Cytokine RT-qPCR and ddPCR for immunological investigations of the endangered Australian sea lion (Neophoca cinerea) and other mammals

Measurement of cytokine gene expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is used widely to assess the immune system of animals and to identify biomarkers of disease, but its application is limited in wildlife species due to a lack of species-specific reagents. The free-ranging endangered Australian sea lion (Neophoca cinerea) experiences significant clinical disease and high pup mortality due to intestinal hookworm infection. Developing immunological tools specific to the species will aid in the assessment of drivers of disease and its impact in population demographics. This study describes the development and validation of cross-reactive RT-qPCR assays to measure five important cytokines involved in innate and Th1/Th2 responses (IL-6, TNFα, IFNγ, IL-4 and IL-10) in unstimulated blood samples from a range of different mammalian species including the Australian sea lion. All RT-qPCR assays efficiencies ranged between 87% (Ovis aries TNFα) and 111% (Bos taurus IL-10) and had strong linearity (R(2)). IL-4 and IFNγ gene expression for N. cinerea fell below the dynamic range (and therefore quantifiable limits) of RT-qPCR assays but were able to be quantified using the novel droplet digital PCR (ddPCR). This study delivers new immunological tools for eco-immunologists studying cytokine gene expression in wildlife species and is to our knowledge, the first cytokine ddPCR approach to be reported in a pinniped species.