Chicken Spleen Research Articles

Aberrant Gene Expression of Selenoproteins in Chicken Spleen Lymphocytes Induced by Mercuric Chloride.

Mercury (Hg) is a heavy metal widely distributed in ecological environment, poisoning the immune system of humans and animals. Selenium (Se) is an essential microelement and selenoproteins involved in the procedure of Se antagonizing organ toxicity induced by heavy metals. The aim of this research was to investigate the changes of gene expression profile of selenoproteins induced by mercuric chloride (HgCl2) in chicken spleen lymphocytes. We established cytotoxicity model of chicken spleen lymphocytes by HgCl2 exposure, the messenger RNA (mRNA) expression levels of 25 selenoproteins in spleen lymphocytes were analyzed by real-time quantitative PCR (qPCR), and the gene expression pattern of selenoproteins was revealed by principal component analysis (PCA). The results showed that the mRNA expression levels of 13 selenoproteins (GPX3, GPX4, TXNRD2, TXNRD3, DIO2, SELENOS, SELENON, SELENOT, SELENOO, SELENOP, SELENOP2, MSRB1, and SEPHS2) were decreased in HgCl2 treatment group, and there was strong positive correlation between these selenoproteins and component 1 as well as component 2 of the PCA. At the same time, the protein expression levels of GPX4, TXNRD1, TXNRD2, SELENOM, SELENOS, and SELENON were detected by Western blotting, which were consistent with the changes of gene expression. The results showed that the expression levels of selenoproteins were aberrant in response to HgCl2 toxicity. The information presented in this study provided clues for further research on the interaction between HgCl2 and selenoproteins, and the possible mechanism of immune organ toxicity induced by HgCl2.

TMT-based quantitative proteomic analysis reveals the spleen regulatory network of dexamethasone-induced immune suppression in chicks

Stress-induced immunosuppression is one of the most widespread problems in the poultry industry. Understanding the molecular regulatory mechanism of immunosuppression induced by stress in the chicken spleen would provide a scientific foundation for the prevention of stress reactions and antistress molecular breeding in poultry. To assess the protein expression profile of spleen tissue in a stress-included immunosuppression model, we performed a TMT-based proteomic analysis of chicken spleen tissue in a Dex-induced immunosuppression model (group C) and a control group (group A). We identified 590 differentially abundant proteins (DAPs) in chicken spleen tissue. These DAPs were significantly enriched in the following functional categories: ECM-receptor interaction, DNA replication, p53 signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway. Integrative analysis of the proteome and our previous transcriptome data revealed 62 DAPs showing correlations with the expression of their encoding mRNAs. Complementary proteome- and transcriptome-level analyses revealed a complex molecular network of stress-included immunosuppression. DPP4 and ALDH1A3 were the most significantly upregulated DAPs. GBP and OASL were identified as important nodes in the network related to stress-induced immunosuppression. The candidate genes identified in this study may be useful for the marker-based breeding of new chicken varieties with reduced stress levels. SignificanceThis study provides a large amount of new information about the spleen proteome of the Dex-induced immunosuppression in chicks, as well as the correlation of transcriptome and proteome. Analysis of this resource has enabled us to examine mechanism of protein and transcript diversification, which expands the understanding of the complexity of the mechanism of stress-induced immunosuppression.

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Prolactin affects the disappearance of ALV-J viremia in vivo and inhibits viral infection

Based on the RNA-seq data of chicken spleen tissues infected with J subgroup avian leukosis virus (ALV-J), we found that prolactin (PRL) gene was one of differentially expressed gene. We measured ALV-J viremia and PRL levels in the plasma of two groups of ALV-J-infected adult chickens. Furthermore, recombinant chicken PRL (cPRL) was used to assess how cPRL affects ALV-J virus replication both in vivo and in vitro. The results showed that PRL levels in the plasma of adult chickens infected with ALV-J were lower than those of uninfected chickens, and that the difference was more significant in the avian leukemia pathological apparent changes. Notably, the fluctuations in PRL levels might influence the disappearance of ALV-J viremia in chickens. The in vitro results showed that preincubating DF-1 cells with cPRL before ALV-J infection elicited the best antiviral effects. Moreover, these effects were not dose-dependent. in vivo, injection of cPRL into ALV-J-infected chicks could reduce the levels of viremia at the 14 days post infection (dpi). Additionally, the expression of the interferon-stimulated genes oligoadenylate synthetase-like (OSAL) and vasoactive intestinal peptide (VIP) increased, and that of the proinflammatory cytokine-encoding TNTα, IL-1β, and IL-6 genes decreased in the spleens of ALV-J-infected chicks injected with cPRL, leading to inhibition of viral replication at the 7 dpi. Collectively, our data demonstrated that PRL plays an important antiviral role in the immune response to ALV-J infection. This is the first report of the relationship between ALV-J infection and PRL. It is of great significance for the prevention and control of ALV-J.

