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Adaptation of avian pathogenic E. coli (APEC) to changing host environments including virulence factors expression is vital for disease progression. FdeC is an autotransporter adhesin that plays a role in uropathogenic Escherichia coli (UPEC) adhesion to epithelial cells. Expression of fdeC is known to be regulated by environmental conditions in UPEC and Shiga toxin-producing E. coli (STEC). The observation in a previous study that an APEC strain IMT5155 in which the fdeC gene was disrupted by a transposon insertion resulted in elevated adhesion to chicken intestinal cells prompted us to further explore the role of fdeC in infection. We found that the fdeC gene prevalence and FdeC variant prevalence differed between APEC and nonpathogenic E. coli genomes. Expression of the fdeC gene was induced at host body temperature, an infection relevant condition. Disruption of fdeC resulted in greater adhesion to CHIC-8E11 cells and increased motility at 42 °C compared to wild type (WT) and higher expression of multiple transporter proteins that increased inorganic ion export. Increased motility may be related to increased inorganic ion export since this resulted in downregulation of YbjN, a protein known to supress motility. Inactivation of fdeC in APEC strain IMT5155 resulted in a weaker immune response in chickens compared to WT in experimental infections. Our findings suggest that FdeC is upregulated in the host and contributes to interactions with the host by down-modulating motility during colonization. A thorough understanding of the regulation and function of FdeC could provide novel insights into E. coli pathogenesis., (© 2024. The Author(s).)

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella , the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses., Competing Interests: Author ES and LL were employed by the company Jefo Nutrition Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Jefo Nutrition Inc. .Jefo Nutrition Inc. provided research support and played role in the decision to publish and study design, but not in the data collection and analysis. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mitchell, Sutton, Elango, Borowska, Perry, Lahaye, Santin, Arsenault and Vervelde.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Borowska, Sives, Vervelde and Sutton.)

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 ., Competing Interests: Author RH was employed by Cobb-Vantress Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bremner, Kim, Morris, Nolan, Borowska, Wu, Tomley, Blake, Hawken, Kaiser and Vervelde.)

Avian pathogenic Escherichia coli (APEC) cause severe respiratory and systemic disease in chickens, commonly termed colibacillosis. Early immune responses after initial infection are highly important for the outcome of the infection. In this study, the early interactions between GFP -expressing APEC strains of serotypes O1:K1:H7 and O2:K1:H5 and phagocytic cells in the lung of CSF1R -reporter transgenic chickens were investigated. CSF1R -reporter transgenic chickens express fluorescent protein under the control of elements of the CSF1R promoter and enhancer, such that cells of the myeloid lineage can be visualized in situ and sorted. Chickens were separately inoculated with APEC strains expressing GFP and culled 6 h post-infection. Flow cytometric analysis was performed to phenotype and sort the cells that harbored bacteria in the lung, and the response of the sorted cells was defined by transcriptomic analysis. Both APEC strains were mainly detected in CSF1R -transgene neg ( CSF1R -tg neg ) and CSF1R -tg low MHC II neg MRC1L-B neg cells and low numbers of APEC were detected in CSF1R -tg high MHC II pos MRC1L-B pos cells. Transcriptomic and flow cytometric analysis identified the APEC pos CSF1R -tg neg and CSF1R -tg low cells as heterophils and the APEC pos CSF1R -tg high cells as macrophages and dendritic cells. Both APEC strains induced strong inflammatory responses, however in both CSF1R -tg neg/low and CSF1R -tg high cells, many immune related pathways were repressed to a greater extent or less activated in birds inoculated with APEC O2- GFP compared to APEC O1- GFP inoculated birds. Comparison of the immune pathways revealed the aryl hydrocarbon receptor ( AhR ) pathway, IL17 and STAT3 signaling, heterophil recruitment pathways and the acute phase response, are modulated particularly post-APEC O2- GFP inoculation. In contrast to in vivo data, APEC O2- GFP was more invasive in CSF1R -tg high cells in vitro than APEC O1- GFP and had higher survival rates for up to 6 h post-infection. Our data indicate significant differences in the responses induced by APEC strains of prevalent serotypes, with important implications for the design and interpretation of future studies. Moreover, we show that bacterial invasion and survival in phagocyte populations in vitro is not predictive of events in the chicken lung., (Copyright © 2020 Alber, Morris, Bryson, Sutton, Monson, Chintoan-Uta, Borowska, Lamont, Schouler, Kaiser, Stevens and Vervelde.)

