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 methods. 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 immediate 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 stimulation 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.
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Acknowledgements
We thank Tuul Sepp, Ulvi Karu, Marju Männiste, Richard Meitern, Jolanta Vrublevska and Martin Pent for the help with blood sampling and analysing and bird maintenance. Tuul Sepp kindly measured leukocyte concentrations. Discussions with Jan Knight greatly helped us to understand and adjust the Pholasin assay. Two anonymous reviewers provided constructive criticism on the manuscript. The study was financed by Estonian Science Foundation (grant # 7737 to PH), the Estonian Ministry of Education and Science (target-financing project # 0180004s09) and by the European Union through the European Regional Development Fund (Centre of Excellence FIBIR).
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Sild, E., Hõrak, P. Assessment of oxidative burst in avian whole blood samples: validation and application of a chemiluminescence method based on Pholasin. Behav Ecol Sociobiol 64, 2065–2076 (2010). https://doi.org/10.1007/s00265-010-1076-z
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DOI: https://doi.org/10.1007/s00265-010-1076-z