The Effect of Chicken Spleen Transfer Factor on Intestinal Mucosa Immunity Barrier of Laying Hens

Chicken spleen Transfer Factor (TF) is a low-molecular-weight lymphocyte extract composed of polypeptide and nucleotide. However, its role in regulating intestinal structure and function in laying hens has remained largely unknown. 100 one-day-old laying hens were randomly divided into five groups and administered with different doses of TF (0.00 [control], 0.05mL, 0.10mL, 0.25mL and 1.00mL). The results showed that the high dose of TF (1.00mL) improved the intestinal mucosa morphology and strengthened the digestive and absorption function. Furthermore, the histology analysis revealed an increase in the number of intraepithelial lymphocytes and goblet cells. Similarly, the results from ELISA demonstrated an increase in the content of IL-10 in the intestinal tract, while the content of TNF-a showed a decrease in this regard. The RT-PCR assay also demonstrated the upregulation of the relative mRNA expressions of Muc2, TLR-2, and TLR-4 genes. The intestinal antioxidant function was significantly enhanced. In conclusion, high-dose of TF can improve the intestinal mucosa morphology and structure, enhance digestion and absorption functions, enhance the intestinal mucosal barrier immune function and antioxidant function, and up-regulate Muc2, TLR-2 and TLR-4 gene relative expression.

Bacillus subtilis BSH has a protective effect on Salmonella infection by regulating the intestinal flora structure in chickens

Salmonellosis is a worldwide zoonotic disease that poses a serious threat to the reproduction of livestock and poultry and the health of young animals. Probiotics including Bacillus species, have received increasing attention as a substitute for antibiotics. In this study, chicks infected with Salmonella were fed feed supplemented with the BSH to observe the pathological changes in the liver, detect the number of viable bacteria in the liver and spleen, and record the death of the chicks. The results showed that BSH could reduce the pathological changes in the liver and the invasion of Salmonella into the liver and spleen of chicks. In addition, the survival rate of chicks in the BSH experimental group was 60%, while that in the infected control group was 26%, indicating that BSH had a protective effect on chicks infected with Salmonella. Finally, the fecal microflora of 9-day-old chicks was analyzed by 16S rRNA high-throughput sequencing. The results showed that Salmonella infection could cause intestinal flora changes, while BSH could alleviate this change. In addition, BSH also promoted the proliferation of Lactobacillus salivarius in the cecum of chick. This study emphasized that BSH has anti- Salmonella infection effects in chickens and can be used as a candidate microecological preparation strain.

Side Effects of In ovo Given Sunset Yellow FCF on the Embryonic Development of the Spleen by Means of Histological and Enzyme Histochemical Methods

Background: E110 is one of the food colorants which is widely used in dairy products, fast foods, jam and dry beverage powders, aqueous drug solutions, tablets, capsules, toothpastes mouthwashes, hair care products and cosmetics. Doubts have accumulated in recent years that food additives might cause allergic reactions in humans or increase these ailments. In this study, side effects of Sunset yellow FCF (E110) on the embryonic development of chicken spleen were evaluated by means of histological, histomorphometrical and enzyme histochemical methods. Methods: In the study, 250 fertilized broiler eggs obtained from a commercial broodstock were used. Eggs were divided into 5 groups each having 50 eggs. Control eggs were either nontreated or distilled water injected via air sac. The eggs in the experimental groups were injected with 100ng/egg, 500ng/egg and 1.000ng/egg E110 prior to incubation. Blood and spleen samples were taken from randomly selected 10 eggs of each group at 11th, 15th, 18th and 21st days of incubation. Leukocyte formula, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte percentages were determined in the blood samples and embriyonic development of the spleen was assessed in the tissue sections. Results: In the 500ng/egg and 1.000ng/egg E110 injected experimental groups, embryonic development of the spleen retarded, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte rates were significantly depressed. Conclusions: Significant disturbances in the immune system functions of the affected animals might occure at post-natal period of their life.

