The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+ and CD8+ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

Baicalin attenuated Mycoplasma gallisepticum-induced immune impairment in chicken bursa of fabricius through modulation of autophagy and inhibited inflammation and apoptosis.

BACKGROUND: Mycoplasma gallisepticum (MG) is the primary etiologic agent of chronic respiratory disease in poultry. However, the mechanism underlying MG-induced immune dysregulation in chicken is still elusive. Baicalin shows excellent anti-bacterial, anti-inflammatory, anti-carcinogenic and anti-viral properties. In the present study, the preventive effects of baicalin against immune impairment in chicken bursa of fabricius (BF) were studied in an MG infection model. RESULTS: Histopathological examination showed increased inflammatory cell infiltrations and fragmented nuclei in the model group. Ultrastructural analysis revealed the phenomenon of apoptosis in bursal cells, along with the deformation of mitochondrial membrane and swollen mitochondria in the model group. However, these abnormal morphological changes were partially alleviated by baicalin. Meanwhile, baicalin treatment attenuated the level of proinflammatory cytokines, and suppressed nuclear factor-kappa B expression at both protein and mRNA level. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assay showed extensive apoptosis in BF in the model group. The mRNA and protein expression levels of apoptosis-related genes were upregulated in BF, while baicalin treatment significantly alleviated apoptosis in BF. In addition, alterations in mRNA and protein expression levels of autophagy-related genes and mitochondrial dynamics proteins were significantly alleviated by baicalin. Moreover, baicalin treatment significantly attenuated MG-induced decrease in CD8+ cells and reduced bacterial load in chicken BF compared to the model group. CONCLUSIONS: These results suggested that baicalin could effectively inhibit MG-induced immune impairment and alleviate inflammatory responses and apoptosis in chicken BF. © 2020 Society of Chemical Industry.

Marek's disease vaccines-induced differential expression of known and novel microRNAs in primary lymphoid organ bursae of White Leghorn.

Marek's disease (MD) is a contagious disease of domestic chickens caused by MD viruses. MD has been controlled primarily by vaccinations, yet sporadic outbreaks of MD take place worldwide. Commonly used MD vaccines include HVT, SB-1 and CVI988/Rispens and their efficacies are reportedly dependent of multiple factors including host genetics. Our previous studies showed protective efficacy of a MD vaccine can differ drastically from one chicken line to the next. Advanced understanding on the underlying genetic and epigenetic factors that modulate vaccine efficacy would greatly improve the strategy in design and development of more potent vaccines. Two highly inbred lines of White Leghorn were inoculated with HVT and CVI988/Rispens. Bursa samples were taken 26 days post-vaccination and subjected to small RNA sequencing analysis to profile microRNAs (miRNA). A total of 589 and 519 miRNAs was identified in one line, known as line 63, 490 and 630 miRNAs were identified in the other, known as line 72, in response to HVT or CVI988/Rispens inoculation, respectively. HVT and CVI988/Rispens induced mutually exclusive 4 and 13 differentially expressed (DE) miRNAs in line 63 birds in contrast to a non-vaccinated group of the same line. HVT failed to induce any DE miRNA and CVI988/Rispens induced a single DE miRNA in line 72 birds. Thousands of target genes for the DE miRNAs were predicted, which were enriched in a variety of gene ontology terms and pathways. This finding suggests the epigenetic factor, microRNA, is highly likely involved in modulating vaccine protective efficacy in chicken.

T cell subset profile and inflammatory cytokine properties in the gut-associated lymphoid tissues of chickens during infectious bursal disease virus (IBDV) infection.

