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

Abstract

1. Researchers involved in ecology and toxicology, as well as many other aspects of avian biology, use phytohaemagglutinin (PHA) skin testing as a means of evaluating the immune status of individuals. 2. Immune function, 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 ecological zones. 3. Typically this test has been applied by challenging one wing web with the immunostimulant PHA, while the other ‘control’ wing is injected with phosphate buffered saline (PBS). Injection sites on the wing web are measured before and 24 h after injection with PHA or PBS. The immune response is considered to be the difference between the two wings. 4. Results from PHA skin tests conducted on 608 birds in seven studies representing passerines, waterfowl, upland game birds and raptors are examined. 5. Numerous advantages to eliminating the PBS injection as the experimental control are: (i) decrease by half, the time required for testing; (ii) decrease handling‐related stress on the birds (proportional to handling time); (iii) reduce the probability of errors at injection time; (iv) spare the other wing for different tests or uses (e.g. tuberculin DTH testing); and (v) decrease the coefficient of variation that is due to measurement inaccuracies. 6. The only disadvantage identified is that hypersensitive individuals (outliers) could be missed, which in this case represents 2 of 608 individuals.

Sex-specific development of cell-mediated immunity under experimentally altered rearing conditions in blue tit nestlings

“…PHA is a bean extract that has a mitogenic effect on T lymphocytes, and its inoculation stimulates dense accumulation of lymphocytes (Goto et al 1978). When nestlings were 11 days old, 0.2 mg of PHA suspended in 0.04 ml of physiological saline solution was inoculated in the middle of the right wing web (Smits et al 1999). The thickness of the wing web was measured with a pressure-sensitive spessimeter (SM-12, Mitutoyo) prior to and 24 h after the PHA injection with an accuracy of 0.01 mm.…”

Genetics, local environment and health as factors influencing plasma carotenoids in wild American kestrels (Falco sparverius)

“…The thickness of each wing web was measured with a micrometer (nearest 0.001mm) just prior to and 24 h ( § 16 min) after injection. CMI was calculated as the di¡er-ence in wing web swelling between right and left wings (Smits et al 1999).…”

What Makes a Host Profitable? Parasites Balance Host Nutritive Resources against Immunity

“…As an index of nestling immunity, we measured cutaneous immunity in nestlings using the phytohemagglutinin (PHA) skin test. This immunological assay consists of injecting young subcutaneously with the mitogen PHA and measuring the local proliferation of T lymphocytes and granulocytes at the site of injection 24 h later (Smits et al 1999;Martin et al 2006). Host cutaneous immunity (i.e., adaptive and innate immunity) renders the biting site unfavorable for the feeding process in blood-sucking insects (Wikel 1996;Kamhawi et al 2000;Lehane 2005).…”

“…The PC1 and PC2 axes explained 80.8% and 15.2% of the variance, respectively. We assessed the ability of 30-day-old nestlings to mount a cutaneous immune response by injecting them subcutaneously in the wing web with 0.1 mg PHA (Sigma, code L1668) dissolved in 0.02 mL of phosphate-buffered saline and by subsequently measuring wing web swelling at the site of injection h later (Smits et al 1999;Martin 24 ‫ע‬ 1 et al 2006). The same trained person (P. Bize) measured wing web swelling of each nestling with a micrometer (Mitotuyo, ref.…”