Chicken Cecal Tonsils Research Articles

Influences of the short-term dietary carbohydrate restriction on the chicken cecal tonsil

This study aims to clarify influences of the short-term carbohydrate (CHO)restriction on plasmacells in chicken cecal tonsil (CT) using immunohistochemical and morphometrical techniques. Healthy male layer chickens were randomly divided into the control and three experimental groups. The three experimental groups were fed with diets containing 50%, 25%, and 0% CHO of that in the control diet, respectively. Following the experimental period for 7 days, CTs were collected from each chicken. Immunohistochemistry was performed to detect immunoglobulin (Ig) A, G and M. The density of IgA-immunoreactive cells was significantly lower in the lymphoid follicles (LFs) and diffuse lymphoid tissues (DLTs) in CTs of the 0% group compared to those of the other groups. There was a positive correlation between daily CHO intake and the density of IgA-immunoreactive cells in the DLTs and LFs. The density of IgM-immunoreactive cells was significantly decreased as daily CHO intake decreased, and there was a positive correlation between these two factors in both DLTs and LFs. The density of IgG-immunoreactive cells was significantly lower in DLTs of the 0% group than those in the controland 50% groups. The density of IgG-immunoreactive cells was significantly decreased with a decrease in daily CHO intake in DLT but not in LF. These findings demonstrated that dietary CHO had a significant effect on the density of plasma cells in the CTs exhibiting a declining trend with a decrease of ingested CHO volume.

Changes in the gene expression and methylation in chicken cecal tonsils after in ovo administration of bioactive substances

Cecal tonsils are the main organs which generate an immune response and also the part of the GALT, thus they are in the close proximity of the intestinal microbiota and continuously exposed to microbe-associated molecular patterns. GALT developed regulatory and anti-inflammatory mechanisms which eliminate or tolerate microbiota. Bioactive substances in ovo administration ensures an early contact between the GALT and beneficial bacteria, which greatly promotes the development of tolerance. Our previous studies have shown that the administration of bioactive substances in ovo silences gene expression in the cecal tonsils. The research hypothesis assumes that negative silencing of expression is correlated with the level of methylation in the tonsils. Therefore the current study aimed to analyze the global and gene-specific DNA methylation profiles in the cecal tonsils of two distinct chicken genotypes administered in ovo with bioactive substances. Eggs of Ross 308 and Green-legged Partridgelike were stimulated on day 12 of incubation. The injected compounds were: probiotic—Lactococcus lactis subsp. cremoris, prebiotic—galactooligosaccharides, and synbiotic—combination of both. Chickens were sacrificed on d 42 post-hatching. Cecal tonsils was collected, RNA and DNA were isolated and intended to gene expression, gene methylation and global methylation analysis. Cecal tonsils changes were observed in the methylation of 6 genes: SYK, ANGPTL4, TNFRSF14, IKZF1, CYR61, SERPING. Analyzes showed that the suppression of gene expression is related to the level of methylation of individual genes. Based on the results obtained in the cecal tonsils, it can be concluded that the silencing of gene expression is of an epigenetic nature. This is another study aimed at analyzing the relationship between the host, its intestinal microbiota and the possibilities of its programming.

Campylobacter jejuni infection induces dynamic expression of avian host defense peptides in vitro and in vivo

