Abstract
As research supports the role of marine mammals as sentinel species, their immune function is rapidly gaining attention as an indicator of aquatic ecosystem health. Immune system monitoring is a valuable tool in the health assessment of wild animal populations as well as in captive managed animals. Immunological data available for sirenians is not extensive. Before further immune-related research and diagnostic studies can be pursued, a basic definition of parameters of the immune system must first be established in order to make accurate correlations to an array of immune-related variables. The aim of this study was to develop a custom quantitative assay for measuring immunoglobulin G (IgG) in sirenians and define the total baseline circulating IgG levels in several populations of dugongs and manatees. Primarily involved in secondary immune response, IgG protects the body by binding to an array of pathogens, thus providing humoral protection from viral and bacterial infections.
To create a quantitative assay that provided increased reproducibility and allowed for a more rapid analysis of manatee IgG than the previously developed ELISA format, this study incorporated a UF-developed mouse anti-manatee IgG monoclonal antibody into a Bio-Layer Interferometry (BLI) assay using the Octet QKe system (ForteBio, Inc.). Bio-Layer Interferometry is a label-free technology for measuring biomolecular interactions using a novel "dip and read" approach. A survey of 334 serum/plasma samples from sirenian populations in five countries were analyzed, including Florida manatees (n = 173), Antillean manatees (n = 145), and dugongs (n = 16). Total IgG levels were compared among sex, age class, populations within subspecies, and time of collection (year and season). IgG reference ranges were also established for several sirenian populations. Complete blood cell counts were investigated for the Crystal River population of Florida manatees and the Belize population of Antillean manatees to identify potential predictors of total IgG levels. The mean IgG level was 10.92 mg/ml (S.D. = 3.25 mg/ml) for Florida manatees, 8.20 mg/ml (S.D. = 3.12 mg/ml) for Antillean manatees, and 14.64 mg/ml (S.D. = 4.21 mg/ml) for dugongs. Total IgG among several individual populations within the Antillean and Florida subspecies were found to vary significantly. Outliers included one Antillean manatee (rescued as a calf) in rehabilitation (total IgG = 1.53 mg/ml), one entangled/rescued Florida manatee (total IgG = 5.76 mg/ml), and a Florida manatee in rehabilitation with a chronic boat strike injury (total IgG = 24.26 mg/ml). IgG levels often correlate with disease state, reflect the immunocompetence of an individual, and/or reflect exposure to environmental pathogens. These data augment the tools available for assessment of health in this species. Additionally, using the methods from this study, two samples of fetal Florida manatee sera were also analyzed. A mean of 26.01 mg/ml suggests significant passive placental transfer of antibodies from cow to calf, which has been previously undocumented in this species.
Acknowledgements
For the archived tissue samples used in this study, the authors would like to thank Dr. Robert K. Bonde and the U.S. Geological Survey's Southeast Ecological Science Center - Sirenia Project, Dr. James 'Buddy' Powell and Sea to Shore Alliance, Fabia Luna and Dr. Fernanda Löffler Niemeyer Attademo and O Centro Nacional de Pesquisa e Conservação de Mamíferos Aquáticos - CMA/ICMBio, Drs. Chip Deutsch and Martine deWit and the Florida Fish and Wildlife Conservation Commission (FWC), Dr. Antonio Mignucci and the Red Caribeña de Varamientos, and Dr. Janet Lanyon and the University of Queensland's Marine Vertebrate Ecology Research Group (MarVERG). For their technical assistance and advice, the authors would like to thank Michele Halvorson and Dan McElroy of FortéBio, Inc. Research funding is provided through the University of Florida (UF) - Aquatic Animal Health Program at the College of Veterinary Medicine (CVM), FWC, the UF Whitney Marine Laboratory for Marine Bioscience, and the UF-CVM Consolidated Faculty Research Development. This study was conducted under IACUC #201106011, Federal Fish and Wildlife Permit (USFWS) #MA067116-1 and #MA791721, and CITES Permit #11US808447.