Avian primordial germ cells are bipotent for male or female gametogenesis

In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells

A low-tech, cost-effective and efficient method for safeguarding genetic diversity by direct cryopreservation of poultry embryonic reproductive cells

Chickens are an important resource for smallholder farmers who raise locally adapted, genetically distinct breeds for eggs and meat. The development of efficient reproductive technologies to conserve and regenerate chicken breeds safeguards existing biodiversity and secures poultry genetic resources for climate resilience, biosecurity, and future food production. The majority of the over 1600 breeds of chicken are raised in low and lower to middle income countries under resource-limited, small-scale production systems, which necessitates a low-tech, cost-effective means of conserving diversity is needed. Here, we validate a simple biobanking technique using cryopreserved embryonic chicken gonads. The gonads are quickly isolated, visually sexed, pooled by sex, and cryopreserved. Subsequently, the stored material is thawed and dissociated before injection into sterile host chicken embryos. By using pooled GFP and RFP-labelled donor gonadal cells and Sire Dam Surrogate mating, we demonstrate that chicks deriving entirely from male and female donor germ cells are hatched. This technology will enable ongoing efforts to conserve chicken genetic diversity for both commercial and smallholder farmers, and to preserve existing genetic resources at poultry research facilities

Chicken anaemia virus evades host immune responses in transformed lymphocytes

Chicken anaemia virus (CAV) is a lymphotropic virus that causes anaemia and immunosuppression in chickens. Previously, we proposed that CAV evades host antiviral responses in vivo by disrupting T-cell signalling, but the precise cellular targets and modes of action remain elusive. In this study, we examined gene expression in Marek’s disease virus-transformed chicken T-cell line MSB-1 after infection with CAV using both a custom 5K immune-focused microarray and quantitative realtime PCR at 24, 48 and 72 h post-infection. The data demonstrate an intricate equilibrium between CAV and the host gene expression, displaying subtle but significant modulation of transcripts involved in the T-cell, inflammation and NF-kB signalling cascades. CAV efficiently blocked the induction of type-I interferons and interferon-stimulated genes at 72 h. The cell expression pattern implies that CAV subverts host antiviral responses and that the transformed environment of MSB-1 cells offers an opportunistic advantage for virus growth

Analysis of the function of IL-10 in chickens using specific neutralising antibodies and a sensitive capture ELISA

AbstractIn mammals, the inducible cytokine interleukin 10 is a feedback negative regulator of inflammation. To determine the extent to which this function is conserved in birds, recombinant chicken IL-10 was expressed as a secreted human Ig Fc fusion protein (chIL-10-Fc) and used to immunise mice. Five monoclonal antibodies (mAb) which specifically recognise chicken IL-10 were generated and characterised. Two capture ELISA assays were developed which detected native chIL-10 secreted from chicken bone marrow-derived macrophages (chBMMs) stimulated with lipopolysaccharide (LPS). Three of the mAbs detected intracellular IL-10. This was detected in only a subset of the same LPS-stimulated chBMMs. The ELISA assay also detected massive increases in circulating IL-10 in chickens challenged with the coccidial parasite, Eimeria tenella. The same mAbs neutralised the bioactivity of recombinant chIL-10. The role of IL-10 in feedback control was tested in vitro. The neutralising antibodies prevented IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes and increased nitric oxide production in LPS-stimulated chBMMs. The results confirm that IL-10 is an inducible feedback regulator of immune response in chickens, and could be the target for improved vaccine efficacy or breeding strategies

Transcriptomic Profiling of Virus-Host Cell Interactions following Chicken Anaemia Virus (CAV) Infection in an In Vivo Model.

Chicken Anaemia Virus (CAV) is an economically important virus that targets lymphoid and erythroblastoid progenitor cells leading to immunosuppression. This study aimed to investigate the interplay between viral infection and the host's immune response to better understand the pathways that lead to CAV-induced immunosuppression. To mimic vertical transmission of CAV in the absence of maternally-derived antibody, day-old chicks were infected and their responses measured at various time-points post-infection by qRT-PCR and gene expression microarrays. The kinetics of mRNA expression levels of signature cytokines of innate and adaptive immune responses were determined by qRT-PCR. The global gene expression profiles of mock-infected (control) and CAV-infected chickens at 14 dpi were also compared using a chicken immune-related 5K microarray. Although in the thymus there was evidence of induction of an innate immune response following CAV infection, this was limited in magnitude. There was little evidence of a Th1 adaptive immune response in any lymphoid tissue, as would normally be expected in response to viral infection. Most cytokines associated with Th1, Th2 or Treg subsets were down-regulated, except IL-2, IL-13, IL-10 and IFNγ, which were all up-regulated in thymus and bone marrow. From the microarray studies, genes that exhibited significant (greater than 1.5-fold, false discovery rate <0.05) changes in expression in thymus and bone marrow on CAV infection were mainly associated with T-cell receptor signalling, immune response, transcriptional regulation, intracellular signalling and regulation of apoptosis. Expression levels of a number of adaptor proteins, such as src-like adaptor protein (SLA), a negative regulator of T-cell receptor signalling and the transcription factor Special AT-rich Binding Protein 1 (SATB1), were significantly down-regulated by CAV infection, suggesting potential roles for these genes as regulators of viral infection or cell defence. These results extend our understanding of CAV-induced immunosuppression and suggest a global immune dysregulation following CAV infection

