Aimie J. Sarson

Transcriptional analysis of host responses to Marek’s disease virus infection in genetically resistant and susceptible chickens

Mareks disease virus (MDV) is a cell-associated oncogenic herpesvirus that targets B cells and T cells, inducing lymphoid tumours in chickens. Genetic resistance to Mareks disease (MD) is regulated in a polygenic fashion. In this study, we sought to compare the gene expression profiles following infection of birds that are genetically resistant or susceptible to MD (with the B21 and B19 haplotypes respectively at the MHC locus), including comparisons to uninfected controls. On days 4, 7, 14 and 21 post-infection, gene expression profiles in spleen tissue were obtained using a chicken immune-specific microarray. A number of genes showed significant (P <or= 0.05) differential expression across time and treatments. These included the chemokine AH221, B-cell marker Bu-1, IgG, IgA, IgM, MHC class II beta chain, granzyme A (GZMA) and signal transducers and activators of transcription 2 (STAT2) genes. In several comparisons, genes such as GZMA and STAT2 were induced in infected birds regardless of their genetic background. However, only immunoglobulin genes were differentially expressed by >or=2-fold in resistant compared with susceptible infected chickens. IgM and IgG were significantly induced on day 7 post-infection in susceptible chickens compared to resistant birds, whereas both of these genes were repressed in susceptible birds on day 14 post-infection. Overall, gene expression profiles in the chicken spleen observed after MDV infection were dependent on time and host genetic background. These gene expression profiles provide a platform for defining novel candidate genes for resistance or susceptibility to MD.

Marek's Disease Virus–Induced Immunosuppression: Array Analysis of Chicken Immune Response Gene Expression Profiling

Mareks disease (MD) is a lymphoproliferative disease of chickens induced by a highly cell-associated oncogenic alpha-herpesvirus, Mareks disease virus (MDV). MDV replicates in chicken lymphocytes and establishes a latency infection within CD4(+) T cells. Host-virus interaction, immune responses to infection, and transcriptional profiling of chicken gene expression in MD are poorly understood. In this study we conducted a global host gene expression analysis in the splenocytes of MDV-infected chickens using oligonucleotide-based Affymetrix GeneChip Chicken Genome Arrays. These arrays contain probes for more than 32,000 chicken transcripts and most of the known MDV genes and open reading frames. Two-week-old MD-susceptible chickens were inoculated with an oncogenic strain of MDV, and spleen samples were collected 5 and 15 days post-infection (dpi) for RNA isolation and microarray analysis. Array results displayed a significant differential pattern of immune response transcriptome between the two phases of MDV infection. The expression levels of more than 22 immune-response and related genes were downregulated, while the expression levels of at least 58 genes were increased at 5 dpi (cytolytic infection), compared to age-matched control birds. In comparison, out of 73 immune-response and related genes, 67 genes were downregulated, with only 6 genes having higher expression levels at 15 dpi (latency infection). Cytokines, chemokines, MHC molecules and related receptors, and adhesion molecules were among the many MDV-induced downregulated genes that are critical for an effective antiviral immune response. In addition, several apoptosis-associated genes were decreased in expression during latent infection, suggesting an MDV-induced blocking of initiation or progression of programmed cell death processes. These chicken arrays are valuable tools in understanding the molecular mechanisms behind viral pathogenesis and chicken gene expression patterns, and associated biological pathways in response to MDV infection.

Cytokine gene expression in splenic CD4+ and CD8+ T cell subsets of genetically resistant and susceptible chickens infected with Marek's disease virus

T cells from the spleens of B(19)/B(19) and B(21)/B(21) chickens infected with MDV JM-16 strain were fractionated by flow cytometry at 4, 10, 21 days post infection (d.p.i.). The expression of cytokine and viral genes (meq and glycoprotein B (gB)) was measured by real-time RT-PCR. It was determined that CD4(+) and CD8(+) T cells had both become infected with Mareks disease virus (MDV) in both chicken lines. There was significantly higher expression of meq in CD4(+) T cells compared to CD8(+) T cells at 10 and 21 d.p.i. Furthermore, at 10 and 21 d.p.i., there was a tendency for higher expression of meq in both T cell subsets of B(19) chickens compared to those of B(21) chickens. There were temporal changes in the expression of cytokines, interferon (IFN)-gamma, interleukin (IL)-18, IL-6, and IL-10, in various T cell subsets. Among these changes, there was an increase in IL-10 expression in both T cell subsets at different time points, especially in the susceptible line at 10 and 21 d.p.i. Our results indicate that cytokines could be differentially induced by MDV in CD4(+) and CD8(+) T cell subsets and that IL-10 may play a role in the modulation of immune response to MDV. However, an association between cytokine gene expression in T cell subsets and resistance or susceptibility to MD was not established.

