Cytokine Gene Expression in Lung Mononuclear Cells of Chickens Vaccinated with Herpesvirus of Turkeys and Infected with Marek's Disease Virus.

Marek's disease virus (MDV) enters the chicken host through the respiratory system. However, little is known about the host immune responses induced by MDV in the lungs. To characterize these responses, chickens were vaccinated with herpesvirus of turkeys (HVT) and challenged with the RB1B strain of MDV via the respiratory route. Lung mononuclear cells of vaccinated only, challenged only, vaccinated and challenged, as well as age-matched controls were isolated at 4, 10, and 21 days post-infection. Real-time quantitative reverse transcription polymerase chain reaction was used to assess the expression of various cytokines. There was significant upregulation of interferon (IFN)-γ and interleukin (IL)-10 in lung mononuclear cells of HVT-vaccinated and RB1B challenged and unvaccinated and RB1B challenged chickens. However, in lung mononuclear cells isolated from chickens that were vaccinated with HVT but remained uninfected, there was an upregulation of IL-4 and IL-13. This study indicates that MDV- and HVT-associated cytokines expressed by lung mononuclear cells are temporally regulated and that these cytokines may be involved in immunity against the virus.

Avian influenza virus vaccines containing Toll-like receptors 2 and 5 ligand adjuvants promote protective immune responses in chickens.

Vaccination remains a useful means for the control of avian influenza viruses (AIV) in chickens. Current vaccines can protect chickens from morbidity and mortality. However, they do not eliminate virus shedding into the environment. Therefore, novel measures must be considered in order to enhance the immunogenicity of AIV vaccines, such as through the administration of immunostimulatory compounds. One such group of compounds is Toll-like receptor (TLR) ligands, such as bacterial flagellin, as well as synthetic lipopeptides such as Pam3CSK4. The objective of the present study was to assess the adjuvant potential of TLR2 and TLR5 ligands flagellin and Pam3 respectively. Chickens were vaccinated twice with an inactivated H4N6 AIV vaccine, 14 days apart. Antibody-mediated responses were assessed in sera and lacrimal secretions, while cell-mediated immune response was assessed by stimulating splenocytes from vaccinated chickens in vitro with the vaccine antigen. To evaluate vaccine efficacy, chickens were challenged with the H4N6 virus, and virus shedding was assessed on day 7 post-challenge. The results suggest that both ligands significantly enhanced antigen-specific IgY antibodies, while only the Pam3 adjuvant induced greater IgM and IgA antibody levels. Chickens receiving the flagellin adjuvant had significantly higher IgY responses, as well as significantly higher hemagglutination-inhibition antibody titers compared to the no adjuvant control. With respect to cell-mediated responses, splenocytes isolated from chickens that received either TLR ligand adjuvant proliferated in response to an in vitro stimulation with vaccine antigens. Lastly, chickens receiving vaccines containing either flagellin or Pam3 adjuvants were partially protected from an experimental AIV challenge and shed significantly less virus compared to controls. Future studies may be aimed at examining the efficacy of Pam3 and flagellin adjuvants for highly pathogenic AIV strains.

The effects of administration of ligands for Toll-like receptor 4 and 21 against Marek's disease in chickens.

Ligands for Toll-like receptors (TLRs) are known to stimulate immune responses, leading to protection against bacterial and viral pathogens. Here, we aimed to examine the effects of various TLR ligands on the development of Marek's disease in chickens. Specific-pathogen free chickens were treated with a series of TLR ligands that interact with TLR3, TLR9 and TLR21. In a pilot study, it was determined that TLR4 and TLR21 ligands are efficacious, in that they could reduce the incidence of Marek's disease tumors in infected birds. Hence, in a subsequent study, chickens were treated with lipopolysaccharide (LPS) as a TLR4 and CpG oligodeoxynucleotides (ODN) as TLR21 agonists before being challenged with the RB1B strain of Marek's disease virus (MDV) via the respiratory route. The results demonstrated that the administration of LPS or CpG ODN, but not PBS or non-CpG ODN, delayed disease onset and reduced MDV genome copy number in the spleens of infected chickens. Taken together, our data demonstrate that TLR4 and 21 agonists modulate anti-virus innate immunity including cytokine responses in MD-infected chicken and this response can only delay, but not inhibit, disease progression.

