Measuring amphibian immunocompetence: validation of the phytohemagglutinin skin-swelling assay in the cane toad, Rhinella marina


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1. Measuring the degree of skin-swelling induced by intradermal injection of phytohemagglutinin (PHA) is simple, quick and inexpensive, does not require specialized equipment and is easily conducted under field conditions.

2. PHA is perhaps the most frequently used assay of immunocompetence in field studies of birds. However, the method has rarely been used, and never validated, for studies on ectothermic vertebrates.

3. Here, we document its use in an amphibian. In response to PHA injected into a toe web, cane toads exhibited a 35% increase in web thickness 24 h postinjection.

4. Histologically, PHA injection initiated a rapid (<12 h) infiltration of neutrophils, eosinophils and macrophages at the injection site, followed by an influx of lymphocytes by 24 h postinjection. A second exposure to PHA stimulated a faster, more intense swelling response.

5. In cane toads, PHA injection elicits a rapid innate immune response, followed by a secondary response that may reflect cell-mediated immune activity. Both components are easily quantifiable by the degree of skin-swelling.

6. Hence, PHA injection offers a convenient assay to quantify immune function in anurans and could usefully be incorporated into studies on the reasons for global amphibian declines.