Expression Profiles of Genes in Toll-Like Receptor-Mediated Signaling of Broilers Infected with Clostridium perfringens

ABSTRACT Toll-like receptors (TLRs) participate in detecting microbial pattern molecules for activation of the host immune response. We investigated possible roles of TLRs in the chicken response to Clostridium perfringens infection by examining the expression of TLR genes and other genes involved in TLR-mediated signaling within the spleens and ilea of C. perfringens-challenged broilers. Upregulation of a tumor necrosis factor alpha-inducing factor homolog in challenged chickens compared to naïve chickens was observed, regardless of the incidence of necrotic enteritis. In addition, the members of the TLR2 subfamily were found to be most strongly involved in the host response to C. perfringens challenge, although the expression of TLR4 and TLR7 was also upregulated in spleen tissues. While the combination of TLR1.2, TLR2.1, and TLR15 appeared to play a major role in the splenic response, the expression of TLR2.2 and TLR1.1 was positively correlated to the expression of adaptor molecules MyD88, TRAF6, TRIF, and receptor interacting protein 1 in the ileal tissues, demonstrating a dynamic spatial and temporal innate host response to C. perfringens.

Molecular cloning and expression analysis of chicken MyD88 and TRIF genes

Toll-like receptors (TLRs) trigger the innate immune system by responding to specific components of microorganisms. MyD88 and TRIF are Toll/interleukin (IL)-1 (TIR)-domain containing adapters, which play essential roles in TLR-mediated signalling via the MyD88-dependant and -independent pathways, respectively. Genes encoding several TLRs have been identified in the chicken genome, however, elements of their signalling pathways have not been well characterized. Here we describe the cloning of chicken MyD88 and TRIF orthologs, and examine the spatial and temporal expression of these genes. The chicken MyD88 cDNA was shown to have an open reading frame (ORF) of 1104 bp, encoding a predicted protein sequence of 368 aa, 8 aa short of a previously published coding sequence due to a premature stop codon. MyD88 gene expression was detected in each tissue tested except in muscle. The chicken TRIF cDNA possessed an ORF of 2205 bp, encoding a predicted protein sequence of 735 aa, which shared 37.3% similarity and 28.9% identity to human TRIF protein sequence. TRIF was ubiquitously expressed in all tissues.

Gene expression profiling within the spleen of Clostridium perfringens-challenged Broilers fed antibiotic-medicated and non-medicated diets

BackgroundClostridium perfringens (Cp) is a Gram-positive anaerobic bacterium that causes necrotic enteritis (NE) in poultry when it overgrows in the small intestine. NE disease has previously been controlled through the use of growth-promoting antibiotics. This practice was recently banned in European countries, leading to significantly increased incidence of NE threatening the poultry industry. Control strategies and technology as substitutes to dietary antibiotics are therefore urgently required. To develop the substitutes, it is important to understand host immune responses to Cp infection. However, the knowledge is still lacking. We therefore investigated gene expression profiles within immunologically-relevant tissue, the spleen, in order to identify factors that are involved in immunity to NE and have potential as therapeutic targets.ResultsUse of a 44 K Agilent chicken genome microarray revealed significant up-regulation of many immune-associated genes in Cp-challenged chickens, including galectin 3, IFNAR1, IgY-receptor, TCRγ, granzyme A, and mannose-6-P-R, which were subsequently validated by quantitative PCR assays. Functional annotation of differentially expressed genes was conducted using the High Throughput Gene Ontology Functional Annotation database. Medicated and Non-medicated chickens had similar annotation profiles with cell activities and regulation being the most dominant biological processes following Cp infection.ConclusionBroiler chickens demonstrated an intricate and holistic magnitude of host response to Cp challenge and the development of NE. Although the influence of dietary antibiotics appeared to be less significant than the disease process, both had a considerable impact on the host response. Markers previously identified in intestinal inflammatory diseases of other species, including humans, and indicators of enhanced antibody responses, appeared to be involved in the chicken response to Cp challenge. The significance in host immune responses of immune mediators identified from the present study warrants further studies to verify their functions during NE development and to determine their potential application to control NE disease.

Expression of cytotoxicity-associated genes in Marek's disease virus-infected chickens.

Cytotoxic host responses to Mareks disease virus (MDV) have been attributed to both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). However, the mechanisms of cell lysis initiated by these cytotoxic responses during MDV infection are not well defined. Therefore, the current study was aimed at elucidating the molecular mechanisms of host cytotoxic responses to MDV infection by investigating the expression of genes in the cell lysis pathway involving granzyme A. Genes encoding cytolytic proteins, NK lysin, and granzyme A were upregulated during early stages of infection, whereas the genes encoding major histocompatibility complex (MHC) class I and the DNA repair and apoptosis protein, poly(ADP-ribose) polymerase (PARP), were downregulated. These findings shed more light on the mechanisms of host response to MDV infection in chickens.

