Uncovering the core principles of the gut-lung axis to enhance innate immunity in the chicken.

Research in mammals has evidenced that proper colonization of the gut by a complex commensal microbial community, the gut microbiota (GM), is critical for animal health and wellbeing. It greatly contributes to the control of infectious processes through competition in the microbial environment while supporting proper immune system development and modulating defence mechanisms at distant organ sites such as the lung: a concept named 'gut-lung axis'. While recent studies point to a role of the GM in boosting immunity and pathogen resilience also in poultry, the mechanisms underlying this role are largely unknown. In spite of this knowledge gap, GM modulation approaches are today considered as one of the most promising strategies to improve animal health and welfare in commercial poultry production, while coping with the societal demand for responsible, sustainable and profitable farming systems. The majority of pathogens causing economically important infectious diseases in poultry are targeting the respiratory and/or gastrointestinal tract. Therefore, a better understanding of the role of the GM in the development and function of the mucosal immune system is crucial for implementing measures to promote animal robustness in commercial poultry production. The importance of early gut colonization in the chicken has been overlooked or neglected in industrial poultry production systems, where chicks are hampered from acquiring a complex GM from the hen. Here we discuss the concept of strengthening mucosal immunity in the chicken through GM modulation approaches favouring immune system development and functioning along the gut-lung axis, which could be put into practice through improved farming systems, early-life GM transfer, feeding strategies and pre-/probiotics. We also provide original data from experiments with germ-free and conventional chickens demonstrating that the gut-lung axis appears to be functional in chickens. These key principles of mucosal immunity are likely to be relevant for a variety of avian diseases and are thus of far-reaching importance for the poultry sector worldwide.

Assessment of trade-offs between feed efficiency, growth-related traits, and immune activity in experimental lines of layer chickens.

BACKGROUND: In all organisms, life-history traits are constrained by trade-offs, which may represent physiological limitations or be related to energy resource management. To detect trade-offs within a population, one promising approach is the use of artificial selection, because intensive selection on one trait can induce unplanned changes in others. In chickens, the breeding industry has achieved remarkable genetic progress in production and feed efficiency over the last 60 years. However, this may have been accomplished at the expense of other important biological functions, such as immunity. In the present study, we used three experimental lines of layer chicken-two that have been divergently selected for feed efficiency and one that has been selected for increased antibody response to inactivated Newcastle disease virus (ND3)-to explore the impact of improved feed efficiency on animals' immunocompetence and, vice versa, the impact of improved antibody response on animals' growth and feed efficiency. RESULTS: There were detectable differences between the low (R+) and high (R-) feed-efficiency lines with respect to vaccine-specific antibody responses and counts of monocytes, heterophils, and/or T cell population. The ND3 line presented reduced body weight and feed intake compared to the control line. ND3 chickens also demonstrated an improved antibody response against a set of commercial viral vaccines, but lower blood leucocyte counts. CONCLUSIONS: This study demonstrates the value of using experimental chicken lines that are divergently selected for RFI or for a high antibody production, to investigate the modulation of immune parameters in relation to growth and feed efficiency. Our results provide further evidence that long-term selection for the improvement of one trait may have consequences on other important biological functions. Hence, strategies to ensure optimal trade-offs among competing functions will ultimately be required in multi-trait selection programs in livestock.

Multi-species annotation of transcriptome and chromatin structure in domesticated animals.

BACKGROUND: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). RESULTS: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. CONCLUSIONS: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

Avian pathogenic Escherichia coli infection of a chicken lung epithelial cell line.

Virulent strains of Escherichia coli (Avian Pathogenic E. Coli: APEC) can cause initial infection of the respiratory tract in chickens potentially leading to systemic infection called colibacillosis, which remains a major cause of economic losses in the poultry industry. The role of epithelial lung cells as first targets of APEC and in initiating the innate immune response is unclear and was investigated in this study. APEC was able to adhere and subsequently invade cells from the chicken lung epithelial CLEC213 cell line exhibiting pneumocyte type II-like characteristics. Invasion was confirmed using confocal microscopy after infection with GFP-labelled APEC. Moreover, the infection resulted in a significant increase in IL-8 gene expression, a chemo-attractant of macrophages and heterophils. Gene expression of interferon α and ß were not significantly upregulated and chicken Surfactant Protein A, also did not show a significant upregulation on either gene or protein level. The immune response of CLEC213 cells towards APEC was shown to be similar to stimulation with E. coli LPS. These results establish CLEC213 cells as a novel model system for studying bacterial infection of the lung epithelium and show that these cells may play a role in the initial innate response towards bacterial pathogens.

