Abstract

Cytokines are signaling molecules that are most often used by the body to mediate an immune response. These proteins activate, recruit, and regulate cells that are responsible for immune, inflammatory, and subsequent repair processes. This study addressed the need for sensitive, species-specific assays to measure cytokines in Florida manatees. Whole blood samples were collected from 42 free-ranging Florida manatees. RNA was extracted from the peripheral blood mononuclear cells for use in quantitative, real-time PCR assays. Primer/probe sets were designed for analysis of IFN-γ, IL-2, and IL-10, as well as GAPDH and β-actin as housekeeping targets. Using these assays, preliminary baseline PBMC ranges of β-actin, GAPDH, IFN-γ, IL-2, and IL-10 levels in healthy, free-ranging Florida manatees in the Crystal River and Brevard overwintering sites were established.

The average β-actin value for the healthy Florida manatee population sampled was 1.44 x 10⁵ copies/100 ng cDNA, and for GAPDH the value was 2.08 x 10⁴ copies/100 ng cDNA. Copy values of IFN-γ, IL-2, and IL-10 were normalized to GAPDH values for the same manatee sample. The final normalized count value averages for each cytokine were as follows: IFN-γ average = 0.055 copies/100 ng cDNA; IL-2 average = 0.126 copies/100 ng cDNA; and IL-10 average = 0.031 copies/100 ng cDNA.

These are the first assessments of IFN-γ, IL-2, and IL-10 levels in Florida manatee PBMCs using qPCR. Future investigations of manatees from the Brevard and Crystal River overwintering sites undergoing a physiologic stress response now have a baseline value for comparison to a healthy counterpart.

Acknowledgements

The authors wish to thank Drs. Bob Bonde, Maggie Hunter, and Michelle Davis of the USGS Sirenia Project as well as Dr. Mike Walsh and Patrick Thomson of the College of Veterinary Medicine, University of Florida for their assistance with sample collection and processing.

* Presenting author