The PHA Test Reflects Acquired T-Cell Mediated Immunocompetence in Birds

José L. Tella,Jesús A. Lemus,Martina Carrete,Guillermo Blanco,Derya Unutmaz

doi:10.1371/journal.pone.0003295

José L. Tella, Jesús A. Lemus + Show 3 more

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https://doi.org/10.1371/journal.pone.0003295

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Abstract

Backgroundcological 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.MethodologyHere, 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 FindingsOur 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/SignificanceThese 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.

Highlights

Immunocompetence has reached a central focus in evolutionary and behavioural ecology after the general upsurge of interest in host-parasite interactions, to the point of the emergence of immunoecology as a new scientific discipline [1]
Adapted from the poultry science methods used in the seventies [3], the technique consists of subcutaneous injection of the mitogen phytohemagglutinin (PHA) dissolved in phosphate-buffered saline (PBS), usually in the wing patagium, and quantifying concomitant swelling at the site of injection over time
Reflecting its wide use, the simplified protocol proposed by Smits et al [4], which involves avoiding the injection of PBS in the opposite patagium as an unneeded control [2], has been cited ca. 200 times since its publication in 1999 (ISI web of Science, acceded on August 2008)

Summary

Introduction

Immunocompetence has reached a central focus in evolutionary and behavioural ecology after the general upsurge of interest in host-parasite interactions, to the point of the emergence of immunoecology as a new scientific discipline [1]. 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.

Results

Conclusion