A phytohaemagglutinin challenge test to assess immune responsiveness of European tree frog Hyla arborea

In: Amphibia-Reptilia
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  • 1 Université de Lyon, 69000 Lyon, France and Université Lyon 1, UMR CNRS 5023, 69622 Villeurbanne, France

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Immune responsiveness, one measure of individual quality, can be used as a sensitive, non-lethal variable that may be negatively affected in animals exposed to degraded, contaminated or otherwise disturbed areas. One frequently used technique to measure immune responsiveness is the phytohaemagglutinin (PHA) challenge test. Swelling occurring at the injection sites are measured before and 24 h after PHA injection. The immune response is considered to be the difference between the two measures. Although this method is easily performed with wild animals, it has been rarely used on small amphibians. Here, we test the possibility of using a PHA test with the European tree frog, Hyla arborea, and we identify the optimal procedure for measuring immune responsiveness in this species. The results allowed us to simplify the procedure in eliminating phosphate-buffered saline (PBS) injection and reducing the duration of the experiment. Injection of PHA into the leg of H. arborea triggered an immune response with a peak of swelling 14 h after injection. A second injection of PHA into the same animal induced more intense leg swelling. In addition, haematological responses showed that the total number of leucocytes increased after PHA injection. A link between the leg swelling and the total leucocytes count recorded in blood has been found. Consequently, this method may provide a useful tool for predicting the pro-inflammatory capacity of field populations of small amphibians.

  • Akira S., Uematsu S., Takeuchi O. (2006): Pathogen recognition and innate immunity. Cell 124: 783-801.

  • Boughton R.K., Joop G., Armitage S.A.O. (2011): Outdoor immunology: methodological considerations for ecologists. Funct. Ecol. 25: 81-100.

  • Brown G., Shilton C., Shine R. (2011): Measuring amphibian immunocompetence: validation of the phytohemagglutinin skin-swelling assay in the cane toad, Rhinella marina. Methods Ecol. Evol. 2: 341-348.

    • Search Google Scholar
    • Export Citation
  • Carey C., Cohen N., Rollins-Smith L. (1999): Amphibian declines: an immunological perspective. Dev. Comp. Immunol. 23: 459-472.

  • Davis A.K., Maney D.L., Maerz J.C. (2008): The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct. Ecol. 22: 760-772.

    • Search Google Scholar
    • Export Citation
  • Dehnhard N., Quillfeldt P., Hennicke J.C. (2011): Leucocyte profiles and H/L ratios in chicks of red-tailed tropicbirds reflect the ontogeny of the immune system. J. Comp. Physiol. B 181: 641-648.

    • Search Google Scholar
    • Export Citation
  • Demas G.E., Zysling D.A., Beechler B.R., Muehlenbein M.P., French S.S. (2011): Beyond phytohaemagglutinin: assessing vertebrate immune function across ecological contexts. J. Anim. Ecol. 80: 710-730.

    • Search Google Scholar
    • Export Citation
  • Fokidis H.B., Greiner E.C., Deviche P. (2008): Interspecific variation in avian blood parasites and hematology associated with the urban-rural gradient of a desert city. J. Avian Biol. 39: 300-310.

    • Search Google Scholar
    • Export Citation
  • Gervasi S.S., Foufopoulos J. (2008): Costs of plasticity: responses to desiccation decrease post-metamorphic immune function in a pond-breeding amphibian. Funct. Ecol. 22: 100-108.

    • Search Google Scholar
    • Export Citation
  • Gilbertson M.K., Haffner G.D., Drouillard K.G., Albert A., Dixon B. (2003): Immunosuppression in the northern leopard frog (Rana pipiens) induced by pesticide exposure. Environ. Toxicol. Chem. 22: 101-110.

    • Search Google Scholar
    • Export Citation
  • Grasman K. (2002): Assessing immunological function in toxicological studies of avian wildlife. Integr. Comp. Biol. 42: 34-42.

  • Gross W.B., Siegel P.B. (1986): Effects of initial and second periods of fasting on heterophil/lymphocyte ratios and body weight. Avian Dis. 30: 345-346.

    • Search Google Scholar
    • Export Citation
  • Kennedy M., Nager R. (2006): The perils and prospects of using phytohaemagglutinin in evolutionary ecology. Trends Ecol. Evol. 21: 653-655.

