Publications by authors named "Bernd Kaspers"

69 Publications

Molecular cloning and characterisation of chicken IL-18 binding protein.

Dev Comp Immunol 2021 Jan 9;114:103850. Epub 2020 Sep 9.

The Pirbright Institute, Pirbright, Woking, UK; Aviagen Ltd, Newbridge, UK. Electronic address:

The human IL-1 receptor family is comprised of 11 membrane bound or soluble receptors and the IL-18 binding protein (IL-18BP). These receptors are dispersed across seven genomic loci, with the majority at a single locus. Direct orthologues were identified in the chicken at conserved genomic loci; however, the IL-18BP remained absent from the first four builds of the chicken genome sequence. Subsequent assemblies identified the gene at a locus syntenic with mammals; however, these predicted sequences differed between genome builds and contained multiple errors. A partial IL-18BP-like sequence in the NCBI EST database was used to clone the full-length cDNA. A splice variant, which lacks the exon that encodes part of the signal peptide, was also cloned. Human IL-18BP is differentially spliced to produce a number of variants, which are all secreted. By contrast, the spliced chicken isoform was predicted to be intracellular, and we identified similar variants with the same exon missing in a limited number of divergent vertebrate species. Mammalian and viral IL-18BPs inhibit IL-18 activity by directly binding to this cytokine. Full-length and intracellular chicken IL-18BPs were equally effective at inhibiting IL-18-mediated IFN-γ release from an avian B-cell line. Analysis of the predicted chIL-18BP protein sequence revealed two crucial residues, which account for 50% of the binding affinity between human IL-18 and IL-18BP, are conserved in the chicken and a fowlpox-encoded homologue, fpv214. This suggests specific fowlpox viruses used in humans as a vaccine vector have the potential to dampen anti-viral host immune responses.
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http://dx.doi.org/10.1016/j.dci.2020.103850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661785PMC
January 2021

In and Out of the Bursa-The Role of CXCR4 in Chicken B Cell Development.

Front Immunol 2020 14;11:1468. Epub 2020 Jul 14.

Department of Veterinary Sciences, Ludwig-Maximilians-Universität München, Munich, Germany.

In contrast to mammals, early B cell differentiation and diversification of the antibody repertoire in chickens do not take place in the bone marrow but in a specialized gut associated lymphoid tissue (GALT), the bursa of Fabricius. During embryonic development, B cell precursors migrate to the bursa anlage, where they proliferate and diversify their B cell receptor repertoire. Around hatch these diversified B cells start to emigrate from the bursa of Fabricius and populate peripheral lymphoid organs, but very little is known how the migratory processes are regulated. As CXCL12 (syn. SDF-1) and CXCR4 were shown to be essential for the control of B cell migration during the development of lymphoid tissues in mammals, we analyzed expression and function of this chemokine/chemokine-receptor pair in the chicken bursa. We found a strong variation of mRNA abundance of CXCL12 and CXCR4 in different stages of bursa development, with high abundance of CXCL12 mRNA in the bursa anlage at embryonic day 10 (ED10). hybridization demonstrated disseminated CXCL12 expression in the early bursa anlage, which condensed in the developing follicles and was mainly restricted to the follicle cortex post-hatch. Flow cytometric analysis detected CXCR4 protein already on early B cell stages, increasing during bursal development. Post-hatch, a subpopulation with the hallmarks of emigrating B cells became detectable, which had lower CXCR4 expression, suggesting that downregulation of CXCR4 is necessary to leave the CXCL12-high bursal environment. blockade of CXCR4 using AMD3100 at the time of B cell precursor immigration strongly inhibited follicle development, demonstrating that CXCL12 attracts pre-bursal B cells into the bursal anlage. Altogether, we show that CXCL12 and its receptor CXCR4 are important for both populating the bursa with B cells and emigration of mature B cells into the periphery post hatch, and that CXCR4 function in primary B cell organs is conserved between mammals and birds.
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http://dx.doi.org/10.3389/fimmu.2020.01468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381227PMC
July 2020

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6.

J Comp Physiol B 2020 01 21;190(1):75-85. Epub 2020 Jan 21.

Institute of Veterinary Physiology and Biochemistry, Justus-Liebig-University, Frankfurter Strasse 100, 35392, Gießen, Germany.

