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.
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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
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