A toll-like receptor 3 ligand enhances protective effects of vaccination against Marek's disease virus and hinders tumor development in chickens.

Marek's disease (MD) is caused by Marek's disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.

Vaccination with CpG-adjuvanted avian influenza virosomes promotes antiviral immune responses and reduces virus shedding in chickens.

The use of virosomes as a vaccine platform has proven successful against several viruses. Here we examined the protective efficacy of a virosome-based vaccine consisting of avian influenza virus (AIV) A/Duck/Czech/56/H4N6 in chickens against a homologous AIV challenge. Virosomes adjuvanted with CpG-ODN or recombinant chicken interferon (IFN)-γ significantly reduced virus shedding after virus challenge. Furthermore, immunization with virosomes adjuvanted with CpG-ODN increased hemagglutination inhibition (HI) and virus-specific neutralizing serum antibodies, as well as virus-specific serum IgG and mucosal IgA responses. We also found a significant increase in the expression of type I and II interferon genes in the protected birds following virus challenge. In summary, this study demonstrated the ability of virosomes adjuvanted with CpG-ODN to reduce AIV shedding, and elicit virus-specific protective antibody responses in vaccinated birds.

Prophylactic treatment with Toll-like receptor ligands enhances host immunity to avian influenza virus in chickens.

Avian influenza viruses (AIV) pose a threat towards the health of both poultry and humans. To interrupt the transmission of the virus, novel prophylactic strategies must be considered which may reduce the shedding of AIV. One potential is the prophylactic use of Toll-like receptor (TLR) ligands. Many cells of the immune system express TLRs, and cellular responses to TLR stimulation include activation and the production of cytokines. TLR ligands have been employed as prophylactic treatments to enhance host resistance to pathogens both in mammals and chickens. Therefore, the present study was conducted to determine whether TLR ligands may be used prophylactically in chickens to enhance host immunity to AIV. Chickens received intramuscular injections of either low or high doses of the TLR ligands poly I:C, lipopolysaccharide (LPS) and CpG ODN. Twenty-four hours post-treatment, chickens were infected with the low pathogenic avian influenza virus H4N6, and both oropharyngeal and cloacal virus shedding were assessed on days 4 and 7 post-infection. To identify potential correlates of immunity, spleen and lungs were collected on days 2, 4 and 7 post-infection for RNA extraction. The results suggested that all of the TLR ligand treatments induced a significant reduction in virus shedding, with the TLR3 ligand poly I:C conferring the greatest AIV immunity compared to control birds, followed by CpG ODN and LPS. Furthermore, transcriptional analysis of gene expression in the spleen and lungs suggest IFN-α and IL-8 as correlates of immunity conferred by poly I:C, and IFN-γ for CpG ODN and LPS. In conclusion, TLR ligands, have the ability to enhance host immunity against AIV, and future studies should consider exploring the combinatory effects of poly I:C and CpG ODN prophylaxis in conjunction with AIV vaccination.

A Toll-like receptor 3 agonist (polyI:C) elicits innate host responses in the spleen and lungs of chickens.

Double-stranded (ds) RNA interacts with host Toll-like receptor (TLR-3), leading to the induction of anti-viral host responses. The present study was designed to compare different routes of administration of a synthetic dsRNA (polyI:C) for induction of innate responses in chicken spleen and lungs. Chickens were treated with polyI:C via the aerosol, intra-air sac (i.a.s.), and intramuscular (IM) routes. Spleen and lungs were collected at 0, 2, 6, 12, and 24 h post-treatment and the expression of innate defence genes was quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR). There was an up-regulation of interferon (IFN)-ß, TLR-3, and Toll/interleukin 1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF) at 6 h post-treatment in the spleen via IM administration of polyI:C. There was also an increase in the expression of TLR-3 and TRIF in the spleen at 2 h post-treatment via the i.a.s. route. The expression of IFN-α and TRIF was upregulated at 6 h post-treatment via the i.a.s. route in the lungs. Overall, our results indicate that administration of polyI:C can locally and systemically induce the expression of innate response genes depending on the route.

