Discrimination of distinct chicken M cell subsets based on CSF1R expression.

In mammals, a subset of follicle-associated epithelial (FAE) cells, known as M cells, conduct the transcytosis of antigens across the epithelium into the underlying lymphoid tissues. We previously revealed that M cells in the FAE of the chicken lung, bursa of Fabricius (bursa), and caecum based on the expression of CSF1R. Here, we applied RNA-seq analysis on highly enriched CSF1R-expressing bursal M cells to investigate their transcriptome and identify novel chicken M cell-associated genes. Our data show that, like mammalian M cells, those in the FAE of the chicken bursa also express SOX8, MARCKSL1, TNFAIP2 and PRNP. Immunohistochemical analysis also confirmed the expression of SOX8 in CSF1R-expressing cells in the lung, bursa, and caecum. However, we found that many other mammalian M cell-associated genes such as SPIB and GP2 were not expressed by chicken M cells or represented in the chicken genome. Instead, we show bursal M cells express high levels of related genes such as SPI1. Whereas our data show that bursal M cells expressed CSF1R-highly, the M cells in the small intestine lacked CSF1R and both expressed SOX8. This study offers insights into the transcriptome of chicken M cells, revealing the expression of CSF1R in M cells is tissue-specific.

Distribution and spatiotemporal development of organised lymphoid tissues in the chicken intestinal tract.

Chickens exhibit a distinct immune architecture characterised by the absence of draining lymph nodes and the presence of a well-developed mucosa-associated lymphoid tissue. The structure and spatiotemporal development of chicken lymphoid tissues in the intestine are poorly documented. The macroscopically indistinct structure of chicken Peyer's patches has impeded studies into their development. The generation of CSF1R-eGFP reporter transgenic chickens enables visualisation of the development, organisation and extent of chicken lymphoid tissues by unique macroscopic views. CSF1R-eGFP reporter transgenic chickens were used to investigate the distribution and spatiotemporal development of PP and caecal tonsils in embryonic day 18 to 8-week-old chickens. Peyer's patch anlagen are present at ED18 with a similar frequency and distribution pattern observed in 2- and 8-week-old chickens. These findings can support in ovo and post-hatch mucosal vaccination strategies and the development of vaccine delivery systems targeted to the specialized epithelium overlying the Peyer's patches.

Revealing Novel-Strain-Specific and Shared Epitopes of Infectious Bronchitis Virus Spike Glycoprotein Using Chemical Linkage of Peptides onto Scaffolds Precision Epitope Mapping.

The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.

Disentangling the innate immune responses of intestinal epithelial cells and lamina propria cells to Salmonella Typhimurium infection in chickens.

Salmonella enterica serovar Typhimurium (STm) is a major foodborne pathogen and poultry are a key reservoir of human infections. To understand the host responses to early stages of Salmonella infection in poultry, we infected 2D and 3D enteroids, the latter of which contains leukocytes, neurons, and mesenchymal cells that are characteristic of the lamina propria. We infected these enteroids with wild-type (WT STm), a non-invasive mutant lacking the prgH gene (ΔprgH STm), or treated them with STm lipopolysaccharide (LPS) and analyzed the expression of innate immune related genes by qPCR at 4 and 8 h. The localization of the tight junction protein, ZO-1, expression was disrupted in WT STm infected enteroids but not ΔprgH STm or LPS treated enteroids, suggesting a loss of epithelial barrier integrity. The innate immune response to LPS was more pronounced in 2D enteroids compared to 3D enteroids and by 8 hpi, the response in 3D enteroids was almost negligible. However, when STm adhered to or invaded the enteroids, both 2D and 3D enteroids exhibited an upregulation of inflammatory responses. The presence of lamina propria cells in 3D enteroids resulted in the unique expression of genes associated with immune functions involved in regulating inflammation. Moreover, 2D and 3D enteroids showed temporal differences in response to bacterial invasion or adherence. At 8 hpi, innate responses in 3D but not 2D enteroids continued to increase after infection with WT STm, whereas the responses to the non-invasive strain decreased at 8 hpi in both 2D and 3D enteroids. In conclusion, STm infection of chicken enteroids recapitulated several observations from in vivo studies of Salmonella-infected chickens, including altered epithelial barrier integrity based on ZO-1 expression and inflammatory responses. Our findings provide evidence that Salmonella-infected enteroids serve as effective models for investigating host-pathogen interactions and exploring the molecular mechanisms of microbial virulence although the 3D model mimics the host more accurately due to the presence of a lamina propria.

CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Corrigendum: The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

[This corrects the article DOI: 10.3389/fcimb.2023.1067993.].

The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

Introduction: Highly pathogenic avian influenza (HPAI) viruses, such as H5N1, continue to pose a serious threat to animal agriculture, wildlife and to public health. Controlling and mitigating this disease in domestic birds requires a better understanding of what makes some species highly susceptible (such as turkey and chicken) while others are highly resistant (such as pigeon and goose). Susceptibility to H5N1 varies both with species and strain; for example, species that are tolerant of most H5N1 strains, such as crows and ducks, have shown high mortality to emerging strains in recent years. Therefore, in this study we aimed to examine and compare the response of these six species, to low pathogenic avian influenza (H9N2) and two strains of H5N1 with differing virulence (clade 2.2 and clade 2.3.2.1) to determine how susceptible and tolerant species respond to HPAI challenge. Methods: Birds were challenged in infection trials and samples (brain, ileum and lung) were collected at three time points post infection. The transcriptomic response of birds was examined using a comparative approach, revealing several important discoveries. Results: We found that susceptible birds had high viral loads and strong neuro-inflammatory response in the brain, which may explain the neurological symptoms and high mortality rates exhibited following H5N1 infection. We discovered differential regulation of genes associated with nerve function in the lung and ileum, with stronger differential regulation in resistant species. This has intriguing implications for the transmission of the virus to the central nervous system (CNS) and may also indicate neuro-immune involvement at the mucosal surfaces. Additionally, we identified delayed timing of the immune response in ducks and crows following infection with the more deadly H5N1 strain, which may account for the higher mortality in these species caused by this strain. Lastly, we identified candidate genes with potential roles in susceptibility/resistance which provide excellent targets for future research. Discussion: This study has helped elucidate the responses underlying susceptibility to H5N1 influenza in avian species, which will be critical in developing sustainable strategies for future control of HPAI in domestic poultry.

Growth performance, caecal microbiome profile, short-chain fatty acids, and litter characteristics in response to placement on reused litter and combined threonine, arginine and glutamine supplementation to juvenile male broiler chickens.

BACKGROUND: Exposure of broilers to litter microbiome may increase specific amino acid (AA) requirements towards activated immune responses. This may challenge the generality of the ideal protein (IP) concept, in which dietary essential AA to lysine ratios aimed to mimic presumably constant AA to lysine ratios in whole bird requirements. Therefore, we tested the effect of threonine, arginine and glutamine (TAG) supplementation to IP-based control diets (C) on performance, caecal microbiome composition, short-chain fatty acids and litter characteristics of broiler chickens placed on reused litter. RESULTS: Thirty-two pens with ten male broiler chickens each were used in a 2 × 2 factorial arrangement of two diet treatments (with or without TAG supplementation) and two litter treatments (placement on clean or reused litter) for 21 days (n = 8). Caecal contents were analysed for microbiome profile using percent guanine + cytosine (%G + C profile) method and short chain fatty acids. TAG-supplemented birds underperformed compared to C birds (P = 0.002), whereas birds placed on reused litter outperformed those on clean litter (P = 0.047). Diet, reused litter and their interaction impacted the %G + C profile at different ranges. Whilst TAG supplementation reduced bacterial abundance at %G + C 51-56 (P < 0.05), reused litter placement tended to reduce %G + C 23-31 and increase %G + C 56-59 (P < 0.10). However, TAG supplementation reduced bacterial abundance at %G + C 47-51 (P < 0.05) and increased caecal branched chain fatty acids on clean litter only (P = 0.025). Greater levels of propionic acid were observed for C birds placed on reused litter only (P = 0.008). Litter pH was greater for reused litter pens than clean litter pens at day 21 (P < 0.001). In addition, litter moisture content was less for TAG birds and reused litter pens compared to C birds (P = 0.041) and clean litter pens (P < 0.001), respectively. CONCLUSIONS: These data support the view that irrespective of performance benefits arising from bird placement on reused litter, TAG supplementation to IP-formulated baseline rations impaired growth, supported by the lowered abundance of caecal bacteria known to dominate in well-performing birds and greater levels of caecal branched chain fatty acids.

