Simplifying the phytohaemagglutinin skin-testing technique in studies of avian immunocompetence

Measures of immune function in birds, while routine in traditional areas of pathology and physiology of domestic fowl (e.g. Goto et al. 1978; van der Zijpp 1983; Laudert, Sivanandan & Halvorson 1993;Chu et al. 1995), have now been recognized as valuable tools to study behaviour, ecology and toxicology of wild species (e.g. Lochmiller, Vestey & Boran 1993; Dufva & Allander 1995; Saino, Calza & Møller 1997; Svensson & Skarstein 1997; Moreno et al. 1998). Variables related to immune function such as differential and total white blood cell counts, and production of antibody-producing lymphocytes against sheep red blood cells (SRBC) are commonly used but require specialized training or laboratory facilities. An inexpensive, easily applied skin test using phytohaemagglutinin (PHA) as a mitogen, has become a popular, effective test that can be applied with minimal training and without specialized equipment. The skin test provides a measure of the proliferative response potential of circulating T lymphocytes to an injected mitogen. Phytohaemagglutinin has long been recognized for its mitogenic and blastogenic properties (Hungerford et al. 1959). This test has been routinely applied, with some variation in the body sites used, according to a long-established protocol developed in poultry science (Goto et al. 1978). In the seminal work of Goto et al. (1978), histological assessment of skin that had been injected with 75 μg PHA revealed an infiltration by heterophils within 3 h. The transient heterophilia, a non-specific inflammatory reaction to mild antigenic stimulus, was no longer evident 6 h after injection. The main cellular response, observed at 6–12 h after injection, consisted of a prominent perivascular accumulation of Functional Ecology 1999 13, 567–572