Oral treatment of chickens with lactobacilli influences elicitation of immune responses.

Commensal microbes in the intestine are in constant interaction with host cells and play a role in shaping the immune system. Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius are members of the chicken intestinal microbiota and have been shown to induce different cytokine profiles in mononuclear cells in vitro. The objective of the present study was to examine the effects of these bacteria individually or in combination on the induction of antibody- and cell-mediated immune responses in vivo. The birds received lactobacilli weekly via oral gavage starting on day of hatch and subsequently, at 14 and 21 days, were immunized with sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), Newcastle disease virus vaccine, and infectious bursal disease virus vaccine. Antibody responses in serum were measured weekly for 4 weeks beginning on the day of primary immunization. The cell-mediated immune response was evaluated at 21 days postimmunization by measurement of gamma interferon (IFN-γ) production in splenocytes stimulated with inactivated vaccine antigens. L. salivarius-treated birds had significantly more serum antibody to SRBC and KLH than birds that were not treated with probiotics. L. salivarius-treated birds also had decreased cell-mediated immune responses to recall antigen stimulation. L. reuteri treatment did not significantly affect the systemic immune response, while L. acidophilus treatment increased the antibody response to KLH. These results indicate that systemic antibody- and cell-mediated immune responses can be modulated by oral treatment with lactobacilli but that these bacteria may vary in their ability to modulate the immune response.

Interferon-γ influences immunity elicited by vaccines against very virulent Marek's disease virus.

Vaccination of chickens with herpesvirus of turkey (HVT) confers only partial protection against challenge with a very virulent Marek's disease virus (MDV). Here, we evaluated the ability of recombinant chicken interferon-gamma (rChIFN-γ) to enhance protective efficacy of HVT against the very virulent MDV strain, RB1B. The bioactivity of IFN-γ expressed by a plasmid expression vector was confirmed by its ability to stimulate a chicken macrophage cell line (HD11) to produce nitric oxide (NO) in vitro. The administration of HVT with 5µg of pcDNA:chIFN-γ plasmid reduced the incidence of tumor development significantly when compared to vaccinated birds (77.7% in the HVT+empty vector group and 80% in HVT group versus 33.3% in the HVT+chIFN-γ group) and significantly increased IFN-γ expression in the splenocytes of the protected group, suggesting that rChIFN-γ increases the potency of HVT against MDV. Further analysis demonstrated that the protected birds that received HVT vaccine and/or plasmid had lower MDV genome load and lower amounts of transcripts for meq and vIL-8 than in the birds without lesions. Similarly, lower expression of IL-10, IL-18 and IL-6 was observed in the chickens without lesions compared to the chickens that had lesions, suggesting an inverse association between up-regulation of these cytokines and vaccine-induced immunity. In conclusion, IFN-γ can positively influence immunity conferred by HVT vaccination against challenge with a very virulent Marek's disease virus (vvMDV) in chickens.

Enhancement of immunogenicity of a virosome-based avian influenza vaccine in chickens by incorporating CpG-ODN.

Influenza virosomes are virus-like particles, representing a platform for vaccine development. In this study, we examined the immunogenicity of avian influenza virosomes with or without inclusion of recombinant chicken interferon-gamma (rChIFN-γ) or CpG-ODN in chickens. Immunization with virosomes adjuvanted with CpG-ODN elicited the highest haemagglutination inhibition antibody titres, as well as IgG and IgA serum antibody responses. Moreover, Virosomes+CpG-ODN formulation induced an antigen-specific spleen cell proliferation and IFN-γ expression. In conclusion, our results demonstrated that virus-specific antibody- and cell-mediated responses may be induced in chickens immunized with virosomes and these responses can be enhanced by incorporating CpG-ODN in the virosome vaccine formulation.

Immune responses against Marek's disease virus.