While infectious bursal disease virus (IBDV) mainly targets immature B cells and causes T cell infiltration in the bursa of Fabricius (BF) of chickens, the effect of IBDV infection on the properties of T cells and relevant cytokine production in avian gut-associated lymphoid tissues (GALTs) remains unknown. Here, we show that while the CD8+ T cell subset is not affected, IBDV infection decreases the percentage of CD4+ T cells in the cecal tonsil (CT), but not in esophagus tonsil, pylorus tonsil, and Meckel's diverticulum of GALTs, in contrast to BF and spleen, in which the proportion of CD4+ cells increases upon IBDV infection. Further, IBDV infection upregulates IFN-γ, IL-10, and the T cell checkpoint receptor LAG-3 mRNA expression in BF. In contrast, in CTs, IBDV infection significantly increases the production of IFN-ß and CTLA-4 mRNA, while no significant effect is seen in the case of IFN-γ, IL-10 and LAG-3. Together, our data reveal differential modulation of T cell subsets and proinflammatory cytokine production in different lymphoid tissues during the course of IBDV infection.

Assessment of the role of intracloacal inoculation of live infectious bursal disease vaccine in breaking through maternally derived antibodies.

Infectious bursal disease (IBD) remains a potential worldwide threat to the poultry industry despite several vaccination approaches. Because maternally derived antibodies (MDA) constitute a critical problem for IBD vaccination, we examined the efficiency of the intracloacal vaccination approach in breaking through MDA. Experiment 1 determined the ability of the vaccinal strain to multiply in the bursa of Fabricius (BF) in chicks with a high level of MDA. Using real-time polymerase chain reaction, we quantified the strain in the bursae of vaccinated and non-vaccinated chicks. Experiment 2 was performed on three groups of chicks with high levels of MDA: group 1, non-vaccinated non-challenged; group 2, non-vaccinated challenged; and group 3, vaccinated challenged. Seroconversion to IBDV was measured using enzyme-linked immunosorbent assay. Groups 2 and 3 were challenged by vvIBDV at 25 days of age. Experiment 3 studied the effect of early IBD vaccinal strain multiplication on the immune response of vaccinated and non-vaccinated chicks to other vaccines. In experiment 1, the vaccinal strain showed progressive multiplication and reached the detectable titre in BF at 12 h post-vaccination despite high MDA titre. Experiment 2 showed that chicks in group 3 had significant seroconversion against IBDV. After challenge, group 3 showed significant improvements in several measured parameters compared with group 2. Moreover, results of experiment 3 proved that early multiplication of the vaccinal strain in the BF has no significant effect on the immune system or immune response to other vaccines. These results proved the promising success of this IBD vaccination approach.RESEARCH HIGHLIGHTS IBD vaccinal strain succeeded in multiplying in BF after intracloacal inoculation.Vaccinated chicks showed significant seroconversion of IBDV antibody titres.Vaccinated chicks showed a significant protection level against vvIBDV.Early IBD vaccination did not affect the immune response to other vaccines.

The morphological basis of the development of the chick embryo immune system.

The chick immune system is a fundamental model in basic immunology. In birds, the bone marrow derived pluripotent stem cells after entering the circulation, migrate to bursa of Fabricius to benefit from a microenvironment which supports the differentiation and maturation of B lymphocytes by the help of its resident cells and tissues. Delivering sufficient functional B cells is required to maintain their peripheral population and normal peripheral humoral responses. Additionally, bursa acts as an active site for the generation of antibody diversity through gene conversion. Being consisted of 98% B lymphocytes, the organ is occupied by other cell types including T cells, macrophages, eosinophils and mast cells. Thymus, which is an epithelial organ is the main site of T cell development where positive and negative selections contribute to the development of functional and not autoreactive T cell repertoire. Bursectomy and thymectomy are surgical exercises through which the involvement of cells of specific immunity including B cells and T cells can be determined.

Current state-of-the-art in the use of plants for the production of recombinant vaccines against infectious bursal disease virus.

