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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)
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
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)
Change in wing web thickness (mm)
• Peak of swelling generally occurs at 24 hours
1.2
**However, measuring at
more than one time point is
important because the peak
of the curve may shift
(dotted line)
1.0
0.8
0.6
0.4
0.2
0.0
24 hrs
48 hrs
Time after challenge