Phytohaemagglutinin (PHA) Wing Web Swelling Technique for Measuring Avian Immunocompetence Delayed-type hypersensitivity (DTH) –The reaction is antigen specific and causes swelling/thickening and redness at the site of antigen injection in immunized animals (e.g. tuberculosis skin test) Using DTH to measure Cell-mediated immune function –Inoculate with phytohaemagglutinin (PHA) –Acts as a T-cell mitogen; causes proliferation of T-cell populations –Amount of swelling “=“ strength of immune response

Use Phytohaemagglutinin (PHA) (PHA-P form) Derived from Phaseolus vulgaris (red kidney bean) Results in infiltration of lymphocytes and macrophages, and subsequent proliferation of T lymphocytes (T-cells), at site of injection Measures cell-mediated immune function: activation of macrophages, natural killer cells, cytotoxic T-cells and cytokines (NOT antibodies)

PHA 1. Prime Birds with PHA In order for it to be a DTH response, birds must have prior immunological exposure 5-10 days before the actual measurements, birds are “primed” with a subcutaneous injection of 0.25 mg of PHA dissolved in 50 ul of PBS buffer in the right scapular apterium (wing web) Creates immunological “memory”

2. Measure Baseline Wing Web Thickness with a Pressure Sensitive Micrometer Immediately Prior to the Second Injection of PHA Data are presented as the change in wing web thickness so a baseline thickness must be taken Another method is to use the left wing as a “control wing” and inject it with just PBS buffer Studies have demonstrated that either way is valid

PHA 3. Inoculate with PHA 5-10 days after priming and after taking a baseline measurement inject the right wing web a second time with 0.25 mg of PHA in 50 ul PBS This second injection will stimulate the DTH response

4. DTH/Cell-mediated immune response infiltration of lymphocytes and macrophages, and subsequent proliferation of T lymphocytes (T-cells), at site of injection

5. Measure Swelling with a Pressure Sensitive Micrometer Measure swelling at 24 and 48 hrs post-injection Amount of swelling “=“ strength of cell-mediated immune response i.e., more swelling = greater infiltration and proliferation of macrophages and lymphocytes

6. Plot Data as Change in Wing Web Thickness (Swelling – Baseline) Peak of swelling generally occurs at 24 hours **However, measuring at more than one time point is important because the peak of the curve may shift (dotted line)