Research Note: Expression of T cell-related cytokines in chicken cecal and spleen tissues following Eimeria tenella infection in vivo

The T cell-mediated immune response plays an important role in coccidiosis. To reveal the host T cell immune response following Eimeria tenella (E. tenella) infection in chickens, we performed quantitative real-time PCR to analyze the dynamic expression of the Th1-related cytokines IFN-γ, IL-2, and IL-12; the Th17-related cytokines IL-17A, IL-17F, and IL-22; and the Treg-related cytokines IL-10, TGF-β, and CTLA-4 in the cecum and spleen at 0, 2, 4, 6, 8, and 10 d postinfection (dpi). In the cecal tissue, the expression of the Th1-related cytokine IFN-γ was significantly higher at 6 and 8 dpi than at other time points (11.97-fold and 39.78-fold, respectively, compared with 0 dpi, P < 0.05). IL-2 and IL-12 expression was significantly higher at 6 and 8 dpi than at 0, 2 and 10 dpi (P < 0.05). The expression of the Th17-related cytokines IL-17A and IL-17F at 2 and 4 dpi and IL-22 expression at 4 dpi were significantly higher than those at 0, 6, 8 and 10 dpi (P < 0.05). The expression of the Treg-related cytokines IL-10, TGF-β and CTLA-4 was significantly higher at 6 and 8 dpi than at 0, 2 and 4 dpi (P < 0.05). In the spleen, IFN-γ and IL-12 expression peaked at 4 dpi, while IL-2 expression peaked at 10 dpi. IL-17A, IL-17F and IL-22 expression was significantly higher at 2 and 4 dpi than at 0, 6, 8 and 10 dpi (P < 0.05). Treg-related cytokine TGF-β expression was almost unchanged and significantly decreased at only 4 dpi (P < 0.05), while CTLA-4 expression showed an overall decreasing trend from 0 to 8 dpi but increased significantly at 10 dpi (P < 0.05). The expression patterns of three T cell subset-related cytokines were different in the cecum and spleen. Furthermore, Th1 and Treg cells participate in the immune response mainly in the latter stage of coccidia infection (6 and 8 dpi), while Th17 cells play a role mainly in the early stages of infection (2 and 4 dpi). Our data will help to deepen the understanding of the complex T cell immune response after coccidia infection.

Characterization of Conventional Dendritic Cells and Macrophages in the Spleen Using the CSF1R-Reporter Transgenic Chickens

The spleen is a major site for the immunological responses to blood-borne antigens that is coordinated by cells of the mononuclear phagocyte system (MPS). The chicken spleen is populated with a number of different macrophages while the presence of conventional dendritic cells (cDC) has been described. However, a detailed characterization of the phenotype and function of different macrophage subsets and cDC in the chicken spleen is limited. Using theCSF1R-reporter transgenic chickens (CSF1R-tg), in which cells of the MPS express a transgene under the control elements of the chickenCSF1R, we carried out an in-depth characterization of these cells in the spleen. Immunohistological analysis demonstrated differential expression of MRC1L-B by periarteriolar lymphoid sheaths (PALS)-associatedCSF1R-tg+cells. In the chicken's equivalent of the mammalian marginal zone, the peri-ellipsoid white-pulp (PWP), we identified high expression of putative CD11c by ellipsoid-associated cells compared to ellipsoid-associated macrophages. In addition, we identified a novel ellipsoid macrophage subset that expressed MHCII, CD11c, MRC1L-B, and CSF1R but not theCSF1R-tg. In flow cytometric analysis, diverse expression of theCSF1R-tg and MHCII was observed leading to the categorization ofCSF1R-tg cells intoCSF1R-tgdimMHCIIinter−hi,CSF1R-tghiMHCIIhi, andCSF1R-tghiMHCIIintersubpopulations. Low levels of CD80, CD40, MHCI, CD44, and Ch74.2 were expressed by theCSF1R-tghiMHCIIintercells. Functionally,in vivofluorescent bead uptake was significantly higher in theCSF1R-tghiMHCIIhiMRC1L-B+cells compared to theCSF1R-tgdimandCSF1R-tghiMHCIIinterMRC1L-B+subpopulations while LPS enhanced phagocytosis by theCSF1R-tghiMHCIIintersubpopulation. The analysis of bead localization in the spleen suggests the presence of ellipsoid-associated macrophage subsets. In addition, we demonstrated the functionality ofex vivoderivedCSF1R-tg+MRC1L-BnegcDC. Finally, RNA-seq analysis of theCSF1R-tg subpopulations demonstrated that separating theCSF1R-tghisubpopulation into CD11chiand CD11cdimcells enriched for cDC and macrophage lineages, respectively, while theCSF1R-tghiMHCIIintersubpopulation was enriched for red pulp macrophages. However, our analysis could not define the cell lineage of the heterogeneousCSF1R-tgdimsubpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation.