In 4-5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.
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http://dx.doi.org/10.1007/s00360-019-01249-1DOI Listing
January 2020

IFNα and IFNγ Impede Marek's Disease Progression.

Viruses 2019 11 28;11(12). Epub 2019 Nov 28.

Department of Veterinary Sciences, Ludwig-Maximilians-Universität München, 80539 Munich, Germany.

Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease, a malignant lymphoproliferative disease of domestic chickens. While MDV vaccines protect animals from clinical disease, they do not provide sterilizing immunity and allow field strains to circulate and evolve in vaccinated flocks. Therefore, there is a need for improved vaccines and for a better understanding of innate and adaptive immune responses against MDV infections. Interferons (IFNs) play important roles in the innate immune defenses against viruses and induce upregulation of a cellular antiviral state. In this report, we quantified the potent antiviral effect of IFNα and IFNγ against MDV infections in vitro. Moreover, we demonstrate that both cytokines can delay Marek's disease onset and progression in vivo. Additionally, blocking of endogenous IFNα using a specific monoclonal antibody, in turn, accelerated disease. In summary, our data reveal the effects of IFNα and IFNγ on MDV infection and improve our understanding of innate immune responses against this oncogenic virus.
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http://dx.doi.org/10.3390/v11121103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950089PMC
November 2019

After TNF-α, still playing hide-and-seek with chicken genes.

Poult Sci 2019 10;98(10):4373-4374

Department of Veterinary Science, Ludwig-Maximilians-Universität, 80539 Munich, Germany.

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http://dx.doi.org/10.3382/ps/pez307DOI Listing
October 2019

The Long Pentraxin PTX3 Is of Major Importance Among Acute Phase Proteins in Chickens.

Front Immunol 2019 1;10:124. Epub 2019 Feb 1.

Department for Veterinary Sciences, Ludwig-Maximilians-Universität Munich, Munich, Germany.

The expression level of acute phase proteins (APPs) mirrors the health status of an individual. In human medicine, C-reactive protein (CRP), and other members of the pentraxin family are of significant relevance for assessing disease severity and prognosis. In chickens, however, which represent the most common livestock species around the world, no such marker has yet gained general acceptance. The aim of this study was therefore, to characterize chicken pentraxin 3 (chPTX3) and to evaluate its applicability as a general marker for inflammatory conditions. The mammalian and chicken PTX3 proteins were predicted to be similar in sequence, domain organization and polymeric structure. Nevertheless, some characteristics like certain sequence sections, which have varied during the evolution of mammals, and species-specific glycosylation patterns, suggest distinct biological functions. ChPTX3 is constitutively expressed in various tissues but, interestingly, could not be found in splenic tissue samples without stimulation. However, upon treatment with lipopolysaccharide (LPS), PTX3 expression in chicken spleens increased to 95-fold within hours. A search for PTX3 reads in various publicly available RNA-seq data sets of chicken spleen and bursa of Fabricius also showed that PTX3 expression increases within days after experimental infection with viral and bacterial pathogens. An experimental infection with avian pathogenic and qPCR analysis of spleen samples further established a challenge dose-dependent significant up-regulation of chPTX3 in subclinically infected birds of up to over 150-fold as compared to untreated controls. Our results indicate the potential of chPTX3 as an APP marker to monitor inflammatory conditions in poultry flocks.
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http://dx.doi.org/10.3389/fimmu.2019.00124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367253PMC
December 2019

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus.

Proc Natl Acad Sci U S A 2018 11 18;115(45):11603-11607. Epub 2018 Oct 18.

Institut für Virologie, Freie Universität Berlin, 14163 Berlin, Germany;

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.
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http://dx.doi.org/10.1073/pnas.1813964115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233081PMC
November 2018

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge.

Dev Comp Immunol 2018 09 2;86:156-170. Epub 2018 May 2.

ISP, INRA, Université de Tours, 37380, Nouzilly, France. Electronic address:

Mammalian type I interferons (IFNα/β) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNβ act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1β and notably IFNB/IFNβ. Neutralization of IFNβ during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages.
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http://dx.doi.org/10.1016/j.dci.2018.04.025DOI Listing
September 2018

Characterization of Chicken Tumor Necrosis Factor-α, a Long Missed Cytokine in Birds.

Front Immunol 2018 17;9:605. Epub 2018 Apr 17.

