The skin-swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro-inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin-swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin-swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro-inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72-hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro-inflammatory cytokine (interleukin 6 [IL-6] and interferon gamma[IFN-γ]) expression than PHA during peak response (24-h post-treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin-swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin-swelling test results.

Department of Zoology Faculty of Science Charles University Prague Viničná 7 128 44 Praha Czech Republic EU

Department of Zoology Faculty of Science Charles University Prague Viničná 7128 44 Praha Czech Republic EU; Research Facility Studenec Institute of Vertebrate Biology Czech Academy of Sciences Květná 8603 65 Brno Czech Republic EU

Institute of Experimental Medicine Czech Academy of Sciences Vídeňská 1083 142 20 Praha 4 Czech Republic EU

Research Facility Studenec Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic EU

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Adelman, J. S. , Ardia D. R., and Schat K. A.. 2014. Ecoimmunology Pp. 391–411 in Schat K. A., Kaspers B. and Kaiser P., eds. Avian immunology. Academic Press, San Diego, CA.

Akla, N. , Pratt J., and Annabi B.. 2012. Concanavalin‐A triggers inflammatory response through JAK/STAT3 signalling and modulates MT1‐MMP regulation of COX‐2 in mesenchymal stromal cells. Exp. Cell Res. 318:2498–2506. PubMed

Ando, Y. , Yasuoka C., Mishima T., Ikematsu T., Uede T., Matsunaga T., et al. 2014. Concanavalin A‐mediated T cell proliferation is regulated by herpes virus entry mediator costimulatory molecule. In Vitro Cell. Dev. Biol. Anim. 50:313–320. PubMed

Bonforte, R. J. , Glade P. R., Siltzbac L. E., and Topilsky M.. 1972. Phytohemagglutinin skin‐test – possible in‐vivo measure of cell‐mediated immunity. J. Pediatr. 81:775–780. PubMed

Brown, G. P. , Shilton C. M., and 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.

Chen, Y. L. , Peng H., Chen Y. Y., Wei H. M., Sun R., and Tian Z. G.. 2014. CD49a promotes T‐cell‐mediated hepatitis by driving T helper 1 cytokine and interleukin‐17 production. Immunology 141:388–400. PubMed PMC

Cheng, S. , and Lamont S. J.. 1988. Genetic analysis of immunocompetence measures in White Leghorn chicken line. Poult. Sci. 67:989–995. PubMed

Crawley, M. J. 2012. The R book, 2nd ed Wiley, Chichester, U.K.

Desmedt, M. , Rottiers P., Dooms H., Fiers W., and Grooten J.. 1998. Macrophages induce cellular immunity by activating Th1 cell responses and suppressing Th2 cell responses. J. Immunol. 160:5300–5308. PubMed

Ekkel, E. D. , Kuypers A. H., Counotte G. H. M., and Tielen M. J. M.. 1995. The phytohemagglutinin [PHA] skin‐test as an indicator of stress‐induced changes in immune reactivity in pigs. Vet. Q 17:143–146. PubMed

Fernandez‐De‐Mera, I. G. , Hofle U., Vicente J., Garcia A., Rodriguez O., and Gortazar C.. 2006. Optimal dose and timing in phytohaemagglutinin skin‐testing of deer. N. Z. Vet. J. 54:357–359. PubMed

Fernandez‐De‐Mera, I. G. , Vicente J., Hofle U., Rodriguez O., Gaspar‐Lopez E., and Gortazar C.. 2008. The effects of sex and age on phytohaemagglutinin skin‐testing of deer. N. Z. Vet. J. 56:71–73. PubMed

Fernandez‐De‐Mera, I. G. , Vicente J., Hofle U., Fons F. R., Ortiz J. A., and Gortazar C.. 2009. Factors affecting red deer skin test responsiveness to bovine and avian tuberculin and to phytohaemagglutinin. Prev. Vet. Med. 90:119–126. PubMed

Fielding, C. A. , McLoughlin R. M., McLeod L., Colmont C. S., Najdovska M., Grail D., et al. 2008. IL‐6 regulates neutrophil trafficking during acute inflammation via STAT3. J. Immunol. 181:2189–2195. PubMed

Finger, J. W. , Adams A. L., Thomson P. C., Shilton C. M., Brown G. P., Moran C., et al. 2013. Using phytohaemagglutinin to determine immune responsiveness in saltwater crocodiles (Crocodylus porosus). Aust. J. Zool. 61:301–311.

Goto, N. , Kodama H., Okada K., and Fujimoto Y.. 1978. Suppression of phytohemagglutinin skin response in thymectomized chickens. Poult. Sci. 57:246–250. PubMed

Goüy de Bellocq, J. , Krasnov B. R., Khokhlova I. S., Ghazaryan L., and Pinshow B.. 2006a. Immunocompetence and flea parasitism of a desert rodent. Funct. Ecol. 20:637–646.

