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. 2008 Sep 29;3(9):e3295.
doi: 10.1371/journal.pone.0003295.

The PHA test reflects acquired T-cell mediated immunocompetence in birds

José L Tella  1 , Jesús A Lemus, Martina Carrete, Guillermo Blanco
Affiliations

The PHA test reflects acquired T-cell mediated immunocompetence in birds

José L Tella et al. PLoS One. .

Expression of concern in

Abstract

Background: cological immunology requires techniques to reliably measure immunocompetence in wild vertebrates. The PHA-skin test, involving subcutaneous injection of a mitogen (phytohemagglutinin, PHA) and measurement of subsequent swelling as a surrogate of T-cell mediated immunocompetence, has been the test of choice due to its practicality and ease of use in the field. However, mechanisms involved in local immunological and inflammatory processes provoked by PHA are poorly known, and its use and interpretation as an acquired immune response is currently debated.

Methodology: Here, we present experimental work using a variety of parrot species, to ascertain whether PHA exposure produces larger secondary than primary responses as expected if the test reflects acquired immunocompetence. Moreover, we simultaneously quantified T-lymphocyte subsets (CD4(+), CD5(+) and CD8(+)) and plasma proteins circulating in the bloodstream, potentially involved in the immunological and inflammatory processes, through flow cytometry and electrophoresis.

Principal findings: Our results showed stronger responses after a second PHA injection, independent of species, time elapsed and changes in body mass of birds between first and second injections, thus supporting the adaptive nature of this immune response. Furthermore, the concomitant changes in the plasma concentrations of T-lymphocyte subsets and globulins indicate a causal link between the activation of the T-cell mediated immune system and local tissue swelling.

Conclusions/significance: These findings justify the widespread use of the PHA-skin test as a reliable evaluator of acquired T-cell mediated immunocompetence in diverse biological disciplines. Further experimental research should be aimed at evaluating the relative role of innate immunocompetence in wild conditions, where the access to dietary proteins varies more than in captivity, and to ascertain how PHA responses relate to particular host-parasite interactions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Local tissue swelling (black dots) and body mass (white dots) of 125 birds subjected to two successive PHA-skin tests.
Figure 2
Figure 2. Changes in counts of lymphocyte subsets and protein concentrations (μg/L) in the bloodstream from basal levels to those resulting 24 h after a first and a second PHA injection.
Black dots represent experimental birds (injected with PHA) and white dots represent control birds (injected with PBS).
Figure 3
Figure 3. Relationships between local tissue swelling induced by first and second PHA injections and the concomitant changes in circulating lymphocyte subsets.
Regression coefficients and statistical significance are shown for each case.
Figure 4
Figure 4. Relationships between local tissue swelling induced by first and second PHA injections and the concomitant changes in circulating protein concentrations (μg/L).
Regression coefficient and statistical significance are shown for each case.
See this image and copyright information in PMC

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