Frøvoll Tuva Løken, Lybeck Kari, Lund Hege, Makvandi-Nejad Shokouh, Grimholt Unni, das Neves Carlos G, Tryland Morten, Nymo Ingebjørg Helena, Klevar Siv
Section for Virology, Immunology and Parasitology, Norwegian Veterinary Institute, 1433, Ås, Norway.
Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 1433, Ås, Norway.
Acta Vet Scand. 2025 Jun 18;67(1):34. doi: 10.1186/s13028-025-00819-4.
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).
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.
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.
驯鹿（Rangifer tarandus tarandus）放牧依赖于季节性牧场的使用。然而，由于气候变化、人类活动和捕食者等因素，牧场正在减少且碎片化，这使得饲养和围栏的需求日益增加。这改变了疾病的发生情况，导致对疾病调查工具的需求更大。通过测量细胞因子可以了解疾病期间免疫途径的激活情况，但目前尚无适用于驯鹿的商业方法。本研究调查了MILLIPLEX®牛细胞因子磁珠检测法是否可用于检测驯鹿细胞上清液和血清中的白细胞介素（IL）-6、IL-8、IL-10、IL-17、肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）。用丝裂原刺激来自驯鹿（n = 4）和牛（Bos taurus，n = 3）的外周血单核细胞（PBMC）6小时和24小时，然后测量细胞上清液中细胞因子的含量。还分析了驯鹿实验性病毒感染（口疮病毒；ORFV和鹿疱疹病毒2型；CvHV2）后的血清。此外，设计了引物，通过实时聚合酶链反应（qPCR）来测量丝裂原刺激后细胞因子基因的表达。
基于牛磁珠的多重免疫测定法在刺激后检测到驯鹿PBMC上清液中的六种细胞因子中的五种（IL-8、IL-10、IL-17、TNF-α、IFN-γ）。在牛样本中检测到了所有细胞因子。虽然牛样本中的细胞因子浓度通常较高，但对驯鹿上清液的分析表明，与未刺激的PBMC相比，刺激后的上清液中IL-10、IL-17、TNF-α和IFN-γ的浓度显著增加。驯鹿和牛的样本中IL-6均未显示出显著增加，而IL-8仅在刺激6小时后的牛样本中增加。感染CvHV2的驯鹿血清在接种后第4天和第7天显示IFN-γ水平显著升高。除IL-6的引物设计未成功外，所有细胞因子的基因表达在刺激驯鹿PBMC后均增加。
本研究表明基于牛磁珠的多重免疫测定法在测量驯鹿中IL-10、IL-17、TNF-α和IFN-γ浓度方面具有潜力。qPCR适用于测量这些细胞因子和IL-8的基因表达。这些方法可用于表征驯鹿的免疫反应，但需要进一步测试和验证。