To address the need for sensitive high-throughput assays to analyse avian innate and adaptive immune responses, we developed and validated a highly multiplexed qPCR 96.96 Fluidigm Dynamic Array to analyse the transcription of chicken immune-related genes. This microfluidic system permits the simultaneous analysis of expression of 96 transcripts in 96 samples in 6 nanolitre reactions and the 9,216 reactions are ready for interpretation immediately. A panel of 89 genes was selected from an RNA-seq analysis of the transcriptional response of chicken macrophages, dendritic cells and heterophils to agonists of innate immunity and from published transcriptome data. Assays were confirmed to be highly specific by amplicon sequencing and melting curve analysis and the reverse transcription and preamplification steps were optimised. The array was applied to RNA of various tissues from a commercial line of broiler chickens housed at two different levels of biosecurity. Gut-associated lymphoid tissues, bursa, spleen and peripheral blood leukocytes were isolated and transcript levels for immune-related genes were defined. The results identified blood cells as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially transcribed between birds housed under varying biosecurity levels. Conventional qPCR analysis of three differentially transcribed genes confirmed the results from the multiplex qPCR array. A highly multiplexed qPCR-based platform for evaluation of chicken immune responses has been optimised and validated using samples from commercial chickens. Apart from applications in selective breeding programmes, the array could be used to analyse the complex interplay between the avian immune system and pathogens by including pathogen-specific probes, to screen vaccine responses, and as a predictive tool for immune robustness., Competing Interests: We declare that the study received financial and in-kind support from Aviagen Ltd. This included a contribution to the stipend of DB while a doctoral candidate at The University of Edinburgh and in-kind contributions including access to broiler populations and the provision of biological materials. For part of the study KAW was an employee of Aviagen Ltd. and RAB has been employed by the company throughout and to date. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Provision of biological materials derived from Aviagen broilers may be subject to a Material Transfer Agreement covering their secondary uses.

The respiratory tract is a key organ for many avian pathogens as well as a major route for vaccination in the poultry industry. To improve immune responses after vaccination of chickens through increased uptake of vaccines and targeting to antigen presenting cells, a better understanding of the avian respiratory immune system is required. Transgenic MacReporter birds were used expressing a reporter gene (eGFP or mApple) under the control of the CSF1R promoter and enhancer in cells of the mononuclear phagocyte (MNP) lineage to visualize the ontogeny of the lymphoid tissue, macrophages and dendritic cells, in the trachea, lung and air sac of birds from embryonic day 18-63 weeks of age. Small aggregates of CSF1R-transgene + cells start to form at the openings of the secondary bronchi at 1 week of age, indicative of the early development of the organised bronchus-associated lymphoid tissue. Immunohistochemical staining revealed subpopulations of MNPs in the lung, based on expression of CSF1R-transgene, CD11, TIM4, LAMP1, and MHC II. Specialised epithelial cells or M cells covering the bronchus-associated lymphoid tissue expressed CSF1R-transgene and type II pneumocytes expressed LAMP1 suggesting that these epithelial cells are phagocytic and transcytose antigen. Highly organised lymphoid tissue was seen in trachea from 4 weeks onwards. Throughout the air sacs at all ages, CSF1R-transgene + cells were scattered and at later stages, CSF1R-transgene + cells lined capillaries. These results will serve as a base for further functional characterization of macrophages and dendritic cells and their role in respiratory diseases and vaccine responses.

This study aimed to evaluate the response of Harderian gland (HG) cells after in vitro stimulation with class B synthetic oligodeoxyribonucleotides (ODN) containing CpG motifs. This knowledge is of importance for the development of mucosal vaccines for poultry, such as eye-drop or spray vaccines, to determine if class B CpG ODN can act as an vaccine adjuvant or as a prophylactic treatment mainly against respiratory disease viruses. The relative expression of Toll-like receptor 21 (TLR21), interferon (IFN)-γ, interleukin (IL)-1β and IL-10 genes were quantified at 1, 3, 6 and 18 h post-stimulation of HG cells from 5-week-old birds. In addition, it was also investigated if expression of these genes was affected by the age of the birds (differences between 5- and 12-week-old birds), concentrations of ODN or cell preparation method used. Class B CpG ODN induced upregulation of TLR21 and IFN-γ mRNA expression levels at 1h post-stimulation depending on concentration of ODN used but only in HG cells isolated from young birds., (Copyright © 2014 Elsevier B.V. All rights reserved.)