Molecular Characterization of Velogenic Avian Avulavirus Type 1 Isolated from Apparently Healthy Emu Birds: Implications to Viral Maintenance and Spread

Background: Newcastle disease caused by Avian avulavirus type 1 (AAvV-1) is one of the dreadful diseases affecting poultry and other avian species. Wild birds and several domestic birds are recognized as reservoirs of AAvV-1 and probably contribute to the epidemiology of ND in the domesticated poultry. Hence, efforts have been made to understand the virulence and genetic nature of AAvV-1 isolates obtained from apparently healthy Emu birds.Methods: This study details characterization of a velogenic Emu/5 AAvV-1 isolate obtained from an asymptomatic emu flock. Full- length fusion gene was amplified and subsequent phylogenetic analysis was performed. Experimental inoculation of 3-week old chicken with the isolate resulted in virulent ND. Expression of cytokine mRNA levels in spleen of infected chicken at different time points correlated well with the clinical picture, gross and histopathological lesions.Result: To our knowledge this is the first evidence for the role of apparently healthy emu bird acting as a reservoir of velogenic AAvV-1 of subgenotype XIII 2.2 which proved to be highly virulent to chicken. This study further highlights the role of reservoir birds in AAvV-1 transmission and the need for adopting most realistic strategies in counteracting the disease.

Identification and expression analysis of MicroRNAs in chicken spleen in a corticosterone-induced stress model

For investigating the effects of stress on the immune response of chickens, we established a corticosterone (CORT)-induced stress model by exogenous intake of CORT. Control group was fed with a basal diet and the stress model group was fed with a 30 mg/Kg CORT-treated diet in ad libitum conditions for 7 days. Then, we used RNA-seq technology to identify the expression pattern of miRNAs, target genes, and relevant pathways in chicken spleen. Results showed that 71 differentially expressed miRNAs (DEMs) were determined, 9 of which were significantly differentially expressed miRNAs (SDEMs), and 241 target genes of DEMs were predicted. GO annotation and KEGG pathway analysis were carried out to understand the role of the DEMs. Out of 287 significantly enriched GO terms, 37 were stress- or immune-related, such as response to light stimulus, detection of oxidative stress, and immune response in mucosal-associated lymphoid tissue. Out of 85 KEGG pathways, 8 were related to stress or immunity, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, and RLR signaling pathway. We then constructed the interaction networks between target genes from immune-related pathways and their DEMs. The analysis results suggested that some DEMs (gga-miR-17 family, gga-miR-15/16 family, gga-miR-2954 and gga-miR-34b-5p) and target genes (SIKE1, CX3CL1, IL11Ra, PIGR, and CDKN1A) were core miRNAs and genes. This study revealed the dynamic miRNA transcriptome, target genes and related pathways in chicken spleen under CORT-induced stress model, which provided a basis for studying the molecular mechanism of stress affecting immune function.

Molecular Response in Intestinal and Immune Tissues to in Ovo Administration of Inulin and the Combination of Inulin and Lactobacillus lactis Subsp. cremoris.