Goüy de Bellocq, J. , Krasnov B. R., Khokhlova I. S., and Pinshow B.. 2006b. Temporal dynamics of a T‐cell mediated immune response in desert rodents. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 145:554–559. PubMed

Goüy de Bellocq, J. , Porcherie A., Moulia C., and Morand S.. 2007. Immunocompetence does not correlate with resistance to helminth parasites in house mouse subspecies and their hybrids. Parasitol. Res. 100:321–328. PubMed

Hernandez, A. , Yager J. A., Wilkie B. N., Leslie K. E., and Mallard B. A.. 2005. Evaluation of bovine cutaneous delayed‐type hypersensitivity (DTH) to various test antigens and a mitogen using several adjuvants. Vet. Immunol. Immunopathol. 104:45–58. PubMed

Iwakura, Y. , Nakae S., Saijo S., and Ishigame H.. 2008. The roles of IL‐17A in inflammatory immune responses and host defense against pathogens. Immunol. Rev. 226:57–79. PubMed

Jacobsson, H. , and Blomgren H.. 1974. Responses of mouse thymic cells to mitogens. Comparison between Phytohemagglutinin and Concanavalin A. Cell. Immunol. 11:427–441. PubMed

Jaroso, R. , Vicente J., Fernandez‐De‐Mera I. G., Aranaz A., and Gortazar C.. 2010. Eurasian wild boar response to skin‐testing with mycobacterial and non‐mycobacterial antigens. Prev. Vet. Med. 96:211–217. PubMed

Jones, G. 1973. Number of reactive cells in mouse lymphocyte cultures stimulated by phytohemagglutinin, concanavalin‐A or histocompatibility antigen. J. Immunol. 111:914–920. PubMed

Kennedy, M. W. , and Nager R. G.. 2006. The perils and prospects of using phytohaemagglutinin in evolutionary ecology. Trends Ecol. Evol. 21:653–655. PubMed

Leavitt, R. D. , Felsted R. L., and Bachur N. R.. 1977. Biological and biochemical properties of Phaseolus vulgaris isolectins. J. Biol. Chem. 252:2961–2966. PubMed

Licastro, F. , Davis L. J., and Morini M. C.. 1993. Lectins and superantigens – membrane interactions of these compounds with T‐lymphocytes affect immune‐responses. Int. J. Biochem. 25:845–852. PubMed

Lopez‐Jaramillo, F. J. , Gonzalez‐Ramirez L. A., Albert A., Santoyo‐Gonzalez F., Vargas‐Berenguel A., and Otalora F.. 2004. Structure of Concanavalin A at pH 8: bound solvent and crystal contacts. Acta Crystallogr. D Biol. Crystallogr. 60:1048–1056. PubMed

Martin, L. B. , Han P., Lewittes J., Kuhlman J. R., Klasing K. C., and Wikelski M.. 2006. Phytohemagglutinin‐induced skin swelling in birds: histological support for a classic immunoecological technique. Funct. Ecol. 20:290–299.

McLoughlin, R. M. , Witowski J., Robson R. L., Wilkinson T. S., Hurst S. M., Williams A. S., et al. 2003. Interplay between IFN‐gamma and IL‐6 signaling governs neutrophil trafficking and apoptosis during acute inflammation. J. Clin. Invest. 112:598–607. PubMed PMC

Merlo, J. L. , Cutrera A. P., Luna F., and Zenuto R. R.. 2014a. PHA‐induced inflammation is not energetically costly in the subterranean rodent Ctenomys talarum (tuco‐tucos). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 175:90–95. PubMed

Merlo, J. L. , Cutrera A. P., and Zenuto R. R.. 2014b. Inflammation in response to phytohemagglutinin injection in the Talas tuco‐tuco (Ctenomys talarum): implications for the estimation of immunocompetence in natural populations of wild rodents. Can. J. Zool. 92:689–697.

