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 Marek's 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.

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.

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

Marek's disease virus (MDV) is a cell-associated oncogenic herpesvirus that targets B cells and T cells, inducing lymphoid tumours in chickens. Genetic resistance to Marek's 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=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.

Host responses are induced in feathers of chickens infected with Marek's disease virus.

Control measures are ineffective in curtailing Marek's disease virus (MDV) infection and replication in the feather follicle epithelium (FFE). Therefore, vaccinated birds which subsequently become infected with MDV, shed the virulent virus although they remain protected against disease. The present study investigated host responses generated against MDV infection in the feather. We observed that in parallel with an increase in viral genome load and viral replication in the feather, there was a gradual but progressive increase in infiltration of CD4+ and CD8+ T cells into the feather pulp of MDV-infected chickens, starting on day 4 and peaking by day 10 post-infection. Concomitant with infiltration of T cells, the expression of interleukin (IL)-18, IL-6, interferon (IFN)-gamma and major histocompatibility complex class I genes was significantly enhanced in the feather pulp of MDV-infected chickens. The finding that host responses are generated in the feather may be exploited for developing strategies to control MDV infection in the FFE, thus preventing horizontal virus transmission.

Marek's disease virus-induced transient paralysis is associated with cytokine gene expression in the nervous system.

Marek's disease (MD)-associated transient paralysis (TP) was experimentally induced in chickens by intraperitoneal inoculation of RB1B strain of Marek's disease virus (MDV). Between 7 and 11 days post-infection (d.p.i.), neck and limb paralysis was observed in 18% of infected chickens, which was associated with various degrees of edema, vacuolation, perivascular cuffing of mononuclear cells, and glial cell infiltration mainly in the cerebrum, cerebellum, and brain stem. The chickens that were infected but did not progress to develop TP until 12 d.p.i. also had similar lesions suggestive of encephalitis in the cerebrum, cerebellum, and brain stem. Chickens infected with MDV had more interleukin (IL)-6, IL-12, and interferon (IFN)-gamma in their brain tissues compared to uninfected chickens. Moreover, IL-18 was significantly increased in brain tissues of birds showing clinical signs of TP compared to uninfected birds. Importantly, the expression of IL-6, IL-18, and IFN- gamma in brain tissues of MDV-infected chickens with signs of TP was significantly increased compared to that in asymptomatic MDV-infected birds. MDV genome load in the brain of chickens showing clinical signs of TP was higher than that in asymptomatic MDV-infected chickens but was not statistically significant. The lesions in the cervical, thoracic, and lumbar spinal cord segments in MDVinfected chickens were characterized mainly by perivascular cuffing of mononuclear cells irrespective of the group. The expression of mRNA for IL-18 and IFN-gamma genes was not significantly different in spinal cord tissues of chickens with TP compared to clinically normal, MDV-infected and noninfected chickens. These results suggest possible underlying immunologic mechanisms for MDV-induced TP.

Transcriptional analysis of host responses to Marek's disease viral infection.

This study was aimed at investigating the genes that control host responses to Marek's disease virus (MDV). Spleen tissues from infected and age-matched uninfected control chickens were examined 4, 7, 14, and 21 d postinfection for gene expression differences, using both microarray and quantitative real-time polymerase chain reaction (PCR) methodologies. Up to 51% of genes assayed during microarray analysis showed a significant change (p < or = 0.05) in expression after MDV infection, of which cell surface molecules, transcription and signal transduction molecules, housekeeping and metabolism mediators, and cytokines and cytokine receptors were most commonly differentially expressed. Setting a fold change threshold (> or =2), 14 of 84 genes showed differential expression over time. In addition, some genes showed differential expression at more than one time point. For example, the granzyme-A homolog gene remained upregulated in infected chickens, with fold changes of 7.98, 13.91, and 9.07 at 7, 14, and 21 d postinfection, respectively. Other genes that were differentially expressed at more than one time point were invariant chain, IgM, and CD3. Quantitative real-time PCR analysis was used to validate microarray results for a subset of genes showing a :2-fold change in expression. Expression of all but one gene (CD28) was confirmed. Identification of genetic mechanisms initiated by in vivo infection with MDV expands the current understanding of immune response to the virus in addition to host response elements associated with viral pathogenesis.