Intestinal microbiota are a key factor in maintaining good health and production results in chickens. They play an important role in the stimulation of immune responses, as well as in metabolic processes and nutrient digestion. Bioactive substances such as prebiotics, probiotics, or a combination of the two (synbiotic) can effectively stimulate intestinal microbiota and therefore replace antibiotic growth promoters. Intestinal microbiota might be stimulated at the early stage of embryo development in ovo. The aim of the study was to analyze the expression of genes related to energy metabolism and immune response after the administration of inulin and a synbiotic, in which lactic acid bacteria were combined with inulin in the intestines and immune tissues of chicken broilers. The experiment was performed on male broiler chickens. Eggs were incubated for 21 days in a commercial hatchery. On day 12 of egg incubation, inulin as a prebiotic and inulin with Lactobacillus lactis subsp. cremoris as a synbiotic were delivered to the egg chamber. The control group was injected with physiological saline. On day 35 post-hatching, birds from each group were randomly selected and sacrificed. Tissues (spleen, cecal tonsils, and large intestine) were collected and intended for RNA isolation. The gene panel (ABCG8, HNF4A, ACOX2, APBB1IP, BRSK2, APOA1, and IRS2) was selected based on the microarray dataset and biological functions of genes related to the energy metabolism and immune responses. Isolated RNA was analyzed using the RT-qPCR method, and the relative gene expression was calculated. In our experiment, distinct effects of prebiotics and synbiotics following in ovo delivery were manifested in all analyzed tissues, with the lowest number of genes with altered expression shown in the large intestines of broilers. The results demonstrated that prebiotics or synbiotics provide a potent stimulation of gene expression in the spleen and cecal tonsils of broiler chickens. The overall number of gene expression levels and the magnitude of their changes in the spleen and cecal tonsils were higher in the group of synbiotic chickens compared to the prebiotic group.

Assay of extracellular matrix degradation and transmigration of chicken peripheral blood mononuclear cells after infection with genotype VII Newcastle disease virus in vitro

Previous studies showed that, compared to genotype IV Newcastle disease virus (NDV), genotype VII NDV induced extensive extracellular matrix (ECM) degradation by up-regulating the protein expression of matrix metalloproteinase (MMP)-14 in chicken spleens. To investigate potential relationship between MMP-14 function and the ECM degradation, an in vitro peripheral blood mononuclear cells (PBMCs) infection model was established to study the effect of genotype VII NDV (JS5/05) infection on MMP-14 expression, ECM degradation and cell transmigration. The gene and protein expression levels of MMP-14 in NDV-infected chicken PBMCs were measured by quantitative real-time PCR (qRT-PCR) and Western blot, and the subcellular location of MMP-14 was analyzed using immunofluorescence microscopy. A fluorescence-based collagen degradation assay was optimized to measure ECM degradation in PBMCs. Additionally, parameters of a transwell-based transmigration assay were also optimized to determine chemotaxis and transmigration of virus-infected PBMCs. The results showed that JS5/05 up-regulated significantly the expression of MMP-14 in PBMCs at the mRNA and protein levels compared to genotype IV NDV (Herts/33). MMP-14 was transported towards the membrane and accumulated on the cell surface of the JS5/05-infected cells, whereas it remained mainly in the cytoplasm of the Herts/33-infected cells. Collagen degradation assay showed that JS5/05-infected cells exhibited significant collagen degradation compared to the Herts/33-infected cells, and the areas of collagen degradation co-localized with cell surface MMP-14 in the JS5/05-infected cells. The transwell-based transmigration system showed that the transmigration of the JS5/05-infected PBMCs was enhanced significantly compared to the Herts/33-infected cells. These results demonstrated that genotype VII NDV induced up-regulation and surface accumulation of MMP-14 in PBMCs, leading to enhanced ECM degradation and cell migration, and the assays optimized for this study were useful for investigating the regulation of cell behaviour by NDV.

Baseline Immune Cell Composition in Broiler Spleen is Altered by Probiotic Supplementation

Probiotics are supplemented in poultry diets to support beneficial intestinal microbial community members. The alteration of the microbiota due to probiotic supplementation is shown to improve host health, but the underlying changes to immune cell populations have not been well-described in poultry. The objective of this study was to examine the effects of probiotics at two different dietary inclusion levels on the immune cell populations in spleens of unchallenged broiler chickens. Probiotic supplementation did not impact overall percentages of leukocytes, but did alter the composition of cells making up leukocyte subpopulations (<i>P</i>&nbsp;≤ 0.05). Additional research is needed to evaluate how these changes to immune cell populations translate to a modulated immune response under challenge conditions.

