Measuring cytokines in Eurasian tundra reindeer (Rangifer tarandus tarandus) with a bovine bead-based multiplex immunoassay and real-time PCR

Abstract

Background - Reindeer (Rangifer tarandus tarandus) herding is based on access to seasonal pastures. Pastureland is, however, being lost and fragmented due to e.g. climate change, human activities, and predators, creating an increasing need for feeding and fencing. This alters disease occurrence, leading to a greater need for disease investigation tools. Knowledge of the activation of immune pathways during disease can be obtained by measuring cytokines, but no commercial methods are currently available for reindeer. This study investigated whether the MILLIPLEX® Bovine Cytokine Magnetic Bead assay could be used to detect interleukin (IL)-6, IL-8, IL-10, IL-17, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) in reindeer cell supernatants and serum. Peripheral blood mononuclear cells (PBMCs) from reindeer (n = 4) and cattle (Bos taurus, n = 3) were stimulated with mitogens for 6 and 24 h (h) and the quantity of cytokines in cell supernatants was measured. Serum from experimental viral infections in reindeer (Orf virus; ORFV and Varicellovirus cervidalpha2; CvHV2) was also analysed. Additionally, primers were designed to measure cytokine gene expression in response to mitogens by real-time polymerase chain reaction (qPCR).<p>

<p>Results - The bovine bead-based multiplex immunoassay detected five of six cytokines (IL-8, IL-10, IL-17, TNF-α, IFN-γ) in reindeer PBMC supernatants after stimulation. All cytokines were detected in bovine samples. Although cytokine concentrations were generally higher in bovine samples, analysis of reindeer supernatants demonstrated significantly increased IL-10, IL-17, TNF-α and IFN-γ concentrations in supernatants from stimulated compared to unstimulated PBMCs. Neither reindeer nor cattle samples showed a significant increase for IL-6, while IL-8 was increased only in bovine samples after 6 h stimulation. Serum from reindeer infected with CvHV2 showed significantly increased IFN-γ levels on days 4 and 7 post inoculation. Gene expression of all cytokines was increased by stimulation of reindeer PBMCs, except IL-6 for which primer design was unsuccessful.<p>

<p>Conclusions - This study shows the potential of the bovine bead-based multiplex immunoassay for measuring IL-10, IL-17, TNF-α, and IFN-γ concentrations in reindeer. The qPCR is suitable for measuring gene expression of these cytokines and IL-8. These methods may be used to characterise immune responses in reindeer, but further testing and validation are warranted.