Department of Veterinary Science, Ludwig-Maximilians-Universität, Munich, Germany.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-α (chTNF-α) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-α were identified in the genomes of 12 additional avian species including and , and the synteny of the closely adjacent loci with mammalian TNF-α orthologs was demonstrated in the crow () genome. In addition to chTNF-α, we obtained full sequences for homologs of TNF-α receptors 1 and 2 (TNFR1, TNFR2). chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-α expression in CD4 but not in CD8 cells. To gain insights into its biological activity, we generated recombinant chTNF-α in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NFκB-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-α/TNF-α receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems.
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http://dx.doi.org/10.3389/fimmu.2018.00605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913325PMC
June 2019

Infectious bursal disease virus inoculation infection modifies -host interaction in broilers.

Gut Pathog 2018 30;10:13. Epub 2018 Mar 30.

1Clinic for Poultry, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.

Background: is considered as a chicken commensal. The gut microbiota and the immune status of the host may affect its colonization. Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens, which allows secondary pathogens to invade or exacerbates their pathogenesis. To investigate the effect of IBDV-induced immunosuppression on the pathogenesis of , broiler chickens were inoculated with a very virulent (vv) strain of IBDV at 14 days post hatch followed by inoculation at 7 (Experiment A) or 9 (Experiment B) days post virus (IBDV) inoculation.

Results: vvIBDV-infection led to a depression in caecal lamina propria B lymphocytes and the anti--antibody response starting at 14 days post inoculation (pbi). The C. -colonization pattern was comparable between mono-inoculated groups of both experiments, but it varied for vvIBDV +  co-inoculated groups. In Experiment A significant higher numbers of colony forming units (CFU) of were detected in the caecum of co-inoculated birds compared to -mono-inoculated birds in the early phase after -inoculation. In Experiment B the clearance phase was affected in the co-inoculated group with significantly higher CFU at 21 days pbi compared to the mono-inoculated group ( < 0.05). No major differences were seen in numbers local lamina propria T lymphocyte populations between -inoculated groups with or without vvIBDV-infection. Interestingly, both pathogens affected the microbiota composition. The consequences of these microflora changes for the host have to be elucidated further.

Conclusion: Our data suggests that the timing between viral and bacterial infection might affect the outcome of colonization differently. Our results confirm previous studies that anti--antibodies may specifically be important for the clearance phase of the bacteria. Therefore, as vvIBDV is widely distributed in the field, it may have a significant impact on the colonization and shedding rate of in commercial poultry flocks. Subsequently, successful IBDV-control strategies may indirectly also benefit the gut-health of chickens.
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http://dx.doi.org/10.1186/s13099-018-0241-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877392PMC
March 2018

Gene expression in the chicken caecum is dependent on microbiota composition.

Vet Res 2017 12 4;48(1):85. Epub 2017 Dec 4.

Veterinary Research Institute, Hudcova 70, 621 00, Brno, Czech Republic.

Gut microbiota is of considerable importance for each host. Despite this, germ-free animals can be obtained and raised to sexual maturity and consequences of the presence or absence of gut microbiota on gene expression of the host remain uncharacterised. In this study, we performed an unbiased study of protein expression in the caecum of germ-free and colonised chickens. The major difference between these two groups was in the expression of immunoglobulins which were essentially absent in the germ-free chickens. Microbiota also caused a minor decrease in the expression of focal adhesion and extracellular matrix proteins and an increase in the expression of argininosuccinate synthase ASS1, redox potential sensing, fermentative metabolic processes and detoxification systems represented by sulfotransferases SULT1C3 or SULT1E1. Since we also analysed expression in the caecum of E. coli Nissle and E. faecium DSM7134 mono-associated chickens, we concluded that at least immunoglobulin expression and expression of cystathionine synthase (CBS) was dependent on microbiota composition with E. coli Nissle stimulating more immunoglobulin and PIGR expression and E. faecium DSM7134 stimulating more CBS expression. Gut microbiota and its composition therefore affected protein expression in the chicken caecum though except for immunoglobulin production, the remaining differences were unexpectedly low.
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http://dx.doi.org/10.1186/s13567-017-0493-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716255PMC
December 2017

Propagation and titration of infectious bursal disease virus, including non-cell-culture-adapted strains, using ex vivo-stimulated chicken bursal cells.