It is more than a century since Marek's disease (MD) was first reported in chickens and since then there have been concerted efforts to better understand this disease, its causative agent and various approaches for control of this disease. Recently, there have been several outbreaks of the disease in various regions, due to the evolving nature of MD virus (MDV), which necessitates the implementation of improved prophylactic approaches. It is therefore essential to better understand the interactions between chickens and the virus. The chicken immune system is directly involved in controlling the entry and the spread of the virus. It employs two distinct but interrelated mechanisms to tackle viral invasion. Innate defense mechanisms comprise secretion of soluble factors as well as cells such as macrophages and natural killer cells as the first line of defense. These innate responses provide the adaptive arm of the immune system including antibody- and cell-mediated immune responses to be tailored more specifically against MDV. In addition to the immune system, genetic and epigenetic mechanisms contribute to the outcome of MDV infection in chickens. This review discusses our current understanding of immune responses elicited against MDV and genetic factors that contribute to the nature of the response.

Expression profiling of genes associated with regulatory functions of T-cell subsets in Marek's disease virus-infected chickens.

The environment of tumours caused by Marek's disease virus (MDV) in chickens has been shown to have an immunoregulatory phenotype. The objective of the present study was to examine the expression of key T-regulatory markers during various stages of MDV pathogenesis. Specific-pathogen free (SPF) as well as major histocompatibility complex-defined chickens were infected with the RB1B and JM-16 strains of MDV, respectively. CD4(+) and CD8(+) T cells from the spleens of infected as well as age-matched controls were sorted by flow cytometry at 4, 10, and 21 days post infection (d.p.i.). The expression of molecules such as CTLA-4, IL-2aR (CD25), PD-1 and PDL-1 was quantified by real-time, quantitative, reverse-transcription polymerase chain reaction. There was an up-regulation of CTLA-4 in CD4(+) T cells at 4 d.p.i. The expression of PD-1 was also up-regulated in the CD4(+) T-cell subset of SPF birds at 21 d.p.i. Furthermore, the expression of PD-1 was enhanced in CD4(+) and CD8(+) T cells of genetically susceptible chickens, linking this molecule to susceptibility to disease. The expression of CD25 was down-regulated in both SPF and genetically defined birds after infection. This may be a mechanism through which the virus exerts its immunosuppressive effects. In conclusion, the results of the present study provide more insight into immunomodulatory processes that occur in the lymphoid tissues of MDV-infected chickens.

Effects of lactobacilli on cytokine expression by chicken spleen and cecal tonsil cells.

Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius are all normal residents of the chicken gastrointestinal tract. Given the interest in using probiotic bacteria in chicken production and the important role of the microbiota in the development and regulation of the host immune system, the objective of the current study was to examine the differential effects of these bacteria on cytokine gene expression profiles of lymphoid tissue cells. Mononuclear cells isolated from cecal tonsils and spleens of chickens were cocultured with one of the three live bacteria, and gene expression was analyzed via real-time quantitative PCR. All three lactobacilli induced significantly more interleukin 1beta (IL-1beta) expression in spleen cells than in cecal tonsil cells, indicating a more inflammatory response in the spleen than in cecal tonsils. In cecal tonsil cells, substantial differences were found among strains in the capacity to induce IL-12p40, IL-10, IL-18, transforming growth factor beta4 (TGF-beta4), and gamma interferon (IFN-gamma). In conclusion, we demonstrated that L. acidophilus is more effective at inducing T-helper-1 cytokines while L. salivarius induces a more anti-inflammatory response.

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.

Cytokine gene expression in splenic CD4(+) and CD8(+) T-cell subsets of chickens infected with Marek's disease virus.

Specific-pathogen free chickens were infected with the RB1B strain of Marek's disease virus (MDV) and T cells from the spleens of infected as well as age-matched controls were fractionated by flow cytometry at 4, 10, and 21 days post-infection (d.p.i.). Real-time quantitative reverse transcription PCR was used to assess the amount of cytokine transcripts as well as viral genes meq and glycoprotein B (gB). There was an increase in the number of CD4(+) T cells, as well as a significant increase in the expression of the viral meq gene in CD4(+) T cells, which coincided with the presence of tumors in various organs of infected birds. It was also observed that there was a significant upregulation in the amount of the gene expression of interferon (IFN)-gamma, interleukin (IL)-18, and IL-6 at 4 and 21 d.p.i. in CD4(+) and CD8(+) T-cell subsets. The expression of IL-10 was upregulated as well. The outcome of the cytokine milieu inclined towards the induction of a type I immune response at 4 and 21 d.p.i. Our study indicates that MDV-associated cytokine profiles vary in CD4(+) and CD8(+) T-cell subsets, and that cytokines including IFN-gamma, IL-18, IL-6, and IL-10 may play a role in the elicitation of an immune response to MDV.