Infectious bursal disease is a widely spread threatening contagious viral infection of chickens that induces major damages to the Bursa of Fabricius and leads to severe immunosuppression in young birds causing significant economic losses for poultry farming. The etiological agent is the infectious bursal disease virus (IBDV), a non-enveloped virus belonging the family of Birnaviridae. At present, the treatment against the spread of this virus is represented by vaccination schedules mainly based on inactivated or live-attenuated viruses. However, these conventional vaccines present several drawbacks such as insufficient protection against very virulent strains and the impossibility to differentiate vaccinated animals from infected ones. To overcome these limitations, in the last years, several studies have explored the potentiality of recombinant subunit vaccines to provide an effective protection against IBDV infection. In this review, we will give an overview of these novel types of vaccines with special emphasis on current state-of-the-art in the use of plants as "biofactories" (plant molecular farming). In fact, plants have been thoroughly and successfully characterized as heterologous expression systems for the production of recombinant proteins for different applications showing several advantages compared with traditional expression systems (Escherichia coli, yeasts and insect cells) such as absence of animal pathogens in the production process, improved product quality and safety, reduction of manufacturing costs, and simplified scale-up.

Prenatal aromatase inhibition alters postnatal immunity in domestic chickens (Gallus gallus).

In birds, exposure to exogenous testosterone during embryonic development can suppress measures of immune function; however, it is unclear whether these effects are due to direct or indirect action via aromatization. Estradiol (E2) is synthesized from testosterone by the enzyme aromatase, and this conversion is a necessary step in many signaling pathways that are ostensibly testosterone-dependent. Many lines of evidence in mammals indicate that E2 can affect immune function. We tested the hypothesis that some of the immunomodulatory effects observed in response to in ovo testosterone exposure in birds are mediated by conversion to E2 by aromatase, by using fadrozole to inhibit aromatization of endogenous testosterone during a crucial period of embryonic immune system development in domestic chickens (Gallus gallus). We then measured total IgY antibody count, response to PHA challenge, mass of thymus and bursa of Fabricius, and plasma testosterone post-hatch on days 3 and 18. Because testosterone has a reputation for immunosuppression, we predicted that if modulation of an immune measure by testosterone is dependent on aromatization, then inhibition of estrogen production by fadrozole treatment would lead to elevated measures of that parameter. Conversely, if testosterone inhibits an immune measure directly, then fadrozole treatment would likely not alter that parameter. Fadrozole treatment reduced circulating E2 in female embryos, but had no effect on males or on testosterone in either sex. Fadrozole-treated chicks had decreased day 3 plasma IgY antibody titers and a strong trend towards increased day 18 thymic mass. Furthermore, fadrozole treatment generated a positive relationship between testosterone and thymic mass in males, and tended to increase day 18 IgY levels for a given bursal mass in females. There was no effect on PHA response, bursal mass, or plasma testosterone at either age post-hatch. The alteration of several indicators of immune function in fadrozole-treated chicks implicates aromatization as a relevant pathway through which developmental exposure to testosterone can affect immunity in birds.

Arsenic (III) or/and copper (II) exposure induce immunotoxicity through trigger oxidative stress, inflammation and immune imbalance in the bursa of chicken.

The environmental hazards of arsenic (As) and copper (Cu) contamination have swept through quite a few districts worldwide. Whereas, molecular mechanisms involved in As- and Cu-induced immunotoxicity in Gallus gallus bursa of Fabricius (BF) are complex and elusive. Male Hy-line chickens were exposed to arsenic trioxide (As2O3; 30 mg/kg) and copper sulfate (CuSO4; 300 mg/kg) alone or in combination, respectively, to examine the potential ecotoxicity of them. The ions homeostasis and BF index of chicken had distinct changes after As or/and Cu exposure. Moreover, As or/and Cu treatment significantly increased the MDA content and NOS activity, and simultaneously resulted in reductions in CAT and AHR activities. Subsequently, it was further exhibited up-regulations of nuclear factor-κB (NF-κB), inflammatory mediators and pro-inflammation cytokines accompanied by depletion of anti-inflammatory cytokines and severe pathological conditions. Moreover, decreased ratio of IFN-γ/IL-4 and increased level of IL-17 illustrated an imbalance of the immune response. Meanwhile, incremental mRNA transcription and protein levels of heat shock proteins (HSPs) alleviated toxicity caused by As or/and Cu. Importantly, exposure to both contaminants significantly soared the BF injury in comparison with exposure to As or Cu alone. All these results illustrated that exposure to As2O3 or/and CuSO4 elicited BF tissue damage and ions changes, and its severity was associated with prolonged persistence of oxidative damage, accompanied by a dysregulated immune response which played a vital role in inflammatory injury. Additionally, combined management of As2O3 and CuSO4 could exacerbate BF injury.