Campylobacter jejuni is considered as the leading cause of worldwide foodborne bacterial gastroenteritis. Chicken is the main reservoir of C. jejuni. Avian innate immune responses to C. jejuni remain poorly defined. Chicken host defense peptides (HDPs) are the major components of avian innate immune system. This study aimed to characterize the chicken HDPs responses to C. jejuni in vitro and in vivo. In the HD11 macrophage cell line, the HDPs, including AvBD1–2, CATH1–3, AvBD7, AvBD4, and AvBD6, were relatively higher expressed in untreated cells, whereas the expressions were suppressed after C. jejuni infection. In contrast, C. jejuni infection significantly increased the expression of the lower expressed HDPs, such as AvBD3, AvBD5, AvBD8–14, and CATHB1, in untreated cells. In the chicken challenge experiment, the immune tissues of spleens and cecal tonsils were collected from C. jejuni-infected and uninfected chickens at 1, 3 and 15 day post inoculation (DPI). In spleens of C. jejuni-infected chickens, only AvBD14 expression was elevated at 1 DPI. The majority of avian HDPs were significantly up-regulated at 3 DPI and dramatically decreased to the levels of uninfected controls at 15 DPI. In chicken cecal tonsils, only AvBD9 and AvBD14 were significantly up-regulated at 1 DPI with C. jejuni infection. Collectively, C. jejuni infection induced dynamic expression of chicken HDPs in both macrophage HD11 and immune tissues of chickens. Suppression of chicken HDPs expression may be an evasion strategy of C. jejuni for persistent colonization in chicken intestine by circumventing the chicken immune system.

Transcriptomics of chicken cecal tonsils and intestine after infection with low pathogenic avian influenza virus H9N2

Influenza viruses cause severe respiratory infections in humans and birds, triggering global health concerns and economic burden. Influenza infection is a dynamic process involving complex biological host responses. The objective of this study was to illustrate global biological processes in ileum and cecal tonsils at early time points after chickens were infected with low pathogenic avian influenza virus (LPAIV) H9N2 through transcriptome analysis. Total RNA isolated from ileum and cecal tonsils of non-infected and infected layers at 12-, 24- and 72-h post-infection (hpi) was used for mRNA sequencing analyses to characterize differentially expressed genes and overrepresented pathways. Statistical analysis highlighted transcriptomic signatures significantly occurring 24 and 72 hpi, but not earlier at 12 hpi. Interferon (IFN)-inducible and IFN-stimulated gene (ISG) expression was increased, followed by continued expression of various heat-shock proteins (HSP), including HSP60, HSP70, HSP90 and HSP110. Some upregulated genes involved in innate antiviral responses included DDX60, MX1, RSAD2 and CMPK2. The ISG15 antiviral mechanism pathway was highly enriched in ileum and cecal tonsils at 24 hpi. Overall, most affected pathways were related to interferon production and the heat-shock response. Research on these candidate genes and pathways is warranted to decipher underlying mechanisms of immunity against LPAIV in chickens.

Probiotic Lactobacilli Limit Avian Influenza Virus Subtype H9N2 Replication in Chicken Cecal Tonsil Mononuclear Cells

Low pathogenic avian influenza virus (LPAIV) H9N2 poses significant threat to animal and human health. The growing interest in beneficial effects of probiotic bacteria on host immune system has led to research efforts studying their interaction with cells of host immune system. However, the role of lactobacilli in inducing antiviral responses in lymphoid tissue cells requires further investigation. The objective of the present study was to examine the antiviral and immunostimulatory effects of lactobacilli bacteria on chicken cecal tonsils (CT) cells against H9N2 LPAIV. CT mononuclear cells were stimulated with probiotic Lactobacillus spp mixture either alone or in combination with a Toll-like receptor (TLR)21 ligand, CpG oligodeoxynucleotides (CpG). Pre-treatment of CT cells with probiotic lactobacilli, alone or in combination with CpG, significantly reduced H9N2 LPAIV replication. Furthermore, lactobacilli alone elicited cytokine expression, including IL-2, IFN-γ, IL-1β, IL-6, and IL-12, and IL-10, while when combined with CpG, a significantly higher expression of (interferon-stimulated gene (viperin)), IL-12, IL-6, CXCLi2, and IL-1β was observed. However, none of these treatments induced significant changes in nitric oxide production by CT cells. In conclusion, probiotic lactobacilli demonstrated a modulatory effect on CT cells, and this correlated with enhanced antiviral immunity and reduced H9N2 LPAIV viral replication.

Characterization of Chicken Leukocyte Subsets from Lymphatic Tissue by Flow Cytometry.

