METHODS Assessment of oxidative burst in avian whole blood samples: validation and application of a chemiluminescence method based on Pholasin Elin Sild & Peeter Hõrak Received: 11 June 2010 / Revised: 20 September 2010 / Accepted: 4 October 2010 / Published online: 17 October 2010 # Springer-Verlag 2010 Abstract Immunoecology deals with the questions about how immune defences have evolved and are being used and optimized in different environments, ecological settings and lineages. In such research, often only single time point measures and small sample volumes are available, which limits the applicability of traditional immunological meth- ods. Methodological progress in the field thus largely depends on the development and validation of immune assays suitable for ecological studies. Here we validate and apply a novel, Pholasin-based chemiluminescence method for assessment of oxidative burst in the whole blood samples of birds. This assay measures an inducible component of innate immunity by quantifying the immedi- ate extracellular oxidative burst of stimulated phagocytes. The assay procedure is simple, measurement precision is satisfactory and the measurement time is only 6 min. It can be performed on 20 μL (or smaller) blood samples that can be preserved for a few days. Blood of six studied passerine species produced chemiluminescence response to stimula- tion with bacterial lipopolysaccharide (LPS) in vitro. The magnitude of the response depended on the concentration and origin of the LPS. Parameters of this response depended on biological factors such as age of birds and in vivo priming with different antigens such LPS and Brucella abortus antigen suspension. Different parameters of the chemiluminescence response were significantly repeatable over 6-day period. All these properties argue for a great potential applicability of this method in immunoecological research. Keywords Avian innate immunity . Chemiluminescence . Oxidative burst . Passerine birds . Phagocyte priming . Pholasin Introduction Immunoecology is a nascent discipline, viewing immune function in the context of ecology and adaptation (Sheldon and Verhulst 1996; Schulenburg et al. 2009) that asks how immune defences have evolved and are being used and optimized in different environments, ecological settings and lineages. The basic link between immunology and evolution- ary ecology stems from the costs of immunity—energetic, developmental, immunopathological etc. (Klasing et al. 1999; Klasing 2004; Sadd and Schmid-Hempel 2009). If immune responses require resources that could be potentially allocat- ed to life-history traits such as growth and reproduction or sexual display, emergence of trade-offs between immune function and other components of fitness is inevitable (Sheldon and Verhulst 1996; Lochmiller and Deerenberg 2000; Norris and Evans 2000). Assessment of the costs related to immune function has remained a major problem for field biologists (Klasing 2004; Lee 2006; Sadd and Schmid-Hempel 2009), not least due to specific limitations inherent to ecological research. Such limitations include species specificity of many assays, requirements of non-lethality and often availability of only single time point measures and small sample volumes (Matson et al. 2005; Millet et al. 2007). Development and Communicated by J. Graves E. Sild (*) : P. Hõrak Department of Zoology, Institute of Ecology and Earth Sciences, The Centre of Excellence FIBIR, Tartu University, Vanemuise 46, 51014 Tartu, Estonia e-mail: egaju@ut.ee Behav Ecol Sociobiol (2010) 64:2065–2076 DOI 10.1007/s00265-010-1076-z