Ammonia inhalation impaired immune function and mitochondrial integrity in the broilers bursa of fabricius: Implication of oxidative stress and apoptosis.

Ammonia (NH3) is considered as environmental pollutant and toxic agent for animals and humans including poultry. Previous reports demonstrated that NH3 suppressed broilers immunity. However, the harmful effects of NH3 on broilers bursa of fabricius (BF) is still unknown. Functionally, apoptosis is very important for many physiological processes including homeostasis of lymphocyte population. Therefore, the present study was aimed to investigate the underlying mechanisms of NH3 toxicity in the broilers BF. Histological observation showed lymphocyte accumulation, cavities and increased interstitial cells in BF. Ultrastructural observation indicated mitochondrial vacuoles, deformation and disappearance of mitochondrial membranes. Oxidative stress markers (CAT, MDA, H2O2, GGT, GSH-Px and GSH) showed that NH3-induced oxidative stress in BF. Meanwhile, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed increased apoptotic cells. In addition, the mRNA and protein expression of dynamin-related protein 1 (Drp1), mitochondrial fission factor (Mff), mitofusin 1 and 2 (Mfn1 and Mfn2), optic atrophy 1 (Opa1) indicated imbalance between mitochondrial inner and outer membrane and results in mitochondrial dysfunction in broilers BF. The mRNA and protein expression of apoptosis-related genes including Caspase-3, Caspase-9, Caspase-8, Cytochrome-C (Cyt-C), p53, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) were significantly altered in broilers BF. Conclusively, these results displayed that excessive NH3 causes BF damage and mitochondrial dysfunction through oxidative stress and apoptosis in BF and could affect immune function of BF. These findings provide possible therapeutic targets to prevent NH3 induced toxicity in the BF of broilers.

Copper induces oxidative stress with triggered NF-κB pathway leading to inflammatory responses in immune organs of chicken.

Copper (Cu) is a necessary trace mineral due to its biological activity. Excessive Cu can induce inflammatory response in humans and animals, but the underlying mechanism is still unknown. Here, 240 broilers were used to study the effects of excessive Cu on oxidative stress and NF-κB-mediated inflammatory responses in immune organs. Chickens were fed with diet containing different concentrations of Cu (11, 110, 220, and 330 mg of Cu/kg dry matter). The experiment lasted for 49 days. Spleen, thymus, and bursa of Fabricius (BF) on day 49 were collected for histopathological observation and assessment of oxidative stress status. Additionally, the mRNA and protein levels of NF-κB and inflammatory cytokines were also analyzed. The results indicated that excess Cu could increase the number and area of splenic corpuscle as well as the ratio of cortex and medulla in thymus and BF. Furthermore, excessive Cu intake could decrease activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); but increase contents of malondialdehyde (MDA), TNF-α, IL-1, IL-1ß; up-regulate mRNA levels of TNF-α, IFN-γ, IL-1, IL-1ß, IL-2, iNOS, COX-2, NF-κB and protein levels of TNF-α, IFN-γ, NF-κB, p-NF-κB in immune organs. In conclusion, excessive Cu could cause pathologic changes and induce oxidative stress with triggered NF-κB pathway, and might further regulate the inflammatory response in immune organs of chicken.

Thyrotropin-Releasing Hormone (TRH) and Somatostatin (SST), but not Growth Hormone-Releasing Hormone (GHRH) nor Ghrelin (GHRL), Regulate Expression and Release of Immune Growth Hormone (GH) from Chicken Bursal B-Lymphocyte Cultures.

