Summary1. The phytohaemagglutinin (PHA) skin-swelling test is widely used in immunoecology and ecotoxicology to estimate cell-mediated immunity. Although often presumed, the involvement of T cells in generating an immune response to PHA in vivo remains unclear. 2. To investigate the mechanism triggering this response we have compared in zebra finch (Taeniopygia guttata) the immune responses to two PHA isolectins differing in their biological properties and one control protein.3. In Experiment I, we applied PHA-P (the commonly used L and E isolectin mixture) into one wing-web and bovine serum allbumin (BSA) into the other. The swelling response to PHA-P was significantly stronger than to BSA confirming that the reaction is governed by PHA-specific properties. 4. In Experiment II, purified PHA-L (the T-cell-stimulating isolectin) and PHA-E (the erythroagglutinating isolectin) were compared. Contrary to our expectations, PHA-E induced a significantly stronger swelling response than PHA-L. Histological analysis revealed significantly higher quantities of erythrocytes and thrombocytes in PHA-E-treated patagia. Nevertheless, there was a positive correlation between the magnitudes of these swellings. 5.In Experiment III, we tested whether the results obtained by the application of PHA-P and pure PHA-L differ. Here, we failed to find any significant difference between these two preparations in their immunostimulatory activity. Magnitudes of the PHA-L-and PHA-P-induced swellings were positively correlated. 6. To the best of our knowledge this is the first study to compare the biological activity of purified PHA fractions in vivo and also the first to show the importance of erythroagglutination in the development of an inflammatory response to PHA-P. 7. Our results indicate that the skin-swelling test using PHA-P reliably mirrors the individual general proinflammatory potential. However, the immunological background of the test is highly complex and the test results cannot be interpreted as measurements of the adaptive immunity or T-cell activity. This interpretational change importantly alters our view on the test results regarding the costs of the response or the evolutionary immunological adaptations.

BackgroundToll-like receptors (TLR) are essential activators of the innate part of the vertebrate immune system. In this study, we analysed the interspecific variability of three TLR (bacterial-sensing TLR4 and TLR5 and viral-sensing TLR7) within the Galloanserae bird clade, investigated their phylogeny, assessed their structural conservation and estimated site-specific selection pressures.ResultsPhysiochemical properties varied according to the TLR analysed, mainly with regards to the surface electrostatic potential distribution. The predicted ligand-binding features (mainly in TLR4 and TLR5) differed between the avian proteins and their fish and mammalian counterparts, but also varied within the Galloanserae birds. We identified 20 positively selected sites in the three TLR, among which several are topologically close to ligand-binding sites reported for mammalian and fish TLR. We described 26, 28 and 25 evolutionarily non-conservative sites in TLR4, TLR5 and TLR7, respectively. Thirteen of these sites in TLR4, and ten in TLR5 were located in functionally relevant regions. The variability appears to be functionally more conserved for viral-sensing TLR7 than for the bacterial-sensing TLR. Amino-acid positions 268, 270, 343, 383, 444 and 471 in TLR4 and 180, 183, 209, 216, 264, 342 and 379 in TLR5 are key candidates for further functional research.ConclusionsHost-pathogen co-evolution has a major effect on the features of host immune receptors. Our results suggest that avian and mammalian TLR may be differentially adapted to pathogen-derived ligand recognition. We have detected signatures of positive selection even within the Galloanserae lineage. To our knowledge, this is the first study to depict evolutionary pressures on Galloanserae TLR and to estimate the validity of current knowledge on TLR function (based on mammalian and chicken models) for non-model species of this clade.Electronic supplementary materialThe online version of this article (doi:10.1186/s12711-014-0072-6) contains supplementary material, which is available to authorized users.

Researchers interested in ecological immunology face substantial methodological problems: 1) most immunological approaches are diffi cult to perform in free-living animals, 2) in some of the applicable methods the immunological background of the test remains unclear. Th e latter is also true for the phytohaemagglutinin (PHA) skin-swelling test, a trait of cell-mediated immunity commonly measured in ecology. A lack of direct evidence documenting the immunological processes in the tissue limits our understanding of the mechanism triggering the response to PHA. Understanding of this mechanism is, nonetheless, crucial for us to uncover the nature of ecological costs and benefi ts of investments into the response. As knowledge of cytokine signalling in the tissue may clarify the response mechanism, in our study we investigated the association between the PHA-induced skin-swelling and tissue cytokine expression in males of grey partridge Perdix perdix . In PHA-challenged birds we assessed expression of nine cytokines (IL-1 β , IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, TGF-β , IFN-γ ) in wing-web skin during an early stage of the immune response. We examined the relationship between the magnitude of tissue swelling and cytokine expression. Contrary to some earlier expectations we did not fi nd any diff erential expression of T-cell growth factor, IL-2, in the tissue. Hence, T-cell proliferation at the time of the swelling measurement is unlikely. We detected diff erential expression in Th 17 pro-infl ammatory (IL-1 β , IL-6) and anti-infl ammatory (TGF-β ) cytokines. Th e PHA-induced swelling response was only weakly linked to the expression of TGF-β . We also found relationships between the PHA-induced swelling response and phenotypic traits of the birds; the PHA swelling was positively associated with the extent of melanin-based breast ornamentation and negatively related to body size. Our results might suggest that variation in swelling is infl uenced by total numbers of responding cells rather than by diff erences in signalling. Moreover, we revealed signifi cant correlations in expression of IL-1 β , IL-6 and TGF-β . Th ese fi ndings are the fi rst to show on the molecular level that the PHA skin-swelling test actually measures infl ammation process which is part of innate immune defence and not the adaptive immune response (as assumed if the test was the refl ection of T-cell proliferation).

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Toll-like receptors (TLRs) are a cornerstone of vertebrate innate immunity. In this study, we identified orthologues of TLR4, TLR5 and TLR7 (representing both bacterial- and viral-sensing TLRs) in the grey partridge (Perdix perdix), a European Galliform game bird species. The phylogeny of all three TLR genes follows the known phylogeny of Galloanserae birds, placing grey partridge TLRs (PePeTLRs) in close proximity to their turkey and pheasant orthologues. The predicted proteins encoded by the PePeTLR genes were 843, 862-863 and 1,047 amino acids long, respectively, and clearly showed all TLR structural features. To verify functionality in these genes we mapped their tissue-expression profiles, revealing generally high PePeTLR4 and PePeTLR5 expression in the thymus and absence of PePeTLR4 and PePeTLR7 expression in the brain. Using 454 next-generation sequencing, we then assessed genetic variation within these genes for a wild grey partridge population in the Czech Republic, EU. We identified 11 nucleotide substitutions in PePeTLR4, eight in PePeTLR5 and six in PePeTLR7, resulting in four, four and three amino acid replacements, respectively. Given their locations and chemical features, most of these non-synonymous substitutions probably have a minor functional impact. As the intraspecific genetic variation of the three TLR genes was low, we assume that either negative selection or a bottleneck may have reduced TLR population variability in this species.