Longitudinal evidence for immunosenescence and inflammaging in free-living great tits.

The first-line effector mechanisms of immune defence, including inflammation and oxidative burst, contribute significantly to host-pathogen resistance. Whether these immune responses undergo age-related changes in birds remains unknown. Here, we tracked selected inflammatory parameters in 54 free-living great tits (Parus major) of known age, captured repeatedly over three consecutive years, with the aims to investigate long-term repeatability and age-dependent changes in cellular oxidative burst responsiveness upon in vitro stimulation with bacterial lipopolysaccharide (LPS), and to identify its relationships with leukotriene B4 (LTB4) levels and haematological traits. In addition, we linked these immunological traits to selected physiological markers (antioxidants and oxidative stress markers). LTB4 levels increased with age and we have shown a similar non-significant tendency also for absolute granulocyte counts, indicating propagating chronic inflammation over the bird's lifetime, consistent with the inflammaging hypothesis. In contrast, cellular oxidative burst followed a quadratic trend of dependency on age with a peak in midlife individuals, in line with the immunosenescence hypothesis. Interestingly, LTB4 levels were positively associated with general oxidative damage, but negatively with antioxidant glutathione peroxidase activity, indicating links to redox balance. This longitudinal study demonstrates the contrasting patterns of age-related changes in background and acute markers of pro-inflammatory immunity contributing to immunosenescence in birds and thus provides basis for interpretation of the tested inflammatory markers in cross-cohort datasets.

Domestic Fowl Breed Variation in Egg White Protein Expression: Application of Proteomics and Transcriptomics.

Avian egg white is essential for protecting and nourishing bird embryos during their development. Being produced in the female magnum, variability in hen oviduct gene expression may affect egg white composition in domestic chickens. Since traditional poultry breeds may represent a source of variation, in the present study we describe the egg white proteome (mass spectrometry) and corresponding magnum transcriptome (high-throughput sequencing) for 20 hens from five domestic fowl breeds (large breeds: Araucana, Czech golden pencilled, Minorca; and small breeds: Booted bantam, Rosecomb bantam). In total, we identified 189 egg white proteins and 16391 magnum-expressed genes. The majority of egg white protein content comprised proteins with an antimicrobial function. Despite general similarity, Between-class Principal Component Analysis revealed significant breed-specific variability in protein abundances, differentiating especially small and large breeds. Though we found strong association between magnum mRNA expression and egg white protein abundance across genes, coinertia analysis revealed no transcriptome/proteome costructure at the individual level. Our study is the first to show variation in protein abundances in egg white across chicken breeds with potential effects on egg quality, biosafety, and chick development. The observed interindividual variation probably results from post-transcriptional regulation creating a discrepancy between proteomic and transcriptomic data.

Different breeds, different blood: Cytometric analysis of whole blood cellular composition in chicken breeds.

While haematological variation is well known in birds, variation in avian breeds (distinct morphotypes of the same species) remains unexplored. Poultry breeds, in particular, may show interesting evolutionary patterns and economically-relevant physiological differences. We performed a comparative examination of blood cellular composition in five chicken (Gallus gallus domesticus) breeds: Araucana, Booted bantam, Czech, Minorca and Rosecomb bantam. In standard-environment-reared hens whole-blood flow cytometry revealed remarkable differences in most erythrocyte- and leukocyte-related parameters. We identified two extremes: Czech, a European breed, with a low heterophil/lymphocyte (H/L) ratio and high CD4+ levels, and Araucana, a South-American breed, with a high H/L ratio and high relative monocyte count. Such variation may reflect a combination of artificial and natural selection acting on health- and stress-related traits in domestic populations. Different breeds have evolved different immunological adaptations reflecting their original need to fight pathogens and physiological constraint resulting from dissimilar physiological trade-offs.

Application of Concanavalin A during immune responsiveness skin-swelling tests facilitates measurement interpretation in mammalian ecology.

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.

The relationship between health and cell-mediated immunity measured in ecology: Phytohaemagglutinin skin-swelling test mirrors blood cellular composition.

The phytohaemagglutinin (PHA) skin-swelling test, a common ecological method for measuring immune responsiveness, uses a subcutaneous injection of PHA to induce an inflammatory response with metrically measurable swelling. Both the immunological basis of this response and the role of pre-exposure physiology remain poorly understood, however, preventing predictions of costs and benefits of immunity investments revealed by the test and their relationship to life-history traits. In this study, we measure PHA-triggered tissue swelling, cellular infiltration into inflamed tissue, and haematological changes in zebra finches Taeniopygia guttata, with the aim of identifying those cell types involved in activation of PHA-induced skin swelling. We show that the inflammation underlying the swelling response is dependent on initial haematological state and alters the composition of circulating leukocytes. Injection of PHA decreased heterophil and monocyte frequency and increased basophil frequency, suggesting changes in leukocyte trafficking and haematopoiesis. Higher initial basophil and lymphocyte frequencies in peripheral blood were associated with increased swelling. High pre-treatment blood lymphocyte levels led to intense lymphocyte migration into inflamed tissue, while high initial basophil levels resulted in lower cellular infiltration. Our results show two possible directions in the association between pre-exposure health and swelling response: (i) a lymphocyte-based pathway that leads to higher responsiveness in high-quality healthy individuals capable of higher energy and resource investment, or (ii) higher responsiveness in diseased individuals with initially elevated basophil levels and pre-activated immunity. Haematological investigation, therefore, facilitates interpretation of PHA skin-swelling test results in ecological immunology, explaining ca. 20 % of variation in skin-swelling data. J. Exp. Zool. 323A: 767-777, 2015. © 2015 Wiley Periodicals, Inc.