It is known that growth hormone (GH) is expressed in immune cells, where it exerts immunomodulatory effects. However, the mechanisms of expression and release of GH in the immune system remain unclear. We analyzed the effect of growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), ghrelin (GHRL), and somatostatin (SST) upon GH mRNA expression, intracellular and released GH, Ser133-phosphorylation of CREB (pCREBS133), intracellular Ca2+ levels, as well as B-cell activating factor (BAFF) mRNA expression in bursal B-lymphocytes (BBLs) cell cultures since several GH secretagogues, as well as their corresponding receptors (-R), are expressed in B-lymphocytes of several species. The expression of TRH/TRH-R, ghrelin/GHS-R1a, and SST/SST-Rs (Subtypes 1 to 5) was observed in BBLs by RT-PCR and immunocytochemistry (ICC), whereas GHRH/GHRH-R were absent in these cells. We found that TRH treatment significantly increased local GH mRNA expression and CREB phosphorylation. Conversely, SST decreased GH mRNA expression. Additionally, when added together, SST prevented TRH-induced GH mRNA expression, but no changes were observed in pCREBS133 levels. Furthermore, TRH stimulated GH release to the culture media, while SST increased the intracellular content of this hormone. Interestingly, SST inhibited TRH-induced GH release in a dose-dependent manner. The coaddition of TRH and SST decreased the intracellular content of GH. After 10 min. of incubation with either TRH or SST, the intracellular calcium levels significantly decreased, but they were increased at 60 min. However, the combined treatment with both peptides maintained the Ca2+ levels reduced up to 60-min. of incubation. On the other hand, BAFF cytokine mRNA expression was significantly increased by TRH administration. Altogether, our results suggest that TRH and SST are implicated in the regulation of GH expression and release in BBL cultures, which also involve changes in pCREBS133 and intracellular Ca2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.

The immunomodulatory functions and molecular mechanism of a new bursal heptapeptide (BP7) in immune responses and immature B cells.

The bursa of Fabricius (BF) is the acknowledged central humoural immune organ unique to birds and plays a vital role in B lymphocyte development. In addition, the unique molecular immune features of bursal-derived biological peptides involved in B cell development are rarely reported. In this paper, a novel bursal heptapeptide (BP7) with the sequence GGCDGAA was isolated from the BF and was shown to enhance the monoclonal antibody production of a hybridoma. A mouse immunization experiment showed that mice immunized with an AIV antigen and BP7 produced strong antibody responses and cell-mediated immune responses. Additionally, BP7 stimulated increased mRNA levels of sIgM in immature mouse WEHI-231 B cells. Gene microarray results confirmed that BP7 regulated 2465 differentially expressed genes in BP7-treated WEHI-231 cells and induced 13 signalling pathways and various immune-related functional processes. Furthermore, we found that BP7 stimulated WEHI-231 cell autophagy and AMPK-ULK1 phosphorylation and regulated Bcl-2 protein expression. Finally, chicken immunization showed that BP7 enhanced the potential antibody and cytokine responses to the AIV antigen. These results suggested that BP7 might be an active biological factor that functions as a potential immunopotentiator, which provided some novel insights into the molecular mechanisms of the effects of bursal peptides on immune functions and B cell differentiation.

Immunomodulatory effects of heat stress and lipopolysaccharide on the bursal transcriptome in two distinct chicken lines.