The Modulating Effect of Dietary Beta-Glucan Supplementation on Expression of Immune Response Genes of Broilers during a Coccidiosis Challenge

Simple SummaryAvian coccidiosis is the leading parasitic disease in the poultry industry and means to control its damages continue to be explored. This study evaluated the feeding effects of a yeast-derived β-glucan on expression of immune response genes in the spleen, thymus, and bursa of commercial broiler chickens during an Eimeria challenge. The study consisted of two dietary treatments (0% or 0.1% β-glucan) each with or without a coccidiosis challenge. There were significant effects from dietary β-glucan, Eimeria challenge, and their interaction for several gene targets in the spleen, thymus, and bursa on days 10 and 14 of age. Based on the current results, supplementation of dietary β-glucan in Eimeria-challenged birds enhanced and modulated the expression of immune response genes during coccidiosis.This study investigated the effects of a yeast-derived β-glucan (Auxoferm YGT) supplementation on mRNA expression of immune response genes in the spleen, thymus, and bursa of broiler chickens during a mixed Eimeria infection. Day (d)-old chicks (n = 1440) were fed diets containing 0% or 0.1% YGT. On d 8 post-hatch, half the replicate pens (n = 8) were challenged with a mixed inoculum of E. acervulina, E. maxima, and E. tenella. On d 10 and d 14 post-hatch, the spleen, thymus, and bursa were collected to evaluate mRNA abundance by quantitative real-time PCR. Data were analyzed using PROC GLIMMIX model (2-way interaction) and differences were established by LS-MEANS with significance reported at p ≤ 0.05. In spleen tissues at d 10, expression of interleukin (IL)-10 and inducible nitric oxide synthase (iNOS) were elevated in both 0.1% YGT-fed challenged and non-challenged birds. In thymus tissues at d 14, expression of IL-10, IL-17F, interferon (IFN)-γ, iNOS, and macrophage migration inhibitory factor (MIF) were elevated in challenged birds fed 0.1% YGT. In bursal tissues at d 10 and d 14, expression of IL-10, IFN-γ, iNOS (d 10 only), and MIF were elevated in 0.1% YGT-fed challenged and non-challenged birds. Dietary β-glucan supplementation to chicken diets modulated their immune response to the Eimeria challenge.

Gga-miR-148a-5p-Targeting PDPK1 Inhibits Proliferation and Cell Cycle Progression of Avain Leukosis Virus Subgroup J (ALV-J)-Infected Cells.

Avian leukosis virus subgroup J disease (ALV-J) is a contagious and immunosuppressive avian disease caused by ALV-J virus. Although miRNA participate in various biological processes of tumors, little is known about the potential role of miRNA in ALV-J. Our previous miRNA and RNA sequencing data showed that the expression of gga-miR-148a-5p was significantly different in ALV-J-infected chicken spleens compared with non-infected chickens. The aim of this study was to investigate the functional roles of gga-miR-148a-5p and identify downstream targets regulated by gga-miR-148a-5p in ALV-J-infected chickens. We found that the expression of gga-miR-148a-5p was significantly downregulated during ALV-J infection of chicken embryo fibroblasts (CEF). Dual luciferase reporter assays demonstrated that PDPK1 is a direct target gene of gga-miR-148a-5p. In vitro, overexpression of gga-miR-148a-5p significantly promoted ALV-J-infected CEF cell proliferation, included cell cycle, whereas inhibition of gga-miR-148a-5p had an opposite effect. Inhibition of PDPK1 promoted the proliferation of ALV-J-infected cells but had no effect on the activity of NF-κB. Together, these results suggested that gga-miR-148a-5p targets PDPK1 to inhibit the proliferation and cell cycle of ALV-J-infected CEF cells. Our study provides a new understanding for the tumor mechanism of ALV-J infection.

Developmental and Tissue Patterns of the Basal Expression of Chicken Avian β-Defensins.

