., M.L. Lundqvist, T.A. Romano, M.M. Peden-Adams, P.A. Fair, M.S. Kindy, B.C. Ellis, S. Gattoni-Celli, D.J. McKillen, H.F. Trent, Y.A. Chen, J.S. Aleida, P.S. Gross, R.W. Chapman, and G.W. Warr. 2007. A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions. Dev Comp Imm 31: 520-529.

4.  Sitt T. 2007. A model system for assessing vaccine efficacy in cetacean species. PhD Dissertation, University of California, Davis.

5.  Sitt T., L. Bowen, M.T. Blanchard, B.R. Smith, L.J. Gershwin, B.A. Byrne, and J.L. Stott. 2008. Quantitation of leukocyte gene expression in cetaceans. Dev Comp Imm 32: 1253-9.

6.  Stordeur P., L. Zhou, B. Byl, F. Brohet, W. Burny, D. Groote, T. Poll, M. Goldman. 2003. Immune monitoring in whole blood using real-time PCR. J Imm Methods 276: 69-77.

 

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Chia-Shan Lee
Laboratory for Marine Mammal Immunology, Department of Pathology, Microbiology and Immunology
School of Veterinary Medicine, University of California
Davis, CA, USA


MAIN : Immunology : Transcriptional Gene Signatures
Powered By VIN

Friendly Reminder to Our Colleagues: Use of VIN content is limited to personal reference by VIN members. No portion of any VIN content may be copied or distributed without the expressed written permission of VIN.

Clinicians are reminded that you are ultimately responsible for the care of your patients. Any content that concerns treatment of your cases should be deemed recommendations by colleagues for you to consider in your case management decisions. Dosages should be confirmed prior to dispensing medications unfamiliar to you. To better understand the origins and logic behind these policies, and to discuss them with your colleagues, click here.

Images posted by VIN community members and displayed via VIN should not be considered of diagnostic quality and the ultimate interpretation of the images lies with the attending clinician. Suggestions, discussions and interpretation related to posted images are only that -- suggestions and recommendations which may be based upon less than diagnostic quality information.

CONTACT US

777 W. Covell Blvd., Davis, CA 95616

vingram@vin.com

PHONE

  • Toll Free: 800-700-4636
  • From UK: 01-45-222-6154
  • From anywhere: (1)-530-756-4881
  • From Australia: 02-6145-2357
SAID=27