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens.

It is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC. RESEARCH HIGHLIGHTS Use of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host-pathogen interactions. pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons. pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

The culture of primary duck endothelial cells for the study of avian influenza.

BACKGROUND: Endothelial cells play a major role in highly pathogenic avian influenza (HPAI) virus pathogenesis in gallinaceous poultry species (e.g. chicken, turkey and quail). Upon infection of gallinaceous poultry with HPAI viruses, endothelial cells throughout the body become rapidly infected, leading to systemic dissemination of the virus, disseminated intravascular coagulation, oedema and haemorrhaging. In contrast, the pathogenesis of HPAI viruses in most wild bird species (e.g. duck, goose and gull species) is not associated with endothelial tropism. Indeed, viral antigen is not found in the endothelial cells of most wild bird species following infection with HPAI viruses. This differential endothelial cell tropism in avian species is poorly understood, mainly due to the absence of appropriate cell culture systems. RESULTS: Here, we describe the isolation and purification of primary duck endothelial cells from the aorta or bone marrow of Pekin duck embryos. Cells were differentiated in the presence of vascular endothelial growth factor and, if needed, enriched via fluorescent-activated cell sorting based on the uptake of acetylated low-density lipoprotein. The expression of von Willebrand factor, a key marker of endothelial cells, was confirmed by polymerase chain reaction. Monocultures of duck endothelial cells, either derived from the aorta or the bone marrow, were susceptible to infection with an H5N1 HPAI virus but to a much lesser extent than chicken endothelial cells. CONCLUSIONS: The methods described herein to isolate and purify duck endothelial cells from the aorta or bone marrow could also be applied to obtain microvascular endothelial cells from other tissues and organs, such as the lung or the intestine, and represent a valuable tool to study the pathogenesis of avian viruses.

Major contribution of the RNA-binding domain of NS1 in the pathogenicity and replication potential of an avian H7N1 influenza virus in chickens.

BACKGROUND: Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. However it is still unclear to what extent each of its two structured domains (i.e. RNA-binding domain, RBD, and effector domain, ED) contribute to its various activities. METHODS: To evaluate the respective contributions of the two domains, we genetically engineered two variants of an H7N1 low pathogenicity avian influenza virus harbouring amino-acid substitutions that impair the functionality of either domain. The RBD- and ED-mutant viruses were compared to their wt- counterpart in vivo and in vitro, notably in chicken infection and avian cell culture models. RESULTS: The double substitution R38A-K41A in the RBD dramatically reduced the pathogenicity and replication potential of the virus, whereas the substitution A149V that was considered to abrogate the IFN-antagonistic activity of the effector domain entailed much less effects. While all three viruses initiated the viral life cycle in avian cells, replication of the R38A-K41A virus was severely impaired. This defect was associated with a delayed synthesis of nucleoprotein NP and a reduced accumulation of NS1, which was found to reach a concentration of about 30 micromol.L- 1 in wt-infected cells at 8 h post-infection. When overexpressed in avian lung epithelial cells, both the wt-NS1 and 3841AA-NS1, but not the A149V-NS1, reduced the poly(I:C)-induced activation of the IFN-sensitive chicken Mx promoter. Unexpectedly, the R38A-K41A substitution in the recombinant RBD did not alter its in vitro affinity for a model dsRNA. When overexpressed in avian cells, both the wt- and A149V-NS1s, as well as the individually expressed wt-RBD to a lesser extent, enhanced the activity of the reconstituted viral RNA-polymerase in a minireplicon assay. CONCLUSIONS: Collectively, our data emphasized the critical importance and essential role of the RNA-binding domain in essential steps of the virus replication cycle, notably expression and translation of viral mRNAs.

Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development.

