Expression Profiling of Whole Blood as a Means of Detecting an Immune Response in an Avian Non-Model Species

Several emerging infectious diseases with major implications on biodiversity and human health have their origin in wild animals; still little research has been done on the immune system and its functions in wild populations. To better understand the ecology of infectious diseases and the spread of zoonotic diseases it is crucial to study infectious diseases and the immune system of their hosts in their natural environment. In this study we investigated the potential of using gene expression profiling from whole blood of the main host of avian influenza virus, the mallard (Anas platyrhynchos), as a means of detecting and analysing an immune response. For this purpose we simulated infections to mimic natural infections in mallards using three immunostimulants: lipopolysaccharides, polyinosinic:polycytidylic acid, and inactivated Staphylococcus aureus. A total of 100 blood samples, taken before and 3 hrs, 6 hrs, 12 hrs, and 24 hrs after stimulation, were individually analysed using high throughput Illumina RNA-sequencing and differential gene expression analysis. We found considerable differences in gene expression profiles between stimulated and unstimulated individuals for all treatment groups and time points. More than 50% of the differentially expressed genes could be assigned roles in the regulation of immune functions. Identification of these genes, but also of genes with previously unknown functions, sheds light on the evolution of avian immune systems. Our findings are of major importance for the growing field of eco-immunology because blood samples, in contrast to organs, can easily and repeatedly be obtained in the wild without harming the animal.