Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep

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.