BACKGROUND: Exposure to heat stress suppresses poultry immune responses, which can increase susceptibility to infectious diseases and, thereby, intensify the negative effects of heat on poultry welfare and performance. Identifying genes and pathways that are affected by high temperatures, especially heat-induced changes in immune responses, could provide targets to improve disease resistance in chickens. This study utilized RNA-sequencing (RNA-seq) to investigate transcriptome responses in the bursa of Fabricius, a primary immune tissue, after exposure to acute heat stress and/or subcutaneous immune stimulation with lipopolysaccharide (LPS) in a 2 × 2 factorial design: Thermoneutral + Saline, Heat + Saline, Thermoneutral + LPS and Heat + LPS. All treatments were investigated in two chicken lines: a relatively heat- and disease-resistant Fayoumi line and a more susceptible broiler line. RESULTS: Differential expression analysis determined that Heat + Saline had limited impact on gene expression (N = 1 or 63 genes) in broiler or Fayoumi bursa. However, Thermoneutral + LPS and Heat + LPS generated many expression changes in Fayoumi bursa (N = 368 and 804 genes). Thermoneutral + LPS was predicted to increase immune-related cell signaling and cell migration, while Heat + LPS would activate mortality-related functions and decrease expression in WNT signaling pathways. Further inter-treatment comparisons in the Fayoumi line revealed that heat stress prevented many of the expression changes caused by LPS. Although fewer significant expression changes were observed in the broiler bursa after exposure to Thermoneutral + LPS (N = 59 genes) or to Heat + LPS (N = 146 genes), both treatments were predicted to increase cell migration. Direct comparison between lines (broiler to Fayoumi) confirmed that each line had distinct responses to treatment. CONCLUSIONS: Transcriptome analysis identified genes and pathways involved in bursal responses to heat stress and LPS and elucidated that these effects were greatest in the combined treatment. The interaction between heat and LPS was line dependent, with suppressive expression changes primarily in the Fayoumi line. Potential target genes, especially those involved in cell migration and immune signaling, can inform future research on heat stress in poultry and could prove useful for improving disease resistance.

Transcriptome profile in bursa of Fabricius reveals potential mode for stress-influenced immune function in chicken stress model.

BACKGROUND: The molecular mechanisms underlying stress-influenced immune function of chicken (Gallus Gallus) are not clear. The stress models can be established effectively by feeding chickens corticosterone (CORT) hormone. The bursa of Fabricius is a unique central immune organ of birds. RNA-Seq technology was used to investigate differences in the expression profiles of immune-related genes and associated pathways in the bursa of Fabricius to clarify molecular mechanisms. The aim of this study was to broaden the understanding of the stress-influenced immune function in chickens. RESULTS: Differentially expressed genes (DEGs) in the bursa of Fabricius between experimental group (basal diet with added CORT 30 mg/kg; C_B group) and control group (basal diet; B_B group) were identified by using RNA-seq technology. In total, we found 1434 significant DEGs (SDEGs), which included 199 upregulated and 1235 downregulated genes in the C_B group compared with the B_B group. The immune system process GO term was the top significantly GO term, including MYD88, TLR4, IL15, VEGFA gene and so on. The cytokine-cytokine receptor interaction pathway and the Toll-like receptor signaling pathway were the key pathways affected by stress. The protein-protein interaction (PPI) analysis of the SDEGs showed that VEGFA, MyD88 and IL15 were hub genes and module analysis showed that MYD88, TLR4 and VEGFA play important roles in response to stress. CONCLUSION: This study showed that the VEGFA and ILs (such as IL15) via the cytokine-cytokine receptor interaction pathway, MYD88 and TLR4 via the Toll-like receptor signaling pathway may play important roles in the regulation of immune function under stress condition with CORT administration. The results of this study provide a reference for further studies of the molecular mechanisms of stress-influenced immune function.

Bursal transcriptome profiling of different inbred chicken lines reveals key differentially expressed genes at 3 days post-infection with very virulent infectious bursal disease virus.

Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.

Salmonella infection may alter the expression of toll like receptor 4 and immune related cells in chicken bursa of Fabricius.

