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Birds have a unique organ for primary B lymphopoiesis, the bursa of Fabricius. Thus, avian B cell development can be classified into pre-bursal, bursal and post-bursal stages. During embryonic development, pre-bursal B cell precursors migrate in a single wave into the bursal mesenchyme; cells which express cell surface immunoglobulin as a consequence of productive gene rearrangement subsequently colonize the follicle anlagen. B cells then proliferate rapidly in bursal follicles and diversify their receptor repertoire by means of gene conversion. After hatch, the formerly homogenous follicles separate into two compartments: an outer cortex and a central medulla. Starting around the time of hatch, B cells emigrate from the bursa, predominantly from the follicle cortex, to colonize peripheral lymphoid tissues. In the periphery, naive B cells encounter antigen, undergo a germinal center reaction and differentiate into antibody-secreting plasma cells. Many aspects of these processes are regulated by cytokines. While the function of BAFF and CD40L on peripheral B cells is largely conserved between birds and mammals, both of these TNF family members play unique roles in the bursa of Fabricius.

Marek’s disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells., Author summary Upon MDV entry via the respiratory tract, B-cells are among the first cells to be infected in the lung and allow an efficient amplification of the virus. B-cells ensure the transmission of the virus to activated T-cells in which it replicates and ultimately transforms CD4-positive T-cells. Although playing a pivotal role in the MDV life cycle, the response of B-cells to MDV is currently not fully understood. Here, by using an in vitro infection model of primary bursal B-cells, we show that MDV infection leads to a prolonged B-cell survival resulting from decreased cell proliferation, protection from apoptosis and activation of autophagy. Our study provides new insights into the B-cell response to MDV infection, demonstrating that MDV triggers a senescence-like phenotype in B-cells that could potentiate their role in MDV pathogenesis.

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4(+) T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4(+) and CD8(+) T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

Harvesting of broilers is a stressful event, whether done mechanically or manually. The use of harvesting machines might compromise animal welfare less than manual harvesting methods because it is less influenced by environmental and flock-specific factors. In this paper, a mechanical and a manual harvesting method are compared in regard to stress-induced behavioural and physiological reactions. The stationary person test and the avoidance distance touch test were applied before harvesting to estimate pre-treatment fear of humans in the flocks. We then recorded the behaviour of the flocks during harvesting and evaluated stress-induced behaviour such as wing flapping and escape behaviour. Furthermore, we took blood samples at the abattoir and analysed the corticosterone concentration and heterophil/lymphocyte ratio. In a statistical analysis, all assessed parameters were related to environmental and flock-specific factors, as well as to the risk of lesions such as haematomas and fractures. Our aim was to figure out if the use of a harvesting machine, in this case the Apollo Generation 2, puts less stress on broilers during harvesting than manual catching. The applied behaviour tests indicate the excitability and fear of humans of the flocks but are complex to interpret. Compared with mechanical harvesting, manual harvesting was more influenced by environmental and flock-specific factors such as average weight (odds ratio [OR] = 1.49; 95% confidence interval [CI] [1.11; 2.22]) or catching duration (OR = 1.79; 95% CI [1.44; 2.19]). The results verified a correlation between stress-induced behaviour and the occurrence of lesions. The risk for haematomas was influenced by escape behaviour during manual harvesting (OR = 1.07; 95% CI [1.02; 1.13]) and by bumps against the containers (OR = 1.06; 95% CI [1.05; 1.07]) and flips (OR = 6.23; 95% CI [3.99; 9.27]) during mechanical harvesting. The risk for fractures during mechanical harvesting was influenced by the occurrence of flips (OR = 2.74; 95% CI [1.07; 5.67]). The risk for wing flapping was twice as high during manual harvesting as during mechanical harvesting (OR = 2.11; 95% CI [1.82; 2.44]). The blood parameters showed no correlations with the initial behaviour test results and the assessed stress-induced behaviour during harvesting. Corticosterone concentration was strongly influenced by light intensity (beta = 3.75; 95% CI [2.55; 4.95]) and outdoor temperature (beta = 46.34; 95% CI [39.18; 53.27]) during manual harvesting. The results showed weak points of both harvesting methods, and we offer suggestions to improve animal welfare during harvesting.

Marek’s disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek’s disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells. Electronic supplementary material The online version of this article (10.1186/s13567-018-0526-x) contains supplementary material, which is available to authorized users.

Marek's disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a T(reg) signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVβ1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/β T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system.

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.

Interferons (IFNs) are essential components of the antiviral defense system of vertebrates. In mammals, functional receptors for type III IFN (lambda interferon [IFN-λ]) are found mainly on epithelial cells, and IFN-λ was demonstrated to play a crucial role in limiting viral infections of mucosal surfaces. To determine whether IFN-λ plays a similar role in birds, we produced recombinant chicken IFN-λ (chIFN-λ) and we used the replication-competent retroviral RCAS vector system to generate mosaic-transgenic chicken embryos that constitutively express chIFN-λ. We could demonstrate that chIFN-λ markedly inhibited replication of various virus strains, including highly pathogenic influenza A viruses, in ovo and in vivo , as well as in epithelium-rich tissue and cell culture systems. In contrast, chicken fibroblasts responded poorly to chIFN-λ. When applied in vivo to 3-week-old chickens, recombinant chIFN-λ strongly induced the IFN-responsive Mx gene in epithelium-rich organs, such as lungs, tracheas, and intestinal tracts. Correspondingly, these organs were found to express high transcript levels of the putative chIFN-λ receptor alpha chain (chIL28RA) gene. Transfection of chicken fibroblasts with a chIL28RA expression construct rendered these cells responsive to chIFN-λ treatment, indicating that receptor expression determines cell type specificity of IFN-λ action in chickens. Surprisingly, mosaic-transgenic chickens perished soon after hatching, demonstrating a detrimental effect of constitutive chIFN-λ expression. Our data highlight fundamental similarities between the IFN-λ systems of mammals and birds and suggest that type III IFN might play a role in defending mucosal surfaces against viral intruders in most if not all vertebrates.