Determining the abundance of leukocyte subtypes, including lymphocyte subpopulations, not only in blood but also in lymphatic tissues, is inevitable to assess the immune status of an organism for research purposes. However, nucleated thrombocytes and erythrocytes exacerbate many hematological techniques in avian species. In order to enable a rapid discrimination of leukocyte subsets from lymphatic tissues of chicken, we adapted existing flow cytometric methods for counting leukocytes in chicken blood. We established staining and gating strategies allowing the flow cytometric characterization and enumeration of total leukocytes, thrombocytes, monocytes/macrophages, CD8α+ lymphocytes, CD4+ T cells, γδ T cells, and B cells in chicken spleen and CD8α+ lymphocytes, CD4+ T cells, γδ T cells, and B cells among intraepithelial lymphocytes in chicken cecal tonsils. For this, we prepared single-cell suspensions of spleen and isolated intraepithelial lymphocytes from cecal tonsils without density centrifugation, and performed antibody staining of cells without subsequent washing steps to prevent cell loss and falsification of obtained cell counts. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Surface engineered polyanhydride-based oral Salmonella subunit nanovaccine for poultry.

PurposeSalmonellosis is a severe economic threat in poultry and a public health concern. Currently available vaccines are ineffective, and thus, developing effective oral Salmonella vaccine is warranted. Especially, a potent oral vaccine such as the mucoadhesive polyanhydride nanoparticle (PNP) protects the vaccine cargo and delivers to intestinal immune sites to elicit robust mucosal immunity and mitigate Salmonella colonization and shedding.Materials and methodsWe designed a Salmonella subunit vaccine using PNP containing immunogenic Salmonella outer membrane proteins (OMPs) and flagellar (F) protein-entrapped and surface F-protein-coated PNPs (OMPs-F-PNPs) using a solvent displacement method. Using high-throughput techniques, we characterized the OMPs-F-PNPs physicochemical properties and analyzed its efficacy in layer birds vaccinated orally.ResultsThe candidate vaccine was resistant in acidic microenvironment and had ideal physicochemical properties for oral delivery in terms of particle size, charge, morphology, biocompatibility, and pH stability. In vitro, in vivo, and ex vivo studies showed that F-protein surface-anchored nanoparticles were better targeted to chicken immune cells in peripheral blood and splenocytes and intestinal Peyer’s patch sites. In layer chickens inoculated orally with OMPs-F-PNPs, substantially higher OMPs-specific IgG response and secretion of Th1 cytokine IFN-γ in the serum, enhanced CD8+/CD4+ cell ratio in spleen, and increased OMPs-specific lymphocyte proliferation were observed. OMPs-F-PNPs vaccination also upregulated the expression of toll-like receptor (TLR)-2 and -4, TGF-β, and IL-4 cytokines’ genes in chicken cecal tonsils (lymphoid tissues). Importantly, OMPs-F-PNPs vaccine cleared Salmonella cecal colonization in 33% of vaccinated birds.ConclusionThis pilot in vivo study demonstrated the targeted delivery of OMPs-F-PNPs to ileum mucosal immune sites of chickens and induced specific immune response to mitigate Salmonella colonization in intestines.

Gene expression profiling of chicken cecal tonsils and ileum following oral exposure to soluble and PLGA-encapsulated CpG ODN, and lysate of Campylobacter jejuni

