121 research outputs found

    Chicken CSF2 and IL-4-, and CSF2-Dependent Bone Marrow Cultures Differentiate into Macrophages over time

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    Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC

    Age-related differences in the respiratory microbiota of chickens

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    In this era of next generation sequencing technologies it is now possible to characterise the chicken respiratory microbiota without the biases inherent to traditional culturing techniques. However, little research has been performed in this area. In this study we characterise and compare buccal, nasal and lung microbiota samples from chickens in three different age groups using 16S rRNA gene analysis. Buccal and nasal swabs were taken from birds aged 2 days (n = 5), 3 weeks (n = 5) and 30 months (n = 6). Bronchoalveolar lavage (BAL) samples were also collected alongside reagent only controls. DNA was extracted from these samples and the V2-V3 region of the 16S rRNA gene was amplified and sequenced. Quality control and OTU clustering were performed in mothur. Bacterial DNA was quantified using qPCR, amplifying the V3 region of the 16S rRNA gene. We found significant differences between the quantity and types of bacteria sampled at the three different respiratory sites. We also found significant differences in the composition, richness and diversity of the bacterial communities in buccal, nasal and BAL fluid samples between age groups. We identified several bacteria which had previously been isolated from the chicken respiratory tract in culture based studies, including lactobacilli and staphylococci. However, we also identified bacteria which have not previously been cultured from the respiratory tract of the healthy chicken. We conclude that our study can be used as a baseline that future chicken respiratory microbiota studies can build upon