Toll like receptor 4 (TLR4), eosinophils and mast cells play significant role in host immunity during several pathogenic infections. However in vivo tissue expression of TLR4 and distribution pattern of eosinophils and mast cells in chicken bursa of Fabricius (BF) during Salmonella enterica serovar Typhimurium (STm) infection is poorly studied. Therefore, herein, following immunostaining, we found localization of TLR4 in follicular cortex and medulla and its expression was statistical increased after 36 h and 72 h of STm stimulation. Chromotrope 2R staining revealed that eosinophils were mostly distributed in follicular cortex, inter-follicular spaces and in or around blood vessels and their number in BF were statistical increased after 72 h of STm stimulation. The presence of eosinophils was confirmed using immunostaining with anti-rabbit eosinophil cationic protein antibody. Toluidine blue stained mast cells were mostly distributed in connective tissues between inter-follicular spaces while some were also present in follicular cortex of BF. However, STm stimulation illustrated non-significant effect on the number of mast cells or their de-granulation, instead their number were gradually decreased in BF with advancement in age of chickens. Hence, this study provided novel information about in vivo tissue distribution of TLR4, eosinophils and mast cells in BF during STm infection.

Bursal immunopathology responses of specific-pathogen-free chickens and red jungle fowl infected with very virulent infectious bursal disease virus.

Very virulent infectious bursal disease virus (vvIBDV) targets B lymphocytes in the bursa of Fabricius (BF), causing immunosuppression and increased mortality rates in young birds. There have been few studies on the host immune response following vvIBDV infection at different inoculum doses in chickens with different genetic backgrounds. In this study, we characterized the immune responses of specific-pathogen-free (SPF) chickens and Malaysian red jungle fowl following infection with vvIBDV strain UPM0081 at 103.8 and 106.8 times the 50% embryo infectious dose (EID50). The viral burden, histopathological changes, immune cell populations, and expression of immune-related genes were measured and compared between infected and uninfected bursa at specific intervals. The populations of KUL1+, CD3+CD4+ and CD3+CD8+ cells were significantly increased in both types of chickens at 3 dpi, and there was significant early depletion of IgM+ B cells at 1 dpi in the red jungle fowl. vvIBDV infection also induced differential expression of genes that are involved in Th1 and pro-inflammatory responses, with groups receiving the higher dose (106.8 EID50) showing earlier expression of IFNG, IL12B, IL15, IL6, CXCLi2, IL28B, and TLR3 at 1 dpi. Although both chicken types showed equal susceptibility to infection, the red jungle fowl were clinically healthier than the SPF chickens despite showing more depletion of IgM+ B cells and failure to induce IFNB activation. In conclusion, high-dose vvIBDV infection caused an intense early host immune response in the infected bursa, with depletion of IgM+ B cells, bursal lesions, and cytokine expression as a response to mitigate the severity of the infection.

Hydrogen sulfide inhalation-induced immune damage is involved in oxidative stress, inflammation, apoptosis and the Th1/Th2 imbalance in broiler bursa of Fabricius.

Hydrogen sulfide (H2S) is widely accepted to be a signaling molecule that exhibits some potentially beneficial therapeutic effects at physiological concentrations. At elevated levels, H2S is highly toxic and has a negative effect on human health and animal welfare. Studies have shown that H2S exposure induces an immune function in mice, but there are few studies of the effect of continuous H2S exposure on immune organs in poultry. In this study, one-day-old broilers were selected and exposed to 4 or 20 ppm of H2S gas for 14, 28 and 42 days of age. After exposure, the bursa of Fabricius (BF) was harvested. The results showed that continuous H2S exposure reduced the body weight, abdominal fat percentage, and antibody titer in broilers. H2S exposure also decreased mRNA expression of IgA, IgM and IgG in the broiler BF. A histological study revealed obvious nuclear debris, and a few vacuoles in the BF, and an ultrastructural study revealed mitochondrial and nuclear damage to BF cells after H2S exposure for 42 d. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay suggested H2S exposure remarkably increased the number of TUNEL positive nuclei and significantly increased apoptotic index. The expression of apoptotic genes also confirmed that H2S inhalation damaged the broiler BF. Increased cytokines and reduced antioxidant responses were detected in the BF after exposure to H2S. Cytokines promoted inflammation and caused a Th1/Th2 imbalance. We suggest that continuous H2S intoxication triggers oxidative stress, inflammation, apoptosis and a Th1/Th2 imbalance in the BF, leading to immune injury in broilers.