Avian Pathol 2018 Apr 8;47(2):179-188. Epub 2017 Nov 8.

a OIE Reference Laboratory for Gumboro Disease, Avian and Rabbit Virology Immunology and Parasitology Unit (VIPAC), French Agency for Food, Environmental and Occupational Heath Safety (ANSES) , Ploufragan , France.

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.
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http://dx.doi.org/10.1080/03079457.2017.1393044DOI Listing
April 2018

Influence of the Gut Microbiota Composition on Campylobacter jejuni Colonization in Chickens.

Infect Immun 2017 11 18;85(11). Epub 2017 Oct 18.

University of Veterinary Medicine Hannover, Clinic for Poultry, Hannover, Germany

The -host interaction may be affected by the host's gut microbiota through competitive exclusion, metabolites, or modification of the immune response. To understand this interaction, colonization and local immune responses were compared in chickens with different gut microbiota compositions. Birds were treated with an antibiotic cocktail (AT) (experiments 1 and 2) or raised under germfree (GF) conditions (experiment 3). At 18 days posthatch (dph), they were orally inoculated either with 10 CFU of or with diluent. Cecal as well as systemic colonization, T- and B-cell numbers in the gut, and gut-associated tissue were compared between the different groups. Significantly higher numbers of CFU of were detected in the cecal contents of AT and GF birds, with higher colonization rates in spleen, liver, and ileum, than in birds with a conventional gut microbiota ( < 0.05). Significant upregulation of T and B lymphocyte numbers was detected in cecum, cecal tonsils, and bursa of Fabricius of AT or GF birds after inoculation compared to the respective controls ( < 0.05). This difference was less clear in birds with a conventional gut microbiota. Histopathological gut lesions were observed only in -inoculated AT and GF birds but not in microbiota-colonized -inoculated hatchmates. These results demonstrate that the gut microbiota may contribute to the control of colonization and prevent lesion development. Further studies are needed to identify key players of the gut microbiota and the mechanisms behind their protective role.
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http://dx.doi.org/10.1128/IAI.00380-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649013PMC
November 2017

Tissue and time specific expression pattern of interferon regulated genes in the chicken.

BMC Genomics 2017 03 28;18(1):264. Epub 2017 Mar 28.

Department for Veterinary Science, University of Munich, Munich, Germany.

Background: Type I interferons are major players against viral infections and mediate their function by the induction of Interferon regulated genes (IRGs). Recently, it became obvious that these cytokines have a multitude of additional functions. Due to the unique features of the chickens' immune system, available data from mouse models are not easily transferable; hence we performed an extensive analysis of chicken IRGs.

Results: A broad database search for homologues to described mammalian IRGs (common IRGs, cIRGs) was combined with a transcriptome analysis of spleen and lung at different time points after application of IFNα. To apply physiological amounts of IFN, half-life of IFN in the chicken was determined. Interestingly, the calculated 36 min are considerably shorter than the ones obtained for human and mouse. Microarray analysis revealed many additional IRGs (newly identified IRGs; nIRGs) and network analysis for selected IRGs showed a broad interaction of nIRGs among each other and with cIRGs. We found that IRGs exhibit a highly tissue and time specific expression pattern as expression quality and quantity differed strongly between spleen and lung and over time. While in the spleen for many affected genes changes in RNA abundance peaked already after 3 h, an increasing or plateau-like regulation after 3, 6 and 9 h was observed in the lung.

Conclusions: The induction or suppression of IRGs in chickens is both tissue and time specific and beside known antiviral mechanisms type I IFN induces many additional cellular functions. We confirmed many known IRGs and established a multitude of so far undescribed ones, thus providing a large database for future research on antiviral mechanisms and additional IFN functions in non-mammalian species.
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http://dx.doi.org/10.1186/s12864-017-3641-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371264PMC
March 2017

Transient and Prolonged Response of Chicken Cecum Mucosa to Colonization with Different Gut Microbiota.

PLoS One 2016;11(9):e0163932. Epub 2016 Sep 29.

Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic.

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163932PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042506PMC
September 2016

Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens.

Eur J Immunol 2016 09 2;46(9):2137-48. Epub 2016 Aug 2.

Crystal Bioscience Inc, Emeryville, CA, USA.