Comparative Analysis of Different Inbred Chicken Lines Highlights How a Hereditary Inflammatory State Affects Susceptibility to Avian Influenza Virus.

Evidence suggests that susceptibility to avian influenza A virus in chickens is influenced by host genetics, but the mechanisms are poorly understood. A previous study demonstrated that inbred line 0 chickens are more resistant to low-pathogenicity avian influenza (LPAI) infection than line CB.12 birds based on viral shedding, but the resistance was not associated with higher AIV-specific IFNγ responses or antibody titres. In this study, we investigated the proportions and cytotoxic capacity of T-cell subpopulations in the spleen and the early immune responses in the respiratory tract, analysing the innate immune transcriptome of lung-derived macrophages following in vitro stimulation with LPAI H7N1 or the TLR7 agonist R848. The more susceptible C.B12 line had a higher proportion of CD8αß+ γδ and CD4+CD8αα+ αVß1 T cells, and a significantly higher proportion of the CD8αß+ γδ and CD8αß+ αVß1 T cells expressed CD107a, a surrogate marker of degranulation. Lung macrophages isolated from line C.B12 birds expressed higher levels of the negative regulator genes TRIM29 and IL17REL, whereas macrophages from line 0 birds expressed higher levels of antiviral genes including IRF10 and IRG1. After stimulation with R848, the macrophages from line 0 birds mounted a higher response compared to line C.B12 cells. Together, the higher proportion of unconventional T cells, the higher level of cytotoxic cell degranulation ex vivo and post-stimulation and the lower levels of antiviral gene expression suggest a potential role of immunopathology in mediating susceptibility in C.B12 birds.

Temporal transcriptome profiling of floating apical out chicken enteroids suggest stability and reproducibility.

Enteroids are miniature self-organising three-dimensional (3D) tissue cultures which replicate much of the complexity of the intestinal epithelium. We recently developed an apical-out leukocyte-containing chicken enteroid model providing a novel physiologically relevant in vitro tool to explore host-pathogen interactions in the avian gut. However, the replicate consistency and culture stability have not yet been fully explored at the transcript level. In addition, causes for the inability to passage apical-out enteroids were not determined. Here we report the transcriptional profiling of chicken embryonic intestinal villi and chicken enteroid cultures using bulk RNA-seq. Comparison of the transcriptomes of biological and technical replicate enteroid cultures confirmed their high level of reproducibility. Detailed analysis of cell subpopulation and function markers revealed that the mature enteroids differentiate from late embryonic intestinal villi to recapitulate many digestive, immune and gut-barrier functions present in the avian intestine. These transcriptomic results demonstrate that the chicken enteroid cultures are highly reproducible, and within the first week of culture they morphologically mature to appear similar to the in vivo intestine, therefore representing a physiologically-relevant in vitro model of the chicken intestine.

Genetic variation in chicken interferon signalling pathway genes in research lines showing differential viral resistance.

Avian viruses of economic interest are a significant burden on the poultry industry, affecting production traits and resulting in mortality. Furthermore, the zoonosis of avian viruses risks pandemics developing in humans. Vaccination is the most common method of controlling viruses; however current vaccines often lack cross-protection against multiple strains of each virus. The mutagenicity of these viruses has also led to virulent strains emerging that can overcome the protection offered by vaccines. Breeding chickens with a more robust innate immune response may help in tackling current and emerging viruses. Understanding the genetic evolution of different lines will thus provide a useful tool in helping the host in the fight against pathogens. This study focuses on the interferon genes and their receptors in different chicken lines that are known to be more resistant or susceptible to particular avian viruses. Comparing genotypic differences in these core immune genes between the chicken lines may explain the phenotypic differences observed and aid the identification of causative variations. The relative resistance/susceptibility of each line to viruses of interest (Marek's disease virus, infectious bursal disease, infectious bronchitis virus and avian influenza virus) has previously been determined. Here we identify single nucleotide polymorphisms in interferons and downstream genes. Functional prediction tools were used to identify variants that may be affecting protein structure, mRNA secondary structure or transcription factor and micro-RNA binding sites. These variants were then considered in the context of the research lines and their distribution between phenotypes. We highlight 60 variants of interest in the interferon pathway genes that may account for susceptibility/resistance to viral pathogens.

