Marek's disease virus-induced immunosuppression: array analysis of chicken immune response gene expression profiling.

Marek's disease (MD) is a lymphoproliferative disease of chickens induced by a highly cell-associated oncogenic alpha-herpesvirus, Marek's 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.

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

Resistance to Marek's 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.

Expression profiles of genes in Toll-like receptor-mediated signaling of broilers infected with Clostridium perfringens.

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.

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

BACKGROUND: Clostridium 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. RESULTS: Use 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, TCR gamma, 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. CONCLUSION: Broiler 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 Marek's 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.

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.

Construction of a microarray specific to the chicken immune system: profiling gene expression in B cells after lipopolysaccharide stimulation.

The objective of this study was to profile gene expression in cells of the chicken immune system. A low-density immune-specific microarray was constructed that contained genes with known functions in the chicken immune system, in addition to chicken-expressed sequence tags (ESTs) homologous with mammalian immune system genes, which were systematically characterized by bioinformatic analyses. Genes and ESTs that met the annotation criteria were amplified and placed on a microarray. The microarray contained 84 immune system gene elements. As a means of calibration, the microarray was then used to examine gene expression in chicken B cells after lipopolysaccharide stimulation. Differential gene expression was observed at 6, 12, and 24 h but not at 48 h after stimulation. The results were validated by semiquantitative polymerase chain reaction. The microarray showed a high degree of reproducibility, as demonstrated by intra- and interassay correlation coefficients of 0.97 and 0.95, respectively. Thus, the low-density microarray developed in this study may be used as a tool for monitoring gene expression in the chicken immune system.