Since the discovery of antibody-producing B cells in chickens six decades ago, chickens have been a model for B-cell development in gut-associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL(-/-) ) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy-chain-only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4-week-old IgL(-/-) chickens, and antigen-specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy-chain-only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B-cell development in a gut-associated lymphoid tissue species.
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http://dx.doi.org/10.1002/eji.201546171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113765PMC
September 2016

Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales.

mSphere 2016 Jan-Feb;1(1). Epub 2015 Dec 2.

Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover, Germany.

The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins.
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http://dx.doi.org/10.1128/mSphere.00028-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863622PMC
June 2016

Neurotropism of Saffold virus in a mouse model.

J Gen Virol 2016 06 9;97(6):1350-1355. Epub 2016 Mar 9.

de Duve Institute, Université Catholique de Louvain, VIRO B1.74.07, 74 Avenue Hippocrate, Brussels B-1200, Belgium.

Saffold virus (SAFV) is a highly seroprevalent human Cardiovirus discovered recently. No clear association between SAFV infection and human disease has been established. Rare infection cases, however, correlated with neurological symptoms. To gain insight into the pathogenesis potential of the virus, we performed experimental mouse infection with SAFV strains of genotypes 2 and 3 (SAFV-2 and SAFV-3). After intraperitoneal infection, both strains exhibited a typical Cardiovirus tropism. Viral load was most prominent in the pancreas. Heart, spleen, brain and spinal cord were also infected. In IFN-receptor 1 deficient (IFNAR-KO) mice, SAFV-3 caused a severe encephalitis. The virus was detected by immunohistochemistry in many parts of the brain and spinal cord, both in neurons and astrocytes, but astrocyte infection was more extensive. In vitro, SAFV-3 also infected astrocytes better than neurons in mixed primary cultures. Astrocytes were, however, very efficiently protected by IFN-α/β treatment.
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http://dx.doi.org/10.1099/jgv.0.000452DOI Listing
June 2016

Preliminary results on intersexual differences in gene expression of chemokine K203 in mononuclear cells of chicken.

Acta Vet Hung 2016 03;64(1):56-64

Department of Veterinary Sciences, Institute for Animal Physiology, Ludwig-Maximilians-University , Munich , Germany.

Sex steroid levels increase during sexual maturation and cause alterations in many physiological and morphological traits. Some of these changes may be connected with age-dependent and intersexual differences in the immune system. This topic is still insufficiently understood, especially in avian species, partially due to methodological limitations. In this study we measured the gene expression of proinflammatory cytokines (IL-1β, IL-6, IL-18) and chemokines [K60 (IL-8-like chicken chemokine - CXCLi1), CAF (IL-8-like chicken chemokine - CXCLi2), and K203] in mononuclear cells isolated from blood and spleen after in vitro stimulation with lipopolysaccharide (LPS). Samples were collected from chickens at two ages (from pullets before sexual maturity and from sexually mature egglaying hens). After LPS stimulation, a substantial increase was recorded in the gene expression of IL-6 and K203. All other measured genes were expressed at low levels in mononuclear cells irrespective of cell sources. We found a trend toward intersexual differences in K203 expression, but the expression of other cytokines and chemokines did not differ between the two sexes. The effect of stimulation was more pronounced in monocytes than in spleen macrophages, mainly in IL-6, IL-1β and K203 gene expression. Our findings represent a basis for further studies on the effects exerted by sexual hormones on the immune phenotype of birds.
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http://dx.doi.org/10.1556/004.2016.006DOI Listing
March 2016

Estimation of dynamic metabolic activity in micro-tissue cultures from sensor recordings with an FEM model.

Med Biol Eng Comput 2016 May 22;54(5):763-72. Epub 2015 Aug 22.

Heinz Nixdorf-Lehrstuhl für Medizinische Elektronik, Technische Universität München, Theresienstr. 90/N3, 80333, Munich, Germany.

We estimated the dynamic cell metabolic activity and the distribution of the pH value and oxygen concentration in tissue samples cultured in vitro by using real-time sensor records and a numerical simulation of the underlying reaction-diffusion processes. As an experimental tissue model, we used chicken spleen slices. A finite element method model representing the biochemical processes and including the relevant sensor data was set up. By fitting the calculated results to the measured data, we derived the spatiotemporal values of the pH value, the oxygen concentration and the absolute metabolic activity (extracellular acidification and oxygen uptake rate) of the samples. Notably, the location of the samples in relation to the sensors has a great influence on the detectable metabolic rates. The long-term vitality of the tissue samples strongly depends on their size. We further discuss the benefits and limitations of the model.
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http://dx.doi.org/10.1007/s11517-015-1367-7DOI Listing
May 2016

In vitro model for lytic replication, latency, and transformation of an oncogenic alphaherpesvirus.

