Sassu Elena L, Kangethe Richard T, Settypalli Tirumala Bharani K, Chibssa Tesfaye Rufael, Cattoli Giovanni, Wijewardana Viskam
Animal Production and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria.
Vet Immunol Immunopathol. 2020 Sep;227:110092. doi: 10.1016/j.vetimm.2020.110092. Epub 2020 Jul 9.
The establishment of a panel of immune markers is of paramount importance to understand the different transcription patterns of infectious diseases in livestock. The array of commercially available immunological assays for cattle and sheep is currently limited, due to the lack of antibodies for these species. Even though SYBR Green based real time quantitative PCR (qPCR) is the most commonly used method to study cytokine transcription in ruminants, a lack of standardization impairs its implementation in the study of different ruminant diseases. In order to obtain reliable qPCR results, several variables need to be considered: choice of reference genes for optimal normalization, variation of annealing temperature among primer sets, and assay specificity and sensitivity. In this study, we developed and validated a panel of immune markers in bovine and ovine samples using SYBR Green based qPCR in a cost-effective way with multiple primer sets optimised to amplify at a common thermal cycling temperature. Twenty primer sets were designed to quantify immune markers (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, TNF-α, IFN-γ, IFN-α, Ki-67, NFkB-65, TLR-3, TLR-4, TLR-8 and Rig-1) in ovine and bovine templates. For optimal normalization and selection of suitable reference genes, primer sets that measure the transcription of five reference genes were also included in the panel. The amplification efficiency, linearity and specificity was validated for all target genes. Optimal amplification conditions were achieved in both ovine and bovine samples for all gene targets, with the exception of Ki67. Relative quantification studies were performed on ovine and bovine mRNA obtained from sheep peripheral blood mononuclear cells (PBMCs) stimulated with three different treatments (PMA/Ionomycin, Concanavalin A (Con A) and pokeweed mitogen (PWM)). Pokeweed and ConA efficiently induced gene transcription of most of the targeted genes, while PMA/Ionomycin showed a weaker induction. Finally, we further assessed usability of our panel by running it on bovine monocyte derived dendritic cells (MoDCs) stimulated with different vaccines. Results confirmed the induction of a specific pro-inflammatory gene transcription pattern by rabies vaccine, which resembles the one occurring during viral infection. Altogether, we validated the efficiency and usability of an extended real-time PCR panel that gives the possibility to rapidly measure a broad spectrum of ovine and bovine immune markers by using a single set of reagents and protocol thus representing a valid and cost-effective tool for research purposes.
建立一组免疫标志物对于了解家畜传染病的不同转录模式至关重要。由于缺乏针对牛和羊的抗体，目前可用于牛和羊的市售免疫检测方法有限。尽管基于SYBR Green的实时定量PCR（qPCR）是研究反刍动物细胞因子转录最常用的方法，但缺乏标准化阻碍了其在不同反刍动物疾病研究中的应用。为了获得可靠的qPCR结果，需要考虑几个变量：用于最佳标准化的参考基因的选择、引物组之间退火温度的变化以及检测的特异性和灵敏度。在本研究中，我们开发并验证了一组用于牛和羊样本的免疫标志物，使用基于SYBR Green的qPCR，以具有成本效益的方式，通过优化多个引物组在共同的热循环温度下进行扩增。设计了20个引物组来定量羊和牛模板中的免疫标志物（IL-1b、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、TNF-α、IFN-γ、IFN-α、Ki-67、NFkB-65、TLR-3、TLR-4、TLR-8和Rig-1）。为了进行最佳标准化和选择合适的参考基因，该组中还包括测量五个参考基因转录的引物组。对所有靶基因的扩增效率、线性和特异性进行了验证。除Ki67外，所有基因靶点在羊和牛样本中均实现了最佳扩增条件。对用三种不同处理（佛波酯/离子霉素、刀豆蛋白A（Con A）和商陆有丝分裂原（PWM））刺激的绵羊外周血单个核细胞（PBMC）获得的羊和牛mRNA进行了相对定量研究。商陆和ConA有效地诱导了大多数靶向基因的基因转录，而佛波酯/离子霉素的诱导作用较弱。最后，我们通过在不同疫苗刺激的牛单核细胞衍生树突状细胞（MoDC）上运行该组进一步评估了其可用性。结果证实狂犬病疫苗诱导了特定的促炎基因转录模式，这与病毒感染期间发生的模式相似。总之，我们验证了一个扩展的实时PCR组的效率和可用性，该组能够通过使用一套试剂和方案快速测量广泛的羊和牛免疫标志物，从而代表了一种有效且具有成本效益的研究工具。
