Cookies Policy

This site uses cookies. By continuing to browse the site you are agreeing to our use of cookies.

I accept this policy

Find out more here

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

No metrics data to plot.
The attempt to load metrics for this article has failed.
The attempt to plot a graph for these metrics has failed.
The full text of this article is not currently available.

Brill’s MyBook program is exclusively available on BrillOnline Books and Journals. Students and scholars affiliated with an institution that has purchased a Brill E-Book on the BrillOnline platform automatically have access to the MyBook option for the title(s) acquired by the Library. Brill MyBook is a print-on-demand paperback copy which is sold at a favorably uniform low price.

Access this article

+ Tax (if applicable)
Add to Favorites
You must be logged in to use this functionality

image of Amphibia-Reptilia

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. arboreatriggered 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.

Affiliations: 1: Université de Lyon, 69000 Lyon, France and Université Lyon 1, UMR CNRS 5023, 69622 Villeurbanne, France


Full text loading...


Data & Media loading...

1. Akira S. , Uematsu S. , Takeuchi O. ( 2006): "Pathogen recognition and innate immunity". Cell Vol 124: 783- 801. [Crossref]
2. Boughton R.K. , Joop G. , Armitage S.A.O. ( 2011): "Outdoor immunology: methodological considerations for ecologists". Funct. Ecol. Vol 25: 81- 100. [Crossref]
3. 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. Vol 2: 341- 348. [Crossref]
4. Carey C. , Cohen N. , Rollins-Smith L. ( 1999): "Amphibian declines: an immunological perspective". Dev. Comp. Immunol. Vol 23: 459- 472. [Crossref]
5. 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. Vol 22: 760- 772. [Crossref]
6. 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 Vol 181: 641- 648. [Crossref]
7. 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. Vol 80: 710- 730. [Crossref]
8. 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. Vol 39: 300- 310. [Crossref]
9. Gervasi S.S. , Foufopoulos J. ( 2008): "Costs of plasticity: responses to desiccation decrease post-metamorphic immune function in a pond-breeding amphibian". Funct. Ecol. Vol 22: 100- 108.
10. 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. Vol 22: 101- 110. [Crossref]
11. Grasman K. ( 2002): "Assessing immunological function in toxicological studies of avian wildlife". Integr. Comp. Biol. Vol 42: 34- 42. [Crossref]
12. Gross W.B. , Siegel P.B. ( 1986): "Effects of initial and second periods of fasting on heterophil/lymphocyte ratios and body weight". Avian Dis. Vol 30: 345- 346. [Crossref]
13. Kennedy M. , Nager R. ( 2006): "The perils and prospects of using phytohaemagglutinin in evolutionary ecology". Trends Ecol. Evol. Vol 21: 653- 655. [Crossref]
14. Kinney K.S. , Cohen N. ( 2005): "Increased splenocyte mitogenesis following sympathetic denervation in Xenopus laevis ". Dev. Comp. Immunol. Vol 29: 287- 293. [Crossref]
15. 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 Vol 12: 27- 34. [Crossref]
16. 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. Vol 20: 290- 299. [Crossref]
17. 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 Vol 273: 815- 822. [Crossref]
18. 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. Vol 58: 219- 222. [Crossref]
19. Meylan S. , Haussy C. , Voituron Y. ( 2010): "Physiological actions of corticosterone and its modulation by an immune challenge in reptiles". Gen. Comp. Endocrinol. Vol 169: 158- 166. [Crossref]
20. Owen J.P. , Clayton D.H. ( 2007): "Where are the parasites in the PHA response?" Trends Ecol. Evol. Vol 22: 228- 229. [Crossref]
21. 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. Vol 3: 1091- 1103. [Crossref]
22. R Core Team( 2010): R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. .
23. 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 Vol 8( 12): e84108. [Crossref]
24. Smits J.E. , Bortolotti G.R. , Tella J.L. ( 1999): "Simplifying the phytohaemagglutinin skin-testing technique in studies of avian immunocompetence". Funct. Ecol. Vol 13: 567- 572. [Crossref]
25. Tella J.L. , Lemus J.A. , Carrete M. , Blanco G. ( 2008): "The PHA test reflects acquired T-cell mediated immunocompetence in birds". PLoS ONE Vol 3: e3295. [Crossref]
26. 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 Vol 169: 23- 31. [Crossref]
27. Vinkler M. , Albrecht T. ( 2011): "Handling ‘immunocompetence’ in ecological studies: do we operate with confused terms?" J. Avian Biol. Vol 42: 490- 493. [Crossref]
28. Vinkler M. , Bainova H. , Albrecht T. ( 2010): "Functional analysis of the skin-swelling response to phytohaemagglutinin". Funct. Ecol. Vol 24: 1081- 1086. [Crossref]
29. 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. Vol 45: 43- 50. [Crossref]
30. Vleck C.M. , Vertalino N. , Vleck D. , Bucher T.L. ( 2000): "Stress, corticosterone and heterophil to lymphocyte rations in free-living Adelie penguins". Condor Vol 102: 392- 400. [Crossref]

Article metrics loading...



Can't access your account?
  • Tools

  • Add to Favorites
  • Printable version
  • Email this page
  • Subscribe to ToC alert
  • Get permissions
  • Recommend to your library

    You must fill out fields marked with: *

    Librarian details
    Your details
    Why are you recommending this title?
    Select reason:
    Amphibia-Reptilia — Recommend this title to your library
  • Export citations
  • Key

  • Full access
  • Open Access
  • Partial/No accessInformation