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Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)

February 5, 2018

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher (P<0.05) in manatees from the east coast of Florida than they were from those from the west coast. We found IL-10 and β-actin to be consistent between sites and identified β-actin as a good candidate for use as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

Citation Information

Publication Year 2018
Title Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)
DOI 10.7589/2017-06-139
Authors Jason A. Ferrante, Margaret Hunter, James F.X. Wellehan
Publication Type Article
Publication Subtype Journal Article
Series Title Journal of Wildlife Diseases
Series Number
Index ID 70195029
Record Source USGS Publications Warehouse
USGS Organization Wetland and Aquatic Research Center