Marek's disease virus-induced expression of cytokine genes in feathers of genetically defined chickens.

Marek's disease (MD) vaccines, although effective in reducing lymphoproliferation, cannot control infectious virus production in the feather follicle epithelium (FFE) which is the site of virus shedding. Therefore, we investigated Marek's disease virus (MDV) replication as well as the expression of cytokine genes in feathers of MDV-infected chickens belonging to genetically defined lines (N2a or B(21)/B(21) haplotype-resistant and P2a or B(19)/B(19) haplotype-susceptible). Though there was not a difference in MDV genome load and transcripts between feathers of these chicken lines at 4 and 10 days post-infection (d.p.i.), feathers of resistant chickens carried significantly lower viral genome load and transcripts at 21 d.p.i. Irrespective of genetic background of the chickens examined, MDV replication showed a significant positive correlation with the expression of IFN-gamma gene. The results imply the usefulness of genetic control approach in reducing virulent MDV transmission.

Expression of cytokine genes following pre- and post-hatch immunization of chickens with herpesvirus of turkeys.

Induction of immune response as characterised by expression of cytokine genes in the spleen following immunization of pre- and post-hatch chickens with herpesvirus of turkeys (HVT) vaccine was studied. The pattern of expression of IFN-gamma and IL-10 genes in pre-hatch immunized chickens was different from that observed in post-hatch HVT immunized chickens. This expression pattern of cytokine genes was associated with significantly higher HVT transcripts in pre-hatch immunized chickens than in post-hatch immunized chickens. In conclusion, HVT immunization in chickens, irrespective of the age of immunization, stimulates host response characterised by the expression of cytokine genes, such as IFN-gamma and IL-10 in the spleen. However, the age of immunization appears to influence the temporal pattern of IFN-gamma and IL-10 expression as well as replication of HVT.

Cytokine gene expression patterns associated with immunization against Marek's disease in chickens.

The present study explored the immunological correlates of protection mediated by a live bivalent vaccine consisting of herpesvirus of turkeys (HVT) and SB-1 against infection with the RB1B strain of Marek's disease virus (MDV). Compared to unvaccinated infected chickens, vaccinated protected birds had lower expression of interleukin (IL)-6, IL-10 and IL-18 genes in spleen. However, there was no difference between these two groups of birds in the expression of interferon (IFN)-gamma, IL-4, IL-12 and inducible nitric oxide synthase (iNOS) genes on day 21 post-infection. Furthermore, protection was associated with lower MDV genome load in spleen but not in feather tips, suggesting that vaccination had little or no effect on curtailing virus transmission. In conclusion, vaccination with a bivalent MD vaccine was associated with distinct cytokine expression patterns in spleen and modulation of cytokine responses by the vaccine may play a role in mediation of protection.

Modulation of antibody-mediated immune response by probiotics in chickens.

Probiotic bacteria, including Lactobacillus acidophilus and Bifidobacterium bifidum, have been shown to enhance antibody responses in mammals. The objective of this study was to examine the effects of a probiotic product containing the above bacteria in addition to Streptococcus faecalis on the induction of the chicken antibody response to various antigens, both systemically and in the gut. The birds received probiotics via oral gavage and subsequently were immunized with sheep red blood cells (SRBC) and bovine serum albumin (BSA) to evaluate antibody responses in serum or with tetanus toxoid (TT) to measure the mucosal antibody response in gut contents. Control groups received phosphate-buffered saline. Overall, BSA and SRBC induced a detectable antibody response as early as week 1 postimmunization (p.i.), which lasted until week 3 p.i. Probiotic-treated birds had significantly (P