Abstract

Efforts expended in the treatment and rehabilitation of stranded marine mammals are considerable, especially in terms of limited financial resources, personnel, and housing. Importantly, limited state of the art diagnostic tools are available that may better assess the health of an animal, potentially resulting in reduced rehabilitation time and overall costs. Additional tools may reveal sub-clinical signs of inflammation or evidence for immune response, which may not be revealed during regular medical evaluation, e.g. physical examination, hematology, serum chemistry, and bacterial cultures. Cytokines are important small proteins that help direct a proper immune response to pathogens. These include interferon, interleukin, and growth factors that are secreted by certain cells of the immune system and have an effect on other cells. The present work was conducted to assess and validate the cross-reactivity of the commercially-available Bio-Plex Pro^TM Human Cytokine Th1/Th2 Panel and Millipore Canine Cytokine 13-plex kits, coupled with the Luminex© platform, to measure cytokines in three pinniped species, harbor seals (Phoca vitulina), grey seals (Halichoerus grypus), and harp seals (Pagophilus groenlandicus). The Luminex© multiplex platform, along with commercially available cytokine kits, was chosen as it allows for the simultaneous and rapid (about 3 hours) quantification of large numbers of cytokines in one small sample (12.5 µl for serum or 50 µl for cell culture supernatant). We assessed cross-reactivity by measuring and comparing cytokines in the supernatant of mitogen-stimulated human, canine and pinniped peripheral blood mononuclear cells (PBMC). The human cytokine panel allowed the detection of cytokines in the supernatant of human PBMCs, but not in the three pinniped species studied, with the exception of TNFα. In contrast, the canine cytokine panel kit did cross-react with the majority of cytokines in the three pinniped species tested. Importantly, cytokines that were quantified in pinnipeds included three pro-inflammatory cytokines (IL-6, IL-8 and TNFα), the Th1 cytokine INFγ, and the Th2 cytokine IL-10. Further, the pattern of cytokine production upon different mitogen stimulation and in the absence of stimulation were consistent for pinniped and canine cells, confirming the specificity of the cross-reactivity. Overall, the Luminex© platform and the canine multiplex cytokine kit allowed the successful measurement of potentially clinically important pinniped cytokines. Future work will focus on testing and evaluating the canine cytokine kit as an additional tool to help veterinarians and rehabilitators assess the health status of stranded pinnipeds, with the potential of serum cytokines to help differentiate between animals without and with different infectious diseases.

Acknowledgements

This material is based upon work supported by NOAA's Northeast Region Marine Mammal Grant Program under Award No. NA11NMF4720241.

* Presenting author