BACKGROUND: Eimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide. The life-cycle of E. tenella is monoxenous with the chicken as the exclusive host; infection occurs in caecal epithelial cells. However, in vitro, the complete life-cycle of the parasite has only been propagated successfully in primary chicken kidney cells, which comprise undefined mixed cell populations; no cell line model has been able to consistently support the development of the sexual stages of the parasite. We therefore sought to develop a new model to study E. tenella gametogony in vitro using a recently characterised chicken cell line (CLEC-213) exhibiting an epithelial cell phenotype. METHODS: CLEC-213 were infected with sporozoites from a precocious strain or with second generation merozoites (merozoites II) from wild type strains. Sexual stages of the parasite were determined both at the gene and protein levels. RESULTS: To our knowledge, we show for the first time in CLEC-213, that sporozoites from a precocious strain of E. tenella were able to develop to gametes, as verified by measuring gene expression and by using antibodies to a microgamete-specific protein (EtFOA1: flagellar outer arm protein 1) and a macrogamete-specific protein (EtGAM-56), but oocysts were not observed. However, both gametes and oocysts were observed when cells were infected with merozoites II from wild type strains, demonstrating that completion of the final steps of the parasite cycle is possible in CLEC-213 cells. CONCLUSION: The epithelial cell line CLEC-213 constitutes a useful avian tool for studying Eimeria epithelial cell interactions and the effect of drugs on E. tenella invasion, merogony and gametogony.

Productive replication of avian influenza viruses in chicken endothelial cells is determined by hemagglutinin cleavability and is related to innate immune escape.

Endotheliotropism is a hallmark of gallinaceous poultry infections with highly pathogenic avian influenza (HPAI) viruses and a feature that distinguishes HPAI from low pathogenic avian influenza (LPAI) viruses. Here, we used chicken aortic endothelial cells (chAEC) as a novel in vitro infection model to assess the susceptibility, permissiveness, and host response of chicken endothelial cells (EC) to infections with avian influenza (AI) viruses. Our data show that productive replication of AI viruses in chAEC is critically determined by hemagglutinin cleavability, and is thus an exclusive trait of HPAI viruses. However, we provide evidence for a link between limited (i.e. trypsin-dependent) replication of certain LPAI viruses, and the viruses' ability to dampen the antiviral innate immune response in infected chAEC. Strikingly, this cell response pattern was also detected in HPAI virus-infected chAEC, suggesting that viral innate immune escape might be a prerequisite for robust AI virus replication in chicken EC.

Transcriptomic profiling of a chicken lung epithelial cell line (CLEC213) reveals a mitochondrial respiratory chain activity boost during influenza virus infection.

Avian Influenza virus (AIV) is a major concern for the global poultry industry. Since 2012, several countries have reported AIV outbreaks among domestic poultry. These outbreaks had tremendous impact on poultry production and socio-economic repercussion on farmers. In addition, the constant emergence of highly pathogenic AIV also poses a significant risk to human health. In this study, we used a chicken lung epithelial cell line (CLEC213) to gain a better understanding of the molecular consequences of low pathogenic AIV infection in their natural host. Using a transcriptome profiling approach based on microarrays, we identified a cluster of mitochondrial genes highly induced during the infection. Interestingly, most of the regulated genes are encoded by the mitochondrial genome and are involved in the oxidative phosphorylation metabolic pathway. The biological consequences of this transcriptomic induction result in a 2.5- to 4-fold increase of the ATP concentration within the infected cells. PB1-F2, a viral protein that targets the mitochondria was not found associated to the boost of activity of the respiratory chain. We next explored the possibility that ATP may act as a host-derived danger signal (through production of extracellular ATP) or as a boost to increase AIV replication. We observed that, despite the activation of the P2X7 purinergic receptor pathway, a 1mM ATP addition in the cell culture medium had no effect on the virus replication in our epithelial cell model. Finally, we found that oligomycin, a drug that inhibits the oxidative phosphorylation process, drastically reduced the AIV replication in CLEC213 cells, without apparent cellular toxicity. Collectively, our results suggest that AIV is able to boost the metabolic capacities of its avian host in order to provide the important energy needs required to produce progeny virus.

Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens.

