We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

Ducks and chickens are hosts of avian influenza virus, each with distinctive responses to infection. To understand these differences, we characterized the innate immune response to low-pathogenicity avian influenza virus H7N1 infection in chickens and ducks. Viral RNA was detected in the lungs of chickens from day 0.8 to 7, in ducks mainly at day 4. In both species, viral RNA was detected in the bursa and gut. Infection in chickens resulted in up-regulation of interferon (IFN)-α and IFN-β mRNA, while in the ducks IFN-γ mRNA was strongly up-regulated in the lung and bursa. In chickens and ducks, all investigated pathogen recognition receptor (PRR) mRNAs were up-regulated; however, in the chicken lung Toll-like receptor (TLR)7 and melanoma differentiation-associated protein (MDA)-5 mRNA were strongly induced. TLR3, TLR7 and MDA-5 responses correlated with IFN-α and IFN-β responses in chickens, but in ducks a correlation between IFN-α and TLR7, retinoic acid-inducible gene-I and MDA-5 was absent. We studied the responses of duck and chicken splenocytes to poly(I:C) and R848 analogues to analyse the regulation of PRRs without the interfering mechanisms of the influenza virus. This revealed IFN-α and IFN-γ responses in both species. MDA-5 was only strongly up-regulated in chicken splenocytes, in which time-related PRR responses correlated with the IFN-α and IFN-β response. This correlation was absent in duck splenocytes. In conclusion, chickens and ducks differ in induction of MDA-5, TLR7 and IFN-α mRNA after an influenza virus infection in vivo and after in vitro stimulation with TLR antagonists.

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-L1C C2-Xyl) modifications, and honeybee phospholipase A2, which carries Nglycans that contain the core K K1C C3-Fuc epitope. Evidence is presented that core K K1C C3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core K K1C C3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core K K1C C3-Fuc and L L1C C2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since Nglycans containing the core K K1C C3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.z 1999 Federation of European Biochemical Societies.

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.

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BackgroundMacrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model.ResultsBased upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings.ConclusionsThe data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0121-9) contains supplementary material, which is available to authorized users.

Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells. Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8alpha+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers. Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function. In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens.

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