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Marek's Disease Virus (MDV) is the causative agent of a lymphoproliferative disease, Marek's disease (MD) in chickens. MD is only controlled by mass vaccination; however, immunity induced by MD vaccines is unable to prevent MDV replication and transmission. The herpesvirus of turkey (HVT) vaccine is one of the most widely used MD vaccines in poultry industry. Vaccines can be adjuvanted with Toll-like receptor ligands (TLR-Ls) to enhance their efficacy. In this study, we examined whether combining TLR-Ls with HVT can boost host immunity against MD and improve its efficacy. Results demonstrated that HVT alone or HVT combined with encapsulated CpG-ODN partially protected chickens from tumor incidence and reduced virus replication compared to the control group. However, encapsulated CpG-ODN only moderately, but not significantly, improved HVT efficacy and reduced tumor incidence from 53% to 33%. Further investigation of cytokine gene profiles in spleen and bursa of Fabricius revealed an inverse association between interleukin (IL)-10 and IL-18 expression and protection conferred by different treatments. In addition, the results of this study raise the possibility that interferon (IFN)-β and IFN-γ induced by the treatments may exert anti-viral responses against MDV replication in the bursa of Fabricius at early stage of MDV infection in chickens.

One of the economically important diseases in the poultry industry is Marek's disease (MD) which is caused by Marek's disease virus (MDV). The use of current vaccines provides protection against clinical signs of MD in chickens. However, these vaccines do not prevent the transmission of MDV to susceptible hosts, hence they may promote the development of new virulent strains of MDV. This issue persuaded us to explore alternative approaches to control MD in chickens. Induction of innate responses at the early stage of life in the chicken may help to prevent or reduce MDV infection. Further, prophylactic use of Toll-like receptor ligands (TLR-Ls) has been shown to generate host immunity against infectious diseases. In this regard, encapsulation of TLR-Ls in Poly(d, l-lactic-co-glycolic acid) (PLGA) may further enhance host responses by controlled release of TLR-Ls for an extended period. Hence, in the current study, protective effects of encapsulated TLR4 and TLR21 ligands, LPS and CpG, respectively, were investigated against MD. Results indicated that administration of encapsulated CpG and LPS first at embryonic day (ED) 18, followed by post-hatch at 14 days-post infection (dpi) intramuscularly, diminished tumor incidence by 60% and 42.8%, respectively at 21dpi compared to the MDV only group. In addition, analysis of cytokine gene profiles of interferon (IFN)-α, IFN-β, IFN-γ, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-18 and IL-10 in spleen and bursa of Fabricius at different time points suggests that TLR-Ls possibly triggered host responses through the expression of IL-1β and IL-18 to reduce tumor formation. However, further studies are needed to explore the role of these pro-inflammatory cytokines and other influencing elements like lymphocytes in the hindrance of tumor development by TLR-Ls treatment in chickens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Early responses against viruses, such as avian influenza virus (AIV), may be induced by Toll-like receptor (TLR) pathways. In the present study, an in ovo model was employed to study the antiviral activities of TLR ligands. It was hypothesized that administration of TLR ligands in ovo at the appropriate dose and time can reduce AIV titer in embryonated chicken eggs. Moreover, the study aimed to determine the mechanisms involved in the TLR-mediated antiviral responses in the chorioallantoic membrane (CAM). Embryonated eggs (10-14 day old) were treated with TLR2, 4, 7, and 21 ligands using different doses and times pre- and post-AIV infection. The results revealed that treatment of embryonated chicken eggs with TLR ligands reduced AIV replication. Further analysis showed that TLR ligands induced interferon (IFN)-γ and IFN stimulatory genes in the CAM, which may have played a role in the reduction of the AIV titer. The timing and dose of TLR ligands administration had significant impacts on the outcome of the treated eggs. In conclusion, the present study demonstrated that the in ovo route may be employed to determine the antiviral characteristics of TLR ligands against AIV.

Chicken macrophages express several receptors for recognition of pathogens, including Toll-like receptors (TLRs). TLRs bind to pathogen-associated molecular patterns (PAMPs) derived from bacterial or viral pathogens leading to the activation of macrophages. Macrophages play a critical role in immunity against viruses, including influenza viruses. The present study was designed to test the hypothesis that treatment of chicken macrophages with TLR ligands reduces avian influenza replication. Furthermore, we sought to study the expression of some of the key mediators involved in the TLR-mediated antiviral responses of macrophages. Chicken macrophages were treated with the TLR2, 3, 4, 7 and 21 ligands, Pam3CSK4, poly(I:C), LPS, R848 and CpG ODN, respectively, at different doses and time points pre- and post-H4N6 avian influenza virus (AIV) infection. The results revealed that pre-treatment of macrophages with Pam3CSK4, LPS and CpG ODN reduced the replication of AIV in chicken macrophages. In addition, the relative expression of genes involved in inflammatory and antiviral responses were quantified at 3, 8 and 18 hours post-treatment with the TLR2, 4 and 21 ligands. Pam3CSK4, LPS and CpG ODN increased the expression of interleukin (IL)-1β, interferon (IFN)-γ, IFN-β and interferon regulatory factor (IFR) 7. The expression of these genes correlated with the reduction of viral replication in macrophages. These results shed light on the process of immunity to AIV in chickens.