Lipid mediators are known to play important roles in the onset and resolution phases of the inflammatory response in mammals. The phospholipid platelet-activating factor (PAF) is a pro-inflammatory lipid mediator which participates in vascular- and innate immunity-associated processes by increasing vascular permeability, by facilitating leukocyte adhesion to the endothelium, and by contributing to phagocyte activation. PAF exerts its function upon binding to its specific receptor, PAF receptor (PAFR), which is abundantly expressed in leukocytes and endothelial cells (ECs). In chickens, lipid mediators and their functions are still poorly characterized, and the role of PAF as an inflammatory mediator has not yet been investigated. In the present study we demonstrate that primary chicken macrophages express PAFR and lysophosphatidylcholine acyltransferase 2 (LPCAT2), the latter being essential to PAF biosynthesis during inflammation. Also, exogenous PAF treatment induces intracellular calcium increase, reactive oxygen species release, and increased phagocytosis by primary chicken macrophages in a PAFR-dependent manner. We also show that PAF contributes to the Escherichia coli lipopolysaccharide (LPS)-induced pro-inflammatory response and boosts the macrophage response to E. coli LPS via phosphatidylinositol 3-kinase/Akt- and calmodulin kinase II-mediated intracellular signaling pathways. Exogenous PAF treatment also increases avian pathogenic E. coli intracellular killing by chicken macrophages, and PAFR and LPCAT2 are upregulated in chicken lungs and liver during experimental pulmonary colibacillosis. Finally, exogenous PAF treatment increases cell permeability and upregulates the expression of genes coding for proteins involved in leukocyte adhesion to the endothelium in primary chicken endothelial cells (chAEC). In addition to these vascular phenomena, PAF boosts the chAEC inflammatory response to bacteria-associated molecular patterns in a PAFR-dependent manner. In conclusion, we identified PAF as an inflammation amplifier in chicken macrophages and ECs, which suggests that PAF could play important roles in the endothelium-innate immunity interface in birds during major bacterial infectious diseases such as colibacillosis.

Defining Mononuclear Phagocyte Subset Homology Across Several Distant Warm-Blooded Vertebrates Through Comparative Transcriptomics.

Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey.

Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella Enteritidis.

Salmonella Gallinarum and Salmonella Enteritidis are genetically closely related however associated with different pathologies. Several studies have suggested that S. Gallinarum is less invasive in vitro than S. Enteritidis. In this study we confirm that the S. Gallinarum strains tested were much less invasive than the S. Enteritidis strains tested in cells of avian or human origin. In addition, the S. Gallinarum T3SS-1-dependent ability to invade host cells was delayed by two to three hours compared to S. Enteritidis, indicating that T3SS-1-dependent entry is less efficient in S. Gallinarum than S. Enteritidis. This was neither due to a decreased transcription of T3SS-1 related genes when bacteria come into contact with cells, as transcription of hilA, invF and sipA was similar to that observed for S. Enteritidis, nor to a lack of functionality of the S. Gallinarum T3SS-1 apparatus as this apparatus was able to secrete and translocate effector proteins into host cells. In contrast, genome comparison of four S. Gallinarum and two S. Enteritidis strains revealed that all S. Gallinarum genomes displayed the same point mutations in each of the main T3SS-1 effector genes sipA, sopE, sopE2, sopD and sopA.

Protection patterns in duck and chicken after homo- or hetero-subtypic reinfections with H5 and H7 low pathogenicity avian influenza viruses: a comparative study.

Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza.

Shortening the unstructured, interdomain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens.

Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in 2000 that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbour a deletion of aa 80-84 in the unstructured region of the non-structural (NS) protein NS1 linking its RNA-binding domain to its effector domain. NS segments harbouring this mutation have since been found in non-H5N1 viruses and we asked whether this 5 aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the WT and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrated that the increased replicative potential conferred by this small deletion was a general feature not restricted to NS1 from H5N1 viruses and suggested that viruses acquiring this mutation may be selected positively in the future.

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

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.

Characterization of the sialic acid binding activity of influenza A viruses using soluble variants of the H7 and H9 hemagglutinins.