Advances, challenges and future applications of avian intestinal in vitro models.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and probiotic microorganisms, as well as its role in the spread of infectious diseases in both the developing and developed world. A paucity of physiologically relevant models has limited research in this essential field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The intestine is characterized by a complex cellular composition with numerous functions, unique dynamic locations and interdependencies making this organ challenging to recreate in vitro. This review illustrates the in vitro tools that aim to recapitulate this intestinal environment; from the simplest cell lines, which mimic select features of the intestine but lack anatomical and physiological complexity, to the more recently developed complex 3D enteroids, which recreate more of the intestine's intricate microanatomy, heterogeneous cell populations and signalling gradients. We highlight the benefits and limitations of in vitro intestinal models and describe their current applications and future prospective utilizations in intestinal biology and pathology research. We also describe the scope to improve on the current systems to include, for example, microbiota and a dynamic mechanical environment, vital components which enable the intestine to develop and maintain homeostasis in vivo. As this review explains, no one model is perfect, but the key to choosing a model or combination of models is to carefully consider the purpose or scientific question.

Establishment of bovine 3D enteroid-derived 2D monolayers.

Three-dimensional (3D) intestinal enteroids are powerful in vitro models for studying intestinal biology. However, due to their closed structure direct access to the apical surface is impeded, limiting high-throughput applications of exogenous compounds and pathogens. In this study, we describe a method for generating confluent 2D enteroids from single-cell suspensions of enzymatically-dissociated ileum-derived bovine 3D enteroids. Confluent monolayers were first achieved using IntestiCult media but to establish a defined, cost-effective culture media, we also developed a bovine enteroid monolayer (BEM) medium. The monolayers cultured in BEM media proliferated extensively and formed confluent cell layers on both Matrigel-coated plastic plates and transwell inserts by day 3 of culture. The 2D enteroids maintained the epithelial cell lineages found in 3D enteroids and ileum tissue. In addition, the monolayers formed a functional epithelial barrier based on the presence of the adherens and tight junction proteins, E-cadherin and ZO-1, and electrical resistance across the monolayer was measured from day 3 and maintained for up to 7 days in culture. The method described here will provide a useful model to study bovine epithelial cell biology with ease of access to the apical surface of epithelial cells and has potential to investigate host-pathogen interactions and screen bioactive compounds.

Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time.

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

Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer.

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Evaluation of a Campylobacter jejuni N-glycan-ExoA glycoconjugate vaccine to reduce C. jejuni colonisation in chickens.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and handling or consumption of contaminated poultry meat is the key source of infection. Glycoconjugate vaccines containing the C. jejuni N-glycan have been reported to be partially protective in chickens. However, our previous studies with subunit vaccines comprising the C. jejuni FlpA or SodB proteins with up to two or three C. jejuni N-glycans, respectively, failed to elicit significant protection. In this study, protein glycan coupling technology was used to add up to ten C. jejuni N-glycans onto a detoxified form of Pseudomonas aeruginosa exotoxin A (ExoA). The glycoprotein, G-ExoA, was evaluated for efficacy against intestinal colonisation of White Leghorn chickens by C. jejuni strains M1 and 11168H relative to unglycosylated ExoA. Chickens were challenged with the minimum dose required for reliable colonisation, which was 102 colony-forming units (CFU) for strain M1 and and 104 CFU for strain 11168H. Vaccine-specific serum IgY was detected in chickens vaccinated with both ExoA and G-ExoA. However, no reduction in caecal colonisation by C. jejuni was observed. While the glycan dose achieved with G-ExoA was higher than FlpA- or SodB-based glycoconjugates that were previously evaluated, it was lower than that of glycoconjugates where protection against C. jejuni has been reported, indicating that protection may be highly sensitive to the amount of glycan presented and/or study-specific variables.

Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field.

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV's major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.

