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.

建立免疫标记物组对于了解家畜传染病的不同转录方式至关重要。由于缺乏针对这些物种的抗体，目前用于牛和羊的可商购的免疫学分析的阵列受到限制。尽管基于SYBR Green的实时定量PCR（qPCR）是研究反刍动物中细胞因子转录的最常用方法，但是缺乏标准化会妨碍其在研究各种反刍动物疾病中的实施。为了获得可靠的qPCR结果，需要考虑几个变量：选择参考基因以实现最佳标准化，引物组之间退火温度的变化以及测定的特异性和灵敏度。

在这项研究中，我们使用基于SYBR Green的qPCR以具有成本效益的方式开发并验证了一组牛和绵羊样品中的免疫标记，并优化了多个引物组以在常见的热循环温度下扩增。设计了20套引物来定量免疫标记（IL-1b，IL-2，IL-4，IL-5，IL-6，IL-10，IL-12，IL-13，IL-15，IL-18，绵羊和牛模板中的IL-23，TNF-α，IFN-γ，IFN-α，Ki-67，NFkB-65，TLR-3，TLR-4，TLR-8和Rig-1）。为了优化归一化和选择合适的参考基因，面板中还包括测量五个参考基因转录的引物组。验证了所有靶基因的扩增效率，线性和特异性。除Ki67外，所有基因靶标的绵羊和牛样品均达到了最佳扩增条件。对通过三种不同处理（PMA /伊诺霉素，伴刀豆球蛋白A（Con A）和商陆有丝分裂原（PWM））刺激的绵羊外周血单核细胞（PBMC）获得的绵羊和牛mRNA进行了相对定量研究。商陆和ConA有效地诱导了大多数目标基因的基因转录，而PMA /伊诺霉素的诱导则较弱。最后，我们通过在用不同疫苗刺激的牛单核细胞衍生的树突状细胞（MoDC）上运行该面板，进一步评估了该面板的可用性。结果证实狂犬病疫苗诱导了一种特定的促炎基因转录模式，类似于病毒感染期间发生的一种。

总之，我们验证了扩展的实时PCR面板的效率和可用性，该面板可通过使用一套试剂和规程快速测量多种绵羊和牛免疫标记，从而代表了一种有效且具有成本效益的工具用于研究目的。