A phytohaemagglutinin challenge test to assess immune responsiveness of European tree frog Hyla arborea

Abstract

Immune responsiveness, one measure of individual quality, can be used as a sensitive, non-lethal variable that may be negatively affected in animals exposed to degraded, contaminated or otherwise disturbed areas. One frequently used technique to measure immune responsiveness is the phytohaemagglutinin (PHA) challenge test. Swelling occurring at the injection sites are measured before and 24 h after PHA injection. The immune response is considered to be the difference between the two measures. Although this method is easily performed with wild animals, it has been rarely used on small amphibians. Here, we test the possibility of using a PHA test with the European tree frog, Hyla arborea, and we identify the optimal procedure for measuring immune responsiveness in this species. The results allowed us to simplify the procedure in eliminating phosphate-buffered saline (PBS) injection and reducing the duration of the experiment. Injection of PHA into the leg of H. arborea triggered an immune response with a peak of swelling 14 h after injection. A second injection of PHA into the same animal induced more intense leg swelling. In addition, haematological responses showed that the total number of leucocytes increased after PHA injection. A link between the leg swelling and the total leucocytes count recorded in blood has been found. Consequently, this method may provide a useful tool for predicting the pro-inflammatory capacity of field populations of small amphibians.