Ortiz, N. N. , Gerdol M., Stocchi V., Marozzi C., Randelli E., Bernini C., et al. 2014. T cell transcripts and T cell activities in the gills of the teleost fish sea bass (Dicentrarchus labrax). Dev. Comp. Immunol. 47:309–318. PubMed

Piálek, J. , Vyskočilová M., Bímová B., Havelková D., J. Piálková , Dufková P., et al. 2008. Development of unique house mouse resources suitable for evolutionary studies of speciation. J. Hered. 99:34–44. PubMed

Powell, P. C. 1980. The influence of erythrocytes on the stimulation of chicken lymphocytes by phytohemagglutinin. Avian Pathol. 9:465–470. PubMed

R Core Team (2014). R: A language and environment for statistical computing R Foundation for Statistical Computing, Vienna, Austria: URL http://www.R-project.org/

Rousselet, E. , Levin M., Gebhard E., Higgins B. M., DeGuise S., and Godard‐Codding C. A. J.. 2013. Evaluation of immune functions in captive immature loggerhead sea turtles (Caretta caretta). Vet. Immunol. Immunopathol. 156:43–53. PubMed

Scheller, J. , Chalaris A., Schmidt‐Arras D., and Rose‐John S.. 2011. The pro‐ and anti‐inflammatory properties of the cytokine interleukin‐6. Biochim. Biophys. Acta 1813:878–888. PubMed

Sheldon, B. C. , and Verhulst S.. 1996. Ecological immunology: costly parasite defences and trade‐offs in evolutionary ecology. Trends Ecol. Evol. 11:317–321. PubMed

Smits, J. E. , Bortolotti G. R., and Tella J. L.. 1999. Simplifying the phytohaemagglutinin skin‐testing technique in studies of avian immunocompetence. Funct. Ecol. 13:567–572.

Stadecker, M. , and Leskowitz S.. 1974. Cutaneous basophil response to mitogens. J. Immunol. 113:496–500. PubMed

Stobo, J. D. , and Paul W. E.. 1973. Functional heterogeneity of murine lymphoid‐cells.3. Differential responsiveness of T cells to phytohemagglutinin and Concanavalin A as a probe for T cell subsets. J. Immunol. 110:362–375. PubMed

Tollington, S. , Greenwood A., Jones C. G., Hoeck P., Chowrimootoo A., Smith D., et al. 2015. Detailed monitoring of a small but recovering population reveals sublethal effects of disease and unexpected interactions with supplemental feeding. J. Anim. Ecol. 84:969–977. PubMed PMC

Trosan, P. , Svobodova E., Chudickova M., Krulova M., Zajicova A., and Holan V.. 2012. The key role of Insulin‐like growth factor I in limbal stem cell differentiation and the corneal wound‐healing process. Stem Cells Dev. 21:3341–3350. PubMed PMC

Turmelle, A. S. , Ellison J. A., Mendonca M. T., and McCracken G. F.. 2010. Histological assessment of cellular immune response to the phytohemagglutinin skin test in Brazilian free‐tailed bats (Tadarida brasiliensis). J. Comp. Physiol. B. 180:1155–1164. PubMed PMC

Vinkler, M. , Bainova H., and Albrecht T.. 2010. Functional analysis of the skin‐swelling response to phytohaemagglutinin. Funct. Ecol. 24:1081–1086.

Vinkler, M. , Schnitzer J., Munclinger P., and Albrecht T.. 2012. Phytohaemagglutinin skin‐swelling test in scarlet rosefinch males: low‐quality birds respond more strongly. Anim. Behav. 83:17–23.

Vinkler, M. , Svobodova J., Gabrielova B., Bainova H., and Bryjova A.. 2014. Cytokine expression in phytohaemagglutinin‐induced skin inflammation in a galliform bird. J. Avian Biol. 45:43–50.

Weiss, A. , Shields R., Newton M., Manger B., and Imboden J.. 1987. Ligand‐receptor interactions required for commitment to the activation of the Interleukin‐2 gene. J. Immunol. 138:2169–2176. PubMed

Weissbrod, A. , Shapiro A., Vasserman G., Edry L., Dayan M., Yitzhaky A., et al. 2013. Automated long‐term tracking and social behavioural phenotyping of animal colonies within a semi‐natural environment. Nat. Commun. 4:2018. PubMed

Wiley, C. , Qvarnstrom A., and Gustafsson L.. 2009. Effects of hybridization on the immunity of collared Ficedula albicollis and pied flycatchers F‐hypoleuca, and their infection by haemosporidians. J. Avian Biol. 40:352–357.

Williams, R. M. , and Benacerraf B.. 1972. Genetic‐control of thymus‐derived cell function.1. In‐vitro DNA synthetic response of normal mouse spleen‐cells stimulated by mitogens Concanavalin A and phytohemagglutinin. J. Exp. Med. 135:1279–1292. PubMed PMC

Xu, D. L. , and Wang D. H.. 2010. Fasting suppresses T cell‐mediated immunity in female Mongolian gerbils (Meriones unguiculatus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 155:25–33. PubMed

Zhang, Z. Q. , and Zhao Z. J.. 2015. Correlations between phytohemagglutinin response and leukocyte profile, and bactericidal capacity in a wild rodent. Integr. Zool. 10:302–310. PubMed