Binding of influenza viruses to target cells is mediated by the viral surface protein hemagglutinin. To determine the presence of binding sites for influenza A viruses on cells and tissues, soluble hemagglutinins of the H7 and H9 subtype were generated by connecting the hemagglutinin ectodomain to the Fc portion of human immunoglobulin G (H7Fc and H9Fc). Both chimeric proteins bound to different cells and tissues in a sialic acid-dependent manner. Pronounced differences were observed between H7Fc and H9Fc, in the binding both to different mammalian and avian cultured cells and to cryosections of the respiratory epithelium of different virus host species (turkey, chicken and pig). Binding of the soluble hemagglutinins was similar to the binding of virus particles, but showed differences in the binding pattern when compared to two sialic acid-specific plant lectins. These findings were substantiated by a comparative glycan array analysis revealing a very narrow recognition of sialoglycoconjugates by the plant lectins that does not reflect the glycan structures preferentially recognized by H7Fc and H9Fc. Thus, soluble hemagglutinins may serve as sialic acid-specific lectins and are a more reliable indicator of the presence of binding sites for influenza virus HA than the commonly used plant lectins.

Presence of dendritic cells in chicken spleen cell preparations and their functional interaction with the parasite Toxoplasma gondii.

Toxoplasmosis is a worldwide epizootic disease of mammals. Chickens, albeit being less susceptible, can be contaminated in free-range flocks and may have an important role in parasite transmission. Plastic adherence selection of chicken spleen cells enriched 8F2+ (putative chicken CD11c) MHC II+ cells of the myeloid type; however, we did not succeed to separate dendritic cells from macrophages using their feature to become loosely adherent after culture as in mammals. Still we clearly identified dendritic-like cells being morphologically distinguishable from macrophages in the KUL01 (macrophage marker) negative fraction, exhibiting responsiveness to LPS and parasite extracts by developing characteristic cellular protrusions as well as a minor phagocytic incorporation of dead parasites. Live T. gondii tachyzoites were able to invade the two different types of myeloid adherent cells, to replicate, and to induce an overall decrease in the expression of MHC II and co-stimulatory molecules, CD80 and CD40. Our data indicate that dendritic cells in addition to macrophages may have a role in hiding viable replicating T. gondii tachyzoites from the immune system and in shuttling them to different organs in the chicken as previously described for different Apicomplexa infecting mammals.

Deletion of the C-terminal ESEV domain of NS1 does not affect the replication of a low-pathogenic avian influenza virus H7N1 in ducks and chickens.

Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R(225)VESEV(230) at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230aa NS1 (H7N1(230)) and a mutant virus with a truncated NS1 (H7N1(224)). The 6aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1(230) and H7N1(224) viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1(230) virus induced a more severe interstitial pneumonia than did the H7N1(224) virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.

A novel chicken lung epithelial cell line: characterization and response to low pathogenicity avian influenza virus.

Avian influenza virus (AIV) infections of the chicken occur via the respiratory route. Unlike ducks which are considered as a natural AIV reservoir, chickens are highly susceptible to AIV infections and do not possess the RIG-I pattern recognition receptor involved in triggering the antiviral interferon response. To study the chicken innate immune response to AIV in the respiratory tract, we established an epithelial cell line (CLEC213) from lung explants of white leghorn chickens. CLEC213 cells exhibited a polyhedral morphology and formed cohesive clusters bound through tight junctions as assessed by electron microscopy. Expression of E-cadherin but not vimentin could be detected as expected for cells of epithelial origin. In addition, CLEC213 cells showed characteristics similar to those of mammalian type II pneumocytes, including the presence of intracytoplasmic vacuoles filled with a mucopolysaccharide material, alkaline phosphatase activity, transcription of chicken lung collectins genes (cLL and SPA), and some intracytoplasmic lamellar-like bodies. CLEC213 cells showed a constitutive expression level of TLR3 and TLR4 and were responsive to stimulation with the respective agonists, poly (I:C) and LPS: between 4h and 24h after treatment, a strong increase in the expression of IFN-α, IFN-ß and IL-8 genes could be detected. Furthermore, CLEC213 cells supported efficient growth of the low pathogenicity avian influenza virus H6N2 (A/duck/France/05057a/2005) in the presence or the absence of trypsin in the culture media. At 4h post-infection, the H6N2 virus induced highly elevated levels of expression of IFN-α and IL-8, moderately elevated levels of LITAF, TGF-ß4 and CCL5. However, an increase of IFN-ß gene expression could not be detected in response to AIV infection. In conclusion, like mammalian type II pneumocytes, CLEC213 are able to mount a robust cytokine and chemokine immune response to microbial patterns and viral infection. We hypothesize that they could derive from lung atrial granular cells. The involvement of such type of lung epithelial cells in the respiratory tract defence of the chicken can thus be further studied.