Development of a high-throughput platform for evaluation of chicken immune responses

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances

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

Aktywność posłów IX kadencji Sejmu RP na TikToku – analiza zagadnienia

Social media is having a significant impact on socio-political life. The expansion and popularisation of further platforms such as TikTok provides an opportunity to use and implement another communication channel. This property is increasingly being recognised by politicians who are beginning to grasp the potential of the platform. The primary purpose of this article is to present the results of a survey conducted between 1 August and 15 September 2022, the subject of which was the analysis of the activity of the ninth-term deputies sitting in the Sejm of the Republic of Poland. The article posits that the rise of public interest in TikTok may influence the activation of politicians in the space described

Ewolucja wizerunku Wołodymyra Zełeńskiego w prasie ukraińskiej, rosyjskiej i niemieckiej (na wybranych przykładach)

Nowadays Volodymyr Zelenskyi attracts a lot of attention – in Western world, he has become the symbol of Ukrainian resistance against the Russian invasion. A lot of media regularly repeated that the president of Ukraine, although he used to be a professional actor, up against the Russian invasion, has become the man of the state. This paper is the result of researching the evolution of Volodymyr Zelenskyi’s image in Russian, Ukrainian, and German press on examples of – in this order – „Izvestiya”, „Ukrainska Pravda” and „Frankfurter Allgemeine Zeitung”. The authors have researched the press from three representative dates: the day of the second round of the presidential election in Ukraine, one day after the beginning of the Russian invasion on Ukraine, and the day of the first anniversary of the invasion. During the analysis, the method of content analysis was used, with due regard to the quantitative and qualitative research methods, as well as the historical genetic method as the support.Współcześnie poświęca się wiele uwagi Wołodymyrowi Zełeńskiemu, który w świecie zachodnim stał się symbolem ukraińskiego oporu przeciwko rosyjskiej inwazji. W wielu przekazach medialnych powtarzano, że prezydent Ukrainy, którego postać wcześniej interpretowano przede wszystkim w kontekście jego aktorskiej przeszłości zawodowej, w obliczu rosyjskiej inwazji wyrósł na męża stanu. Niniejsza praca ma na celu zbadanie ewolucji wizerunku Wołodymyra Zełeńskiego w prasie ukraińskiej, rosyjskiej i niemieckiej na przykładzie odpowiednio „Ukraińskiej Prawdy”, rosyjskiej „Izwiestiji” oraz niemieckiego „Frankfurter Allgemiene Zeitung”, badając trzy reprezentacyjne daty: dzień drugiej tury wyborów prezydenckich na Ukrainie, dzień po rozpoczęciu przez Rosję inwazji na Ukrainę oraz dzień pierwszej rocznicy tej inwazji. Do analizy wykorzystano metodę analizy zawartości (z uwzględnieniem zarówno metod ilościowych, jak i jakościowych), a także wspomagano się historyczną metodą genetyczną

Chicken intestinal organoids: a novel method to measure the mode of action of feed additives

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 signalling. 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