Campylobacter jejuni (C. jejuni) is a leading bacterial cause of food-borne illness in humans. Contaminated chicken meat is an important source of infection for humans. Chickens are not clinically affected by colonization, and immune responses following natural infection have limited effects on bacterial load in the gut. Induction of intestinal immune responses may possibly lead to a breakdown of the commensal relationship of chickens with Campylobacter. We have recently shown that soluble and poly D, L-lactic-co-glycolic acid (PLGA)-encapsulated CpG oligodeoxynucleotide (ODN) as well as C. jejuni lysate, are effective in reducing the intestinal burden of C. jejuni in chickens; however, the mechanisms behind this protection have yet to be determined. The present study was undertaken to investigate the mechanisms of host responses conferred by these treatments. Chickens were treated orally with soluble CpG ODN, or PLGA-encapsulated CpG ODN, or C. jejuni lysate, and expression of cytokines and antimicrobial peptides was evaluated in cecal tonsils and ileum using quantitative RT-PCR. Oral administration of soluble CpG ODN upregulated the expression of interferon (IFN)-γ, interleukin (IL)-1β, CXCLi2, transforming growth factor (TGF)-β4/1, IL-10 and IL-13, while treatment with PLGA-encapsulated CpG ODN upregulated the expression of IL-1β, CXCLi2, TGF-β4/1, IL-13, avian β-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). C. jejuni lysate upregulated the expression of IFN-γ, IL-1β, TGF-β4/1, IL-13, AvBD1, and CATHL-3. In conclusion, induction of cytokine and antimicrobial peptides expression in intestinal microenvironments may provide a means of reducing C. jejuni colonization in broiler chickens, a key step in reducing the incidence of campylobacteriosis in humans.

Characterization of host responses induced by Toll-like receptor ligands in chicken cecal tonsil cells

The innate responses of cecal tonsils against invading microorganisms are mediated by conserved pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). TLRs expressed by mammalian and avian immune system cells have the capability to recognize pathogen-associated molecular patterns (PAMPs). Although, the role of TLR ligands in innate and adaptive responses in chickens has been characterized in spleen and bursa of Fabricius, considerably less is known about responses in cecal tonsils. The aim of the current study was to assess responses of mononuclear cells from cecal tonsils to treatment with the TLR2, TLR4 and TLR21 ligands, Pam3CSK4, lipopolysaccharide (LPS), and CpG oligodeoxynucleotide (ODN), respectively. All three ligands induced significant up-regulation of interferon (IFN)-γ, interleukin (IL)-1β, IL-6 and CxCLi2/IL-8, whereas no significant changes were observed in expression of IL-13 or the antimicrobial peptides, avian β-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). In general, CpG ODN elicited the highest cytokine responses by cecal tonsil mononuclear cells, inducing significantly higher expression compared to LPS and Pam3CSK4, for IFNγ, IL-1β, IL-6 and CxCLi2 at various time points. These findings suggest the potential use of TLR21 ligands as mucosal vaccine adjuvants, especially in the context of pathogens of the intestinal tract.

Modulation of lipid peroxidation and antioxidant defense system in the chicken cecal tonsil by dietary high fluorine

Modulation of lipid peroxidation and antioxidant defense system in the chicken cecal tonsil by dietary high fluorine

Early immune dynamics following infection with Salmonella enterica serovars Enteritidis, Infantis, Pullorum and Gallinarum: Cytokine and chemokine gene expression profile and cellular changes of chicken cecal tonsils

Salmonella enterica subspecies enterica infection remains a serious problem in a wide range of animals and in man. Poultry-derived food is the main source of human infection with the non-host-adapted serovars while fowl typhoid and pullorum disease are important diseases of poultry. We have assessed cecal colonization and immune responses of newly hatched and older chickens to Salmonella serotypes Enteritidis, Infantis, Gallinarum and Pullorum. S. Enteritidis and S. Infantis colonized the ceca more efficiently than S. Gallinarum and S. Pullorum. Salmonella infection was also associated with increased staining for B-lymphocytes and macrophages in the cecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds stimulated the expression of CXCLi1 and CXCLi2 chemokines in the cecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the cecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes.