    • Search Google Scholar
    • Export Citation
  • Kinney K.S., Cohen N. (2005): Increased splenocyte mitogenesis following sympathetic denervation in Xenopus laevis. Dev. Comp. Immunol. 29: 287-293.

    • Search Google Scholar
    • Export Citation
  • Lobato E., Moreno J., Merino S., Sanz J.J., Arriero E. (2005): Haematological variables are good predictors of recruitment in nestling pied flycatchers (Ficedula hypoleuca). Ecoscience 12: 27-34.

    • Search Google Scholar
    • Export Citation
  • Martin L.B., Han P., Lewittes J., Kuhlman J., Klasing K., Wikelsk M. (2006): Phytohemagglutinin-induced skin swelling in birds: histological support for a classic immunoecological technique. Funct. Ecol. 20: 290-299.

    • Search Google Scholar
    • Export Citation
  • Matson K., Cohen A., Klasing K., Ricklefs R., Scheuerlein A. (2006): No simple answers for ecological immunology: relationships among immune indices at the individual level break down at the species level in waterfowl. Proc. R. Soc. B 273: 815-822.

    • Search Google Scholar
    • Export Citation
  • Merino S., Martinez J., Møller A.P., Sanabria L., De Lope F., Pérez J., Rodrigues-Caabeiro F. (1999): Phytohaemagglutinin injection assay and physiological stress in nestling house martins. Anim. Behav. 58: 219-222.

    • Search Google Scholar
    • Export Citation
  • Meylan S., Haussy C., Voituron Y. (2010): Physiological actions of corticosterone and its modulation by an immune challenge in reptiles. Gen. Comp. Endocrinol. 169: 158-166.

    • Search Google Scholar
    • Export Citation
  • Owen J.P., Clayton D.H. (2007): Where are the parasites in the PHA response? Trends Ecol. Evol. 22: 228-229.

  • Pigeon G., Bélisle M., Garant D., Cohen A., Pelletier F. (2013): Ecological immunology in a fluctuating environment: an integrative analysis of tree swallow nestling immune defense. Ecol. Evol. 3: 1091-1103.

    • Search Google Scholar
    • Export Citation
  • R Core Team (2010): R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/.

    • Search Google Scholar
    • Export Citation
  • Salaberria C., Muriel J., de Luna M., Gil D., Puerta M. (2013): The PHA test as an indicator of phagocytic activity in a passerine birds. PLoS ONE 8 (12): e84108.

    • Search Google Scholar
    • Export Citation
  • Smits J.E., Bortolotti G.R., Tella J.L. (1999): Simplifying the phytohaemagglutinin skin-testing technique in studies of avian immunocompetence. Funct. Ecol. 13: 567-572.

    • Search Google Scholar
    • Export Citation
  • Tella J.L., Lemus J.A., Carrete M., Blanco G. (2008): The PHA test reflects acquired T-cell mediated immunocompetence in birds. PLoS ONE 3: e3295.

    • Search Google Scholar
    • Export Citation
  • Venesky M., Wilcoxen T., Rensel M., Rollins-Smith L., Kerby J., Parris M. (2012): Dietary protein restriction impairs growth, immunity, and disease resistance in southern leopard frog tadpoles. Oecologia 169: 23-31.

    • Search Google Scholar
    • Export Citation
  • Vinkler M., Albrecht T. (2011): Handling ‘immunocompetence’ in ecological studies: do we operate with confused terms? J. Avian Biol. 42: 490-493.

    • Search Google Scholar
    • Export Citation
  • Vinkler M., Bainova H., Albrecht T. (2010): Functional analysis of the skin-swelling response to phytohaemagglutinin. Funct. Ecol. 24: 1081-1086.

    • Search Google Scholar
    • Export Citation
  • Vinkler M., Svobodova J., Gabrielova B., Bainova H., Bryjova A. (2014): Cytokine expression in phytohaemagglutinin-induced skin inflammation in a galliform bird. J. Avian Biol. 45: 43-50.

    • Search Google Scholar
    • Export Citation
  • Vleck C.M., Vertalino N., Vleck D., Bucher T.L. (2000): Stress, corticosterone and heterophil to lymphocyte rations in free-living Adelie penguins. Condor 102: 392-400.

    • Search Google Scholar
    • Export Citation

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