Defensins are a class of antimicrobial peptides in vertebrates that function as the first line of innate immunity with potent antimicrobial and immunomodulatory activities. Fourteen defensins, namely, avian β-defensin 1 to 14 (AvBD1-14), have been identified in chickens. Before characterizing the role of AvBDs in innate immunity during the early development of chickens, we collected tissue segments from the liver, spleen, and gastrointestinal (GI) tract including the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon from broilers at days 1, 3, 7, 14, and 28. After RNA isolation and reverse transcription, we determined the expression levels of the 14 AvBD genes in these tissues during the first 28 days after hatching by real-time PCR. The results suggested the AvBDs were widely expressed in the chicken liver, spleen, and gastrointestinal (GI) tract. Interestingly, we did not detect AvBD11 expressed in the GI tract, even in the liver and spleen. Additionally, AvBDs were differentially expressed in the chicken GI tract. AvBD5 and AvBD14 were expressed most abundantly in the proximal GI tract, especially the esophagus and crop. Moreover, AvBD5, AvBD7, AvBD9, and AvBD14 were expressed in an inverted-V pattern with the peak being the observed expression at days 3, 7, or 14 in the chicken spleen, esophagus, duodenum, and cecum. Other AvBDs presented biphasic or inverted-V expression patterns in different tissues. The expression levels of all detected AvBDs were strengthened after hatching rather than decreasing steadily. Therefore, AvBDs were found to be expressed widely in the chicken liver, spleen, and GI tract and their expression levels were primarily up regulated during the early development of chicken, implying the potential essential roles of AvBDs in early innate defense and infection resistance of chickens.

The role of Meq-vIL8 in regulating Marek's disease virus pathogenesis.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

Effects of ambient temperature on body size and organ development in broilers

The current study investigated the effect of poultry house temperature change on the growth and development of both broiler chickens themselves and the development of their individual organs. Following 42 days of rearing, results showed that the body oblique length, chest width, chest depth, chest angle, keel length, pelvic width, and tibial length of broilers in the low temperature group were significantly lower than those of the control group (P&lt;0.05), while the breast depth of broiler chickens in the high-temperature group was significantly lower than those in the control group (P&lt;0.05). Furthermore, low temperatures significantly increased the function of the heart, liver, spleen and pancreas of broiler chickens (P&lt;0.05). After 7 days of rearing, the bursal index of broilers in the high temperature group was significantly higher than in the control group as well as in the low temperature group (P&lt;0.05). Previous research has established that high and low temperatures significantly affect the growth and development as well as the immunity of broiler chickens, namely that low environmental temperatures have a more adverse effect on broiler chickens than high temperatures. Consequently, using and maintaining high temperatures early in rearing helps to improve the immunity of broilers and improve their performance. The results of our study provides an opportunity to provide both a theoretical and a practical basis for accurate temperature setting in poultry houses for effective breeding of broiler chickens, which will make it possible to increase the productivity of broiler chickens and increase the economic efficiency of the poultry house. Key words: temperature, breeding, body weight, organ development, broilers chickens.

Effect of β-mannanase enzyme supplementation on the morphofunctional state of broiler chickens’ immunocompetent organs

Identifying and eliminating factors that interfere with the nutrient absorption from feed is essential for successful poultry rearing. The nutritional value of feed mixtures used in the poultry industry can be increased by the use of enzyme supplements, in particular β-mannanase (Hemicell), which breaks down the anti-nutrients β-mannans that are present in significant quantities in soy products. For the study, two groups of 50 broiler chickens were formed. In the control group a standard feed mixture from cereals was used, particularly soybeans, and in the experimental group 0.05% β-mannanase enzyme supplement (Hemicell) in addition to the compound feed was used. Production indicators were established (average daily gain, survival rate, feed costs). On the 15th, 25th and 42nd days of rearing, lymphatic organs (cloacal bursa and spleen) were collected from seven chickens of each group, followed by macro, microscopic and morphometric evaluation. It was found that the addition of an enzyme supplement promotes an increase in the production indicators of poultry rearing, reduces feed use, does not affect the macroscopic characteristics of the lymphatic organs. β-mannanase (Hemicell) contributes to the maintenance of an active morphofunctional state of the cloacal bursa and spleen during the entire period of poultry rearing. This was manifested by high indices of the absolute and relative organ masses, a moderate development degree of tissue components and cloacal bursa histological structures (number, shape and average length of lymphatic nodules, density of lymphocytes in the cortex and medulla) and spleen (number and diameter of lymphatic nodules and periarterioal lymphatic sheaths). In the control group, at all stages of rearing, the morphofunctional state of the bursa was found to be lower and signs of immunosuppression were revealed by the end of the rearing period (a sharp decline in the bursa absolute mass, organ mass index, a decrease in the average length of nodules, change in their shape, thinned cortical layer and delymphotisation). The morphofunctional state of the spleen of chickens from the control group remained high throughout the entire study period. So use of the β-mannanase enzyme supplement (Hemicell) while rearing broiler chickens not only increased the efficiency of their rearing but also contributed to maintaining a high morphological and functional state of the cloacal bursa.