Development of gene expression assays measuring immune responses in the spotted hyena (<i>Crocuta crocuta</i>)

<p>As scavengers, spotted hyenas (<i>Crocuta crocuta</i>) are exposed to a wide array of pathogens but exhibit low mortality rates due to infectious disease. This suggests that this species exhibits a unique and robust immune response to pathogens. However, few tools exist to measure cell-mediated immunity (CMI) in hyenas and we aimed to develop a gene expression assay to quantify antigen-specific responses. Whole blood from five <i>Mycobacterium bovis</i>- sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes <i>ACTB</i>, <i>GAPDH</i>, <i>YWHAZ</i> and <i>TBP</i> in these samples was determined as well as the mean fold change in the expression of <i>IFNG</i>, <i>CXCL8</i>, <i>CXCL9</i>, <i>CXCL10</i> and <i>CXCL11</i> in <i>M. bovis</i>-antigen stimulated blood. The expression of <i>YWHAZ</i> and <i>TBP</i> showed greatest stability, and <i>YWHAZ</i> was selected as a reference for further analysis. The expression of <i>CXCL9</i> and <i>CXCL11</i> showed greatest upregulation in antigen-stimulated blood and the assay results for these genes were strongly correlated. The measurement of antigen-induced <i>CXCL9</i> and <i>CXCL11</i> expression, relative to that of <i>YWHAZ</i>, can be used to measure <i>CMI</i> responses to infectious diseases in spotted hyenas.</p