Hofstetter Amelia Ruth, Eberle Kirsten C, Venn-Watson Stephanie K, Jensen Eric D, Porter Tracy J, Waters Theresa E, Sacco Randy E
Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, United States of America.
Translational Medicine and Research Program, National Marine Mammal Foundation, San Diego, California, United States of America.
PLoS One. 2017 Dec 22;12(12):e0189437. doi: 10.1371/journal.pone.0189437. eCollection 2017.
Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.
在人工饲养环境中照顾海豚的兽医以及监测野生海豚种群的研究人员，都使用基于血液的诊断方法来监测宽吻海豚（Tursiops truncatus）的健康状况。定量聚合酶链反应（qPCR）可用于评估外周血单核细胞（PBMC）的细胞因子转录模式。这可以为目前可用的血液检测补充有关免疫状态的信息。要充分实现这一潜力，需要确定宽吻海豚细胞因子基因转录水平的正常范围。我们在七个月的时间里对四只海豚进行了连续采血调查。将PBMC用植物血凝素（1、5和10μg/mL）和伴刀豆球蛋白A（1μg/mL）在体外刺激48小时。使用宽吻海豚特异性引物（IL-1α、IL-1β、IL-1RA、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12p40、IL-13、IL-18、IFN-γ和TNF-α）通过qPCR对这些培养物的RNA进行检测。来自另一只被诊断患有急性肺病的宽吻海豚的两份血样为数据提供了更多视角。我们观察到，有丝分裂原的选择对观察到的基因转录幅度有显著影响。另一方面，大多数检测的细胞因子在动物体内的变化有限。然而，IL-6和IL-12p40在老年和幼年海豚之间存在差异。此外，有丝分裂原反应的幅度将检测的细胞因子分为三组。这些数据为未来研究中目标细胞因子mRNA的选择及其有丝分裂原刺激的细胞因子基因转录的预期范围提供了参考。