Proc Natl Acad Sci U S A 2015 Jun 26;112(23):7279-84. Epub 2015 May 26.

Veterinärwissenschaftliches Department, Institut für Tierphysiologie, Ludwig-Maximilians-Universität München, 80539 Munich, Germany;

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.
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http://dx.doi.org/10.1073/pnas.1424420112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466724PMC
June 2015

Impact of antigenic diversity on laboratory diagnosis of Avian bornavirus infections in birds.

J Vet Diagn Invest 2014 Nov 18;26(6):769-77. Epub 2014 Aug 18.

Institute for Virology, University Medical Center Freiburg, Freiburg, Germany (Zimmermann, Staeheli, Rubbenstroth)Clinic for Birds, Reptiles, Amphibians and Ornamental Fish, Centre for Clinical Veterinary Medicine, University Ludwig Maximilian Munich, Oberschleißheim, Germany (Rinder)Department of Veterinary Sciences, University Ludwig Maximilian Munich, Munich, Germany (Kaspers)

Avian bornaviruses (ABVs) are a group of genetically diverse viruses within the Bornaviridae family that can infect numerous avian species and represent the causative agents of proventricular dilatation disease, an often fatal disease that is widely distributed in captive populations of parrots and related species. The current study was designed to assess the antigenic variability of the family Bornaviridae and to determine its impact on ABV diagnosis by employing fluorescent antibody assays. It was shown that polyclonal rabbit sera directed against recombinant bornavirus nucleoprotein, X protein, phosphoprotein, and matrix protein provided sufficient cross-reactivity for the detection of viral antigen from a broad range of bornavirus genotypes grown in cell culture. In contrast, a rabbit anti-glycoprotein serum and 2 monoclonal antibodies directed against nucleoprotein and phosphoprotein proteins reacted more specifically. Antibodies were readily detected in sera from avian patients infected with known ABV genotypes if cells persistently infected with a variety of different bornavirus genotypes were used for analysis. For all sera, calculated antibody titers were highest when the homologous or a closely related target virus was used for the assay. Cross-reactivity with more distantly related genotypes of other phylogenetic groups was usually reduced, resulting in titer reduction of up to 3 log units. The presented results contribute to a better understanding of the antigenic diversity of family Bornaviridae and further emphasize the importance of choosing appropriate diagnostic tools for sensitive detection of ABV infections.
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http://dx.doi.org/10.1177/1040638714547258DOI Listing
November 2014

No contact transmission of avian bornavirus in experimentally infected cockatiels (Nymphicus hollandicus) and domestic canaries (Serinus canaria forma domestica).

Vet Microbiol 2014 Aug 13;172(1-2):146-56. Epub 2014 May 13.

Institute for Virology, University Medical Center Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany.

Avian bornaviruses (ABV) are the causative agents of proventricular dilatation disease (PDD), a widely distributed disease of parrots. Distinct ABV lineages were also found in various non-psittacine avian species, such as canaries, but the pathogenic role of ABV in these species is less clear. Despite the wide distribution of ABV in captive parrots and canaries, its mode of transmission is poorly understood: both horizontal transmission via the urofaecal-oral route and vertical transmission are discussed to play a role. In this study we investigated pathology and horizontal transmission of ABV in domestic canaries (Serinus canaria forma domestica) and cockatiels (Nymphicus hollandicus), two natural host species commonly used for experimental ABV infections. ABV inoculation resulted in persistent infection of all inoculated animals from both species. ABV-infected cockatiels exhibited PDD-like symptoms, such as neurologic signs or shedding of undigested seeds. In contrast, infected domestic canaries did not develop clinical disease. Interestingly, we did not detect viral RNA in cloacal swabs and organ samples or ABV-specific antibodies in serum samples of contact-exposed sentinel birds from either species at any time during a four months observation period. Our results strongly indicate that horizontal transmission of ABV by direct contact is inefficient in immunocompetent fully fledged domestic canaries and cockatiels.
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http://dx.doi.org/10.1016/j.vetmic.2014.05.011DOI Listing
August 2014

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

PLoS Genet 2014 Jun 5;10(6):e1004417. Epub 2014 Jun 5.