Gene expression profiling of chicken lymphoid cells after treatment with Lactobacillus acidophilus cellular components

Lactobacillus acidophilus has been shown to exert immunostimulating activities in a number of species, including the chicken. To examine the molecular mechanisms of this phenomenon, we investigated spatial and temporal expression of immune system genes in chicken cecal tonsil and spleen mononuclear cells in response to structural constituents of L. acidophilus. Using a low-density chicken immune system microarray, we found that cecal tonsil cells responded more rapidly than spleen cells to the bacterial stimuli, with the most potent stimulus for cecal tonsil cells being DNA and for splenocytes being the bacterial cell wall components. We also discovered that in both splenocytes and cecal tonsil cells, STAT2 and STAT4 genes were highly induced. Given the close interactions between cecal tonsil cells and commensal bacteria, we further examined the involvement of STAT2 and STAT4 signaling pathways in cellular responses to bacterial DNA. Our results revealed that the expression of STAT2, STAT4, IL-18, MyD88, IFN- α, and IFN- γ genes were up-regulated in cecal tonsil cells after treatment with L. acidophilus DNA.

Cytokine gene expression in chicken cecal tonsils following treatment with probiotics and Salmonella infection

Probiotics are currently employed for control of pathogens and enhancement of immune response in chickens. In this study, we investigated the underlying immunological mechanisms of the action of probiotics against colonization of the chicken intestine by Salmonella enterica subsp. enterica serovar Typhimurium ( Salmonella serovar Typhimurium). Birds received probiotics by oral gavage on day 1 of age and, subsequently, received Salmonella serovar Typhimurium on day 2 of age. Cecal tonsils were removed on days 1, 3 and 5 post-infection (p.i.), RNA was extracted and subjected to real-time quantitative RT-PCR for measurement of interleukin (IL)-6, IL-10, IL-12 and interferon (IFN)-γ gene expression. There was no significant difference in IL-6 and IL-10 gene expression in cecal tonsils of chickens belonging to various treatment groups. Salmonella serovar Typhimurium infection resulted in a significant increase in IL-12 expression in cecal tonsils on days 1 and 5 p.i. However, when chickens were treated with probiotics prior to experimental infection with Salmonella, the level of IL-12 expression was similar to that observed in uninfected control chickens. Treatment of birds with probiotics resulted in a significant decrease in IFN-γ gene expression in cecal tonsils of chickens infected with Salmonella compared to the Salmonella-infected birds not treated with probiotics. These findings reveal that repression of IL-12 and IFN-γ expression is associated with probiotic-mediated reduction in intestinal colonization with Salmonella serovar Typhimurium.

The cellular differentiation of M cells from crypt undifferentiated epithelial cells into microvillous epithelial cells in follicle-associated epithelia of chicken cecal tonsils.

To clarify the cellular origin and the fate of M cells, detailed distributions of the epithelial cells were investigated scanning electron microscopically on the follicle-associated epithelia (FAE) of chicken cecal tonsils. The distribution of M cells was closely related with the situation of the crypt orifices in chicken cecal tonsils. In undeveloped cecal tonsils, the intestinal crypts were localized at the periphery of the FAE. In these tonsils, M cells without microvilli (M(0)) were predominantly populated in the basal region of the FAE, whereas goblet cells and microvillous epithelial cells (MV) were more distributed in the middle to the apical region of the FAE. A few M cells with short microvilli were dispersed throughout the FAE. Significantly shrunk MV (MVs) clustered together in transitional portions from the lateral face to the roof of the FAE. In well-developed cecal tonsils, the crypts also opened at the lateral surface in addition to the periphery of the FAE. In these tonsils, the M(0) accumulated densely in the small areas around the crypt orifices exclusively. No sign of exfoliation of apoptotic epithelial cells was found in the M(0)-accumulated areas and at their peripheral boundaries. The MVs were often clustered in the central regions among the crypt orifices in addition to the roof of the FAE. These findings suggest that M cells are directly derived from the undifferentiated crypt epithelial cells, not fall into apoptotic cell death and further differentiate into MV in the FAE of chicken cecal tonsils.

Helper activity of chicken Vβ1 + and Vβ2 + αβ T cells for in vitro lgA antibody synthesis

Chickens have only two T cell receptor variable β gene families: Vβ1 and Vβ2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of Vβ1 + αβ T cells by treatment with a TCR Vβ1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both Vβ1 + and Vβ2 + chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of Vβ1 + T cell-depleted chickens to produce lgA is not caused by the inability of Vβ2 + T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).