Development of Quantitative PCR Assays for Investigation of Immune Function in the Florida Manatee (Trichechus manatus latirostris)
Jason A. Ferrante*, PhD; Linda Archer, BS; Galaxia Cortés-Hinojosa, MV, MSc, PhD; James F.X. Wellehan, Jr., DVM, MS, PhD, DACZM, DACVM (Virology, Bacteriology/Mycology), DECZM (Herpetology)
Trichechid herpesvirus 1 (TrHV1) is found in both healthy and diseased Florida manatees (Trichechus manatus latirostris).2 Herpesvirus reactivation has been detected in experimental situations even when cortisol elevation was not, making the virus a potential biomarker of stress.1 Cytokines function as signaling molecules to mediate an immune response. This study addressed the need for sensitive, species specific assays to measure TrHV1 and cytokines in Florida manatees. Whole blood samples were collected from 42 free-ranging Florida manatees. DNA and RNA were extracted from the buffy coat for use in quantitative, real-time PCR (qPCR) assays. Primer/probe sets were designed for analysis of TrHV1, IFN-γ, IL-2, and IL-10, as well as GAPDH and β-actin, as housekeeping targets. Using these assays, preliminary baseline buffy coat ranges of these targets from manatees in the Crystal River and Brevard overwintering sites were established.
TrHV1 copy numbers ranged between <10 (limit of detection) and 74.2 counts/100 ng DNA, with an average of 40.9±21.2 counts/100 ng. The average β-actin value for the healthy Florida manatee population sampled was 1.44×105 copies/100 ng cDNA, and for GAPDH the value was 2.08×104 copies/100 ng cDNA. Copy values of IFN-γ, IL-2, and IL-10 were normalized to GAPDH values. The final normalized count value averages were as follows: IFN-γ=0.055, IL-2=0.126, and IL-10=0.031 (values in # copies/100 ng cDNA).
Future investigations of manatees from these overwintering sites now have a baseline value for comparison. In addition, these assays may ultimately provide the ability to monitor manatee immunologic processes.
The authors thank the American Association of Zoo Veterinarians for the Zoological Medicine and Wildlife Health Research Grant which made this research possible. Additionally, we wish to acknowledge the U.S. Geological Survey Sirenia Project and the University of Florida Aquatic Animal Health Program, and the Lowry Park Zoo for help with sample collection. Additional funding was provided through the Florida Fish and Wildlife Conservation Commission, the UF Whitney Marine Laboratory for Marine Bioscience, and the University of Florida Aquatic Animal Health program at the College of Veterinary Medicine.
1. Buske-Kirschbaum A, Geiben A, Wermke C, Pirke CM, Hellhammer D. Preliminary evidence for Herpes labialis recurrence following experimentally induced disgust. Psychother Psychosom. 2001;70(2):86–91.