Basel Institute for Immunology, Basel, Switzerland; Department of Pathology, University of Cambridge, Cambridge, United Kingdom; Pirbright Institute (formerly Institute for Animal Health), Compton, United Kingdom; Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.
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http://dx.doi.org/10.1371/journal.pgen.1004417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046983PMC
June 2014

Existence of conventional dendritic cells in Gallus gallus revealed by comparative gene expression profiling.

J Immunol 2014 May 16;192(10):4510-7. Epub 2014 Apr 16.

Centre d'Immunologie de Marseille-Luminy, Aix-Marseille University, UM2, 13288 Marseille Cedex 9, France;

The existence of conventional dendritic cells (cDCs) has not yet been demonstrated outside mammals. In this article, we identified bona fide cDCs in chicken spleen. Comparative profiling of global and of immune response gene expression, morphology, and T cell activation properties show that cDCs and macrophages (MPs) exist as distinct mononuclear phagocytes in the chicken, resembling their human and mouse cell counterparts. With computational analysis, core gene expression signatures for cDCs, MPs, and T and B cells across the chicken, human, and mouse were established, which will facilitate the identification of these subsets in other vertebrates. Overall, this study, by extending the newly uncovered cDC and MP paradigm to the chicken, suggests that these two phagocyte lineages were already in place in the common ancestor of reptiles (including birds) and mammals in evolution. It opens avenues for the design of new vaccines and nutraceuticals that are mandatory for the sustained supply of poultry products in the expanding human population.
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http://dx.doi.org/10.4049/jimmunol.1303405DOI Listing
May 2014

Antiviral activity of lambda interferon in chickens.

J Virol 2014 Mar 26;88(5):2835-43. Epub 2013 Dec 26.

Institute for Virology, University of Freiburg, Freiburg, Germany.

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.
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http://dx.doi.org/10.1128/JVI.02764-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958073PMC
March 2014

Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells.

Proc Natl Acad Sci U S A 2013 Dec 26;110(50):20170-5. Epub 2013 Nov 26.

Research Division, Crystal Bioscience Inc., Emeryville, CA 94608.

Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.
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http://dx.doi.org/10.1073/pnas.1317106110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864345PMC
December 2013

Avian bornaviruses are widely distributed in canary birds (Serinus canaria f. domestica).

Vet Microbiol 2013 Aug 9;165(3-4):287-95. Epub 2013 Apr 9.

Department of Virology, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany.

Avian bornavirus (ABV) was identified in 2008 as the causative agent of proventricular dilatation disease (PDD) in psittacine birds. In addition, ABV variants were detected in wild waterfowl and in a canary bird. PDD-like diseases were also reported in various other avian species, but it remains unknown whether ABV is involved. In this study we detected ABV in 12 of 30 tested canary bird flocks (40%), indicating a wide distribution of ABV in captive canary birds in Germany. Sequence analysis identified several distinct ABV genotypes which differ markedly from the genotypes present in psittacine birds. Some canaries naturally infected with ABV exhibited gastrointestinal and neurological symptoms which resembled PDD in psittacines, while others did not show signs of disease. Canaries experimentally inoculated with ABV developed infections of the brain and various other organs. The experimentally infected canaries transmitted the virus to sentinel birds kept in the same aviary, but did not show any clinical signs during a five month observation period. Embryonated eggs originating from ABV-infected hens contained ABV-specific RNA, but virus could not be re-isolated from embryonic tissue. These results indicate that ABV is widely distributed in canary birds and due to its association to clinical signs should be considered as a potential pathogen of this species.
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http://dx.doi.org/10.1016/j.vetmic.2013.03.024DOI Listing
August 2013

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi.

Vet Res 2013 Feb 11;44:10. Epub 2013 Feb 11.

Institute of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Str, 15, Berlin 14163, Germany.

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.
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http://dx.doi.org/10.1186/1297-9716-44-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598538PMC
February 2013

2D DIGE analysis of the bursa of Fabricius reveals characteristic proteome profiles for different stages of chicken B-cell development.

Proteomics 2013 Jan 5;13(1):119-33. Epub 2012 Dec 5.

Department for Veterinary Science, Institute for Animal Physiology, Ludwig-Maximilians-Universität München, Munich, Germany.

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.
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http://dx.doi.org/10.1002/pmic.201200177DOI Listing
January 2013