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Phytohaemagglutinin (PHA) Wing Web Swelling Technique for Measuring Avian Immunocompetence

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)

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Phytohaemagglutinin (PHA) Wing Web Swelling Technique for Measuring Avian Immunocompetence

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  1. 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

  2. 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)

  3. 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”

  4. 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

  5. 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

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

  7. 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

  8. 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)

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