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

Time after challenge

24 hrs 48 hrs

Ch

ang

e in

win

g w

eb t

hic

knes

s (m

m)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

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)