Transcriptomic analysis of caecal tissue in inbred chicken lines that exhibit heritable differences in resistance to Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans and the handling or consumption of contaminated poultry meat is a key source of infection. Selective breeding of poultry that exhibit elevated resistance to Campylobacter is an attractive control strategy. Here we studied the global transcriptional response of inbred chicken lines that differ in resistance to C. jejuni colonisation at a key site of bacterial persistence. RESULTS: Three-week-old chickens of line 61 and N were inoculated orally with C. jejuni strain M1 and caecal contents and tonsils were sampled at 1 and 5 days post-infection. Caecal colonisation was significantly lower in line 61 compared to line N at 1 day post-infection, but not 5 days post-infection. RNA-Seq analysis of caecal tonsils of both lines revealed a limited response to C. jejuni infection compared to age-matched uninfected controls. In line N at days 1 and 5 post-infection, just 8 and 3 differentially expressed genes (DEGs) were detected (fold-change > 2 and false-discovery rate of < 0.05) relative to uninfected controls, respectively. In the relatively resistant line 61, a broader response to C. jejuni was observed, with 69 DEGs relating to immune regulation, cell signalling and metabolism at 1 day post-infection. However, by day 5 post-infection, no DEGs were detected. By far, the greatest number of DEGs were between uninfected birds of the two lines implying that differential resistance to C. jejuni is intrinsic. Of these genes, several Major Histocompatibility Complex class I-related genes (MHCIA1, MHCBL2 and MHCIY) and antimicrobial peptides (MUC2, AvBD10 and GZMA) were expressed to a greater extent in line N. Two genes within quantitative trait loci associated with C. jejuni colonisation were also more highly expressed in line N (ASIC4 and BZFP2). Quantitative reverse-transcriptase PCR analysis of a subset of transcripts confirmed the RNA-Seq results. CONCLUSIONS: Our data indicate a limited transcriptional response in the caecal tonsils of inbred chickens to intestinal colonisation by Campylobacter but identify a large number of differentially transcribed genes between lines 61 and N that may underlie variation in heritable resistance to C. jejuni.

Kinetics of the Cellular and Transcriptomic Response to Eimeria maxima in Relatively Resistant and Susceptible Chicken Lines.

Eimeria maxima is a common cause of coccidiosis in chickens, a disease that has a huge economic impact on poultry production. Knowledge of immunity to E. maxima and the specific mechanisms that contribute to differing levels of resistance observed between chicken breeds and between congenic lines derived from a single breed of chickens is required. This study aimed to define differences in the kinetics of the immune response of two inbred lines of White Leghorn chickens that exhibit differential resistance (line C.B12) or susceptibility (line 15I) to infection by E. maxima. Line C.B12 and 15I chickens were infected with E. maxima and transcriptome analysis of jejunal tissue was performed at 2, 4, 6 and 8 days post-infection (dpi). RNA-Seq analysis revealed differences in the rapidity and magnitude of cytokine transcription responses post-infection between the two lines. In particular, IFN-γ and IL-10 transcript expression increased in the jejunum earlier in line C.B12 (at 4 dpi) compared to line 15I (at 6 dpi). Line C.B12 chickens exhibited increases of IFNG and IL10 mRNA in the jejunum at 4 dpi, whereas in line 15I transcription was delayed but increased to a greater extent. RT-qPCR and ELISAs confirmed the results of the transcriptomic study. Higher serum IL-10 correlated strongly with higher E. maxima replication in line 15I compared to line C.B12 chickens. Overall, the findings suggest early induction of the IFN-γ and IL-10 responses, as well as immune-related genes including IL21 at 4 dpi identified by RNA-Seq, may be key to resistance to E. maxima.

Inside-out chicken enteroids with leukocyte component as a model to study host-pathogen interactions.

Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host-pathogen interactions. We have developed complex multilobulated 3D chicken enteroids from intestinal embryonic villi and adult crypts. These avian enteroids develop optimally in suspension without the structural support required to produce mammalian enteroids, resulting in an inside-out enteroid conformation with media-facing apical brush borders. Histological and transcriptional analyses show these enteroids comprise of differentiated intestinal epithelial cells bound by cell-cell junctions, and notably, include intraepithelial leukocytes and an inner core of lamina propria leukocytes. The advantageous polarisation of these enteroids has enabled infection of the epithelial apical surface with Salmonella Typhimurium, influenza A virus and Eimeria tenella without the need for micro-injection. We have created a comprehensive model of the chicken intestine which has the potential to explore epithelial and leukocyte interactions and responses in host-pathogen, food science and pharmaceutical research.