Antibody producing B-cells are an essential component of the immune system. In contrast to human and mice where B-cells develop in the bone marrow, chicken B-cells develop in defined stages in the bursa of Fabricius, a gut associated lymphoid tissue. In order to gain a better understanding of critical biological processes like immigration of B-cell precursors into the bursa anlage, their differentiation and final emigration from the bursa we analyzed the proteome dynamics of this organ during embryonic and posthatch development. Samples were taken from four representative developmental stages (embryonic day (ED) 10, ED18, day 2, and day 28) and compared in an extensive 2D DIGE approach comprising six biological replicates per time point. Cluster analysis and PCA demonstrated high reliability and reproducibility of the obtained data set and revealed distinctive proteome profiles for the selected time points, which precisely reflect the differentiation processes. One hundred fifty three protein spots with significantly different intensities were identified by MS. We detected alterations in the abundance of several proteins assigned to retinoic acid metabolism (e.g. retinal-binding protein 5) and the actin-cytoskeleton (e.g. vinculin and gelsolin). By immunohistochemistry, desmin was identified as stromal cell protein associated with the maturation of B-cell follicles. Strongest protein expression difference (10.8-fold) was observed for chloride intracellular channel 2. This protein was thus far not associated with B-cell biology but our data suggest an important function in bursa B-cell development.

Rationale: Cardiac neural crest cells (NCs) contribute to heart morphogenesis by giving rise to a variety of cell types from mesenchyme of the outflow tract, ventricular septum, and semilunar valves to neurons of the cardiac ganglia and smooth muscles of the great arteries. Failure in cardiac NC development results in outflow and ventricular septation defects commonly observed in congenital heart diseases. Cardiac NCs derive from the vagal neural tube, which also gives rise to enteric NCs that colonize the gut; however, so far, molecular mechanisms segregating these 2 populations and driving cardiac NC migration toward the heart have remained elusive. Objective: Stromal-derived factor-1 (SDF1) is a chemokine that mediates oriented migration of multiple embryonic cells and mice deficient for Sdf1 or its receptors, Cxcr4 and Cxcr7 , exhibit ventricular septum defects, raising the possibility that SDF1 might selectively drive cardiac NC migration toward the heart via a chemotactic mechanism. Methods and Results : We show in the chick embryo that Sdf1 expression is tightly coordinated with the progression of cardiac NCs expressing Cxcr4 . Cxcr4 loss-of-function causes delayed migration and enhanced death of cardiac NCs, whereas Sdf1 misexpression results in their diversion from their normal pathway, indicating that SDF1 acts as a chemoattractant for cardiac NCs. These alterations of SDF1 signaling result in severe cardiovascular defects. Conclusions: These data identify Sdf1 and its receptor Cxcr4 as candidate genes responsible for cardiac congenital pathologies in human.

The type I interferon (IFN) system plays an important role in antiviral defense against influenza A viruses (FLUAV), which are natural chicken pathogens. Studies of mice identified the Mx1 protein as a key effector molecule of the IFN-induced antiviral state against FLUAV. Chicken Mx genes are highly polymorphic, and recent studies suggested that an Asn/Ser polymorphism at amino acid position 631 determines the antiviral activity of the chicken Mx protein. By employing chicken embryo fibroblasts with defined Mx-631 polymorphisms and retroviral vectors for the expression of Mx isoforms in chicken cells and embryonated eggs, we show here that neither the 631Asn nor the 631Ser variant of chicken Mx was able to confer antiviral protection against several lowly and highly pathogenic FLUAV strains. Using a short interfering RNA (siRNA)-mediated knockdown approach, we noted that the antiviral effect of type I IFN in chicken cells was not dependent on Mx, suggesting that some other IFN-induced factors must contribute to the inhibition of FLUAV in chicken cells. Finally, we found that both isoforms of chicken Mx protein appear to lack GTPase activity, which might explain the observed lack of antiviral activity.

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo . To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.

The respiratory tract is continuously challenged by aerosols that contain particulate material and non-pathogenic micro-organisms and may also carry pathogens of significant economic importance for the poultry industry, such as MDV, AIV or APEC. To control such pathogens, several layers of non-specific and specific defense mechanisms develop shortly before and after hatch. Cells of the myeloid linage are the first cells to be found throughout the respiratory tract, while the appearance of lymphocytes follows the release patterns of the thymus and bursa of Fabricius. In addition, several organized lymphoid structures exist in the paraocular, nasal and lung tissues. However, compared to disseminated immune cells, the formation of organized lymphoid tissue is significantly delayed, as demonstrated for the bronchus-associated lymphoid tissue (BALT). The morphological features of this system have been described in recent years, but their functional features are still poorly understood.