Cloning and characterisation of the chicken orthologue of dendritic cell-lysosomal associated membrane protein (DC-LAMP)

A cDNA encoding the chicken orthologue of dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208 was cloned by RT-PCR from RNA isolated from mature chicken bone marrow-derived dendritic cells (chBM-DCs). The cloned chicken DC-LAMP (chDC-LAMP) cDNA consists of 1281 nucleotides encoding an open reading frame of 426 amino acids (aa). Comparison of the deduced aa sequence of DC-LAMP with orthologous proteins from human and mouse revealed 27 and 24 identity, respectively. The predicted chDC-LAMP protein shares the characteristic features of LAMP family members. ChDC-LAMP mRNA, unlike its mammalian orthologues, was expressed in a wide range of tissues, at highest levels in the lung. Lymphoid tissues including thymus, spleen, bursa, ceacal tonsil and Meckel's diverticulum had high chDC-LAMP mRNA expression levels. ChDC-LAMP mRNA was expressed in all splenocyte subsets with the highest expression in Bu-1 + B cells and KUL01 + cells, which would include macrophages and DC. ChDC-LAMP mRNA was highly expressed in chBM-DC, whereas expression levels in chicken monocyte-derived macrophages (chMo-Mac) and the HD11 macrophage cell line were significantly lower. Following CD40L stimulation, chDC-LAMP mRNA expression levels were up-regulated in mature chBM-DC, chMo-Mac and HD11 cells whereas lipopolysaccharide (LPS) only up-regulated chDC-LAMP mRNA expression levels in chBM-DC. ChDC-LAMP is not solely expressed on chicken DC but can be used as a marker to differentiate between immature and mature DC. © 2009 Elsevier Ltd. All rights reserved

Characterization of subpopulations of chicken mononuclear phagocytes that express TIM4 and CSF1R

The phosphatidylserine receptor TIM4, encoded by , mediates the phagocytic uptake of apoptotic cells. We applied anti-chicken TIM4 mAbs in combination with reporter transgenes to dissect the function of TIM4 in the chick (). During development in ovo, TIM4 was present on the large majority of macrophages, but expression became more heterogeneous posthatch. Blood monocytes expressed KUL01, class II MHC, and mApple uniformly. Around 50% of monocytes were positive for surface TIM4. They also expressed many other monocyte-specific transcripts at a higher level than TIM4 monocytes. In liver, highly phagocytic TIM4 cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. Although they expressed mRNA, Kupffer cells did not express the -mApple transgene, suggesting that additional transcriptional regulatory elements are required by these cells. By contrast, -mApple was detected in liver TIM4 and TIM4 cells, which were not phagocytic and were more abundant than Kupffer cells. These cells expressed alongside high levels of , , , and other markers associated with conventional dendritic cells in mice. In bursa, TIM4 was present on the cell surface of two populations. Like Kupffer cells, bursal TIM4 phagocytes coexpressed many receptors involved in apoptotic cell recognition. TIM4 cells appear to be a subpopulation of bursal B cells. In overview, TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and reporters distinguished Kupffer cells from an abundant population of dendritic cell-like cells

A low-tech, cost-effective and efficient method for safeguarding genetic diversity by direct cryopreservation of poultry embryonic reproductive cells

Chickens are an important resource for smallholder farmers who raise locally adapted, genetically distinct breeds for eggs and meat. The development of efficient reproductive technologies to conserve and regenerate chicken breeds safeguards existing biodiversity and secures poultry genetic resources for climate resilience, biosecurity, and future food production. The majority of the over 1600 breeds of chicken are raised in low and lower to middle income countries under resource-limited, small-scale production systems, which necessitates a low-tech, cost-effective means of conserving diversity is needed. Here, we validate a simple biobanking technique using cryopreserved embryonic chicken gonads. The gonads are quickly isolated, visually sexed, pooled by sex, and cryopreserved. Subsequently, the stored material is thawed and dissociated before injection into sterile host chicken embryos. By using pooled GFP and RFP-labelled donor gonadal cells and Sire Dam Surrogate mating, we demonstrate that chicks deriving entirely from male and female donor germ cells are hatched. This technology will enable ongoing efforts to conserve chicken genetic diversity for both commercial and smallholder farmers, and to preserve existing genetic resources at poultry research facilities