Transcriptional profiling analysis of host response to Clostridium perfringens infection in broilers

Necrotic enteritis is a disease caused by Clostridium perfringens, which threatens poultry production in the absence of dietary antibiotics. A total number of 600 Ross broilers were reared in 12 pens with each hosting 50 birds. Each 6 pens of birds were fed medicated (bacitracin at 55 mg/kg) or nonmedicated starter diets immediately after the chicks were placed. At d 18, birds were challenged with C. perfringens (10(7) cfu/mL mixed with feed). Spleens were collected from 12 birds of each group (2 birds per pen randomly) at d 18 (before infection), 19, 20, and 22. A low-density chicken immune microarray was used to study gene expression profiling of host response to C. perfringens infection. Six biological replicates (2 birds per biological replicate) for each treatment group were labeled with either Cy5 or Cy3 with dye swap. A total of 24 arrays were used for this study. Gene signal intensity was globally normalized by locally weighted regression and smoothing scatter plots and expressed on a natural log scale. A mixed model including treatment, time, array, subgrid (random effect), dye, and all interactions among treatment and time was used to identify differentially expressed genes between postinfection vs. preinfection, among postinfections, and between medication treatments, at the 5% significance level. The results indicated subtle medication effects on gene expression of these immune-related genes compared with bacterial infection effect. Our findings strongly suggest that both cell-mediated and antibody-mediated immune responses via MHC class I and II systems were actively involved in the host defense against C. perfringens infection in broilers. The unique cytokine signaling pathway and apoptosis cascade found in the study provide a new insight of molecular regulation of host immune response. Collectively, the findings of the present study will shed light on the molecular mechanisms underlying C. perfringens infection in broilers.

Proteomic analysis of host responses to Marek's disease virus infection in spleens of genetically resistant and susceptible chickens.

Resistance to Mareks disease (MD) in chickens is genetically regulated and there are lines of chickens with differential susceptibility or resistance to this disease. The present study was designed to study comparative changes in the spleen proteomes of MD-susceptible B19 and MD-resistant B21 chickens in response to MDV infection. Spleen proteomes were examined at 4, 7, 14 and 21 days post-infection (d.p.i.) using two-dimensional gel electrophoresis and subsequently the protein spots were identified by one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). On average, there were 520+/-27 distinct protein spots on each gel and 1.6+/-0.7% of the spots differed quantitatively in their expression (p< or =0.05 and fold change > or =2) between infected B19 and B21 chickens. There was one spot at 4d.p.i. and three spots each at the rest of the time points, which had a qualitative difference in expression. Most of the differentially expressed proteins at 4 and 7d.p.i. displayed increased expression in B21 chickens; conversely the differentially expressed proteins at 14 and 21d.p.i. showed an increase in expression in B19 chickens. The differentially expressed proteins identified in the present study included antioxidants, molecular chaperones, proteins involved in the formation of cytoskeleton, protein degradation and antigen presentation, signal transduction, protein translation and elongation, RNA processing and cell proliferation. These findings shed light on some of the underlying processes of genetic resistance or susceptibility to MD.

Association of a Marker in the Vitamin D Receptor Gene with Marek’s Disease Resistance in Poultry

Vitamin D is an important immunomodulator that mediates its effect via a nuclear receptor. In this study, we analyzed 3 uncorrelated genetic markers (tag single nucleotide polymorphisms) in the vitamin D receptor gene for association with Mareks disease (MD) resistance. The database consisted of 400 commercial White Leghorn chickens that had been vaccinated with herpes turkey virus and challenged by intraperitoneal injection of the virulent MD virus RB1B. Viral titers in feather tips were determined at weekly intervals for 8 wk, mortality was recorded, and necropsy analyses preformed on all chickens. The 3 genotypes defined by 1 of the markers were associated with significant differences in the viral load (integration of the viral titer over time; P = 3 x 10(-4)). The effect was additive, with the 2 homozygotes differing by a factor of 2. The ranking of the genotypes by viral load, frequency of MD lesions, mortality, and bursal atrophy were consistent. There was no effect on the tissue distribution of MD lesions. The degree of MD resistance in the 9 genotypes defined by the 3 tag single nucleotide polymorphisms was proportional to the frequency of major histocompatibility complex class II-positive peripheral blood leukocytes that had been previously measured in uninfected chickens in a different database.