The innate immune receptors, such as Toll-like receptors (TLRs), are intimately involved in the early sensing of invading microorganisms or their structural components. Engagement of TLRs with their ligands results in activation of several downstream intracellular pathways leading to activation of innate and adaptive immune system cells. It was initially thought that TLRs are primarily expressed by antigen-presenting cells (APCs), such as macrophages and dendritic cells, and that interactions between microbial ligands and TLRs in these cells will indirectly result in activation of cells of the adaptive immune system, especially T cells. However, it has now become evident that TLRs are also expressed by various T cell subsets, such as conventional αβT cells, regulatory T cells, and γδT cells as well as natural killer T cells. Importantly, it appears that at least in some of these T cell subsets, TLRs are functionally active, because stimulation of these cells with TLR agonists in the absence of APCs results in exertion of effector or regulatory functions of T cells. The present review attempts to summarize the recent findings related to TLR expression in different T cell subsets and the direct role of TLRs in the induction and regulation of T cell responses, including those responses that occur at mucosal surfaces. In addition, the potential use of TLR agonists for steering T cell responses as a prophylactic or therapeutic strategy in the context of infectious, allergic or autoimmune diseases is explored.

Transmission of H9N2 avian influenza virus (AIV) can occur in poultry by direct or indirect contact with infected individuals, aerosols, large droplets and fomites. The current study investigated the potential of H9N2 AIV transmission in chickens via a fecal route. Transmission was monitored by exposing naïve chickens to fecal material from H9N2 AIV-infected chickens (model A) and experimentally spiked feces (model B). The control chickens received H9N2 AIV. Results revealed that H9N2 AIV could persist in feces for up to 60–84 h post-exposure (PE). The H9N2 AIV titers in feces were higher at a basic to neutral pH. A higher virus shedding was observed in the exposed chickens of model B compared to model A. We further addressed the efficacy of Toll-like receptor (TLR) ligands to limit transmission in the fecal model. Administration of CpG ODN 2007 or poly(I:C) alone or in combination led to an overall decrease in the virus shedding, with enhanced expression of type I and II interferons (IFNs) and interferon-stimulating genes (ISGs) in different segments of the small intestine. Overall, the study highlighted that the H9N2 AIV can survive in feces and transmit to healthy naïve chickens. Moreover, TLR ligands could be applied to transmission studies to enhance antiviral immunity and reduce H9N2 AIV shedding. [ABSTRACT FROM AUTHOR]

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1β, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens. [ABSTRACT FROM AUTHOR]

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)− 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation. [ABSTRACT FROM AUTHOR]

Gamma delta (γδ) T cells are highly enriched in mucosal barrier sites including intestinal tissues where microbial infections and tumors often originate in mammals. Human γδ T cells recognize stress antigens and microbial signals via their T cell receptor (TCR), natural killer (NK) receptors, and pattern recognition receptors. However, little is known about antigens or ligands capable of stimulating chicken γδ T cells. The results of the present study demonstrated that polyinosinic-polycytidylic acid (poly(I:C)), a Toll-like receptor (TLR)3 ligand, significantly induced upregulation of CD8α molecules on circulating and lung γδ T cells. Moreover, poly(I:C) stimulation induced interferon (IFN)-γ production from splenic and lung CD8α+ γδ T cells while Cytosine-phosphate-Guanine oligodeoxynucleotides (CpG-ODN) 2007, a TLR21 ligand, stimulation induced IFN-γ production by circulating γδ T cells. Neither poly(I:C) nor CpG-ODN 2007 stimulation elicited degranulation of γδ T cells. Additionally, the results revealed that CpG-ODN 2007 induced IFN-γ production from TCR-stimulated γδ T cells sorted from spleen. In our experiments, isopentenyl pyrophosphate (IPP), 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), or zoledronate (Zol) stimulation did not induce IFN-γ production or degranulation in γδ T cells. Taken together, a combination of CpG-ODN 2007 and anti-CD3ε monoclonal antibodies (mAbs) can stimulate chicken γδ T cells and induce production of IFN-γ by these cells while IFN-γ production by γδ T cells induced by stimulation of poly(I:C) needs signals from other cells. These results suggest that chicken γδ T cells can sense invading pathogens via TLRs and produce IFN-γ as a first line of defense. • poly(I:C) stimulation upregulated CD8α molecules on chicken γδ T cells derived from PBMCs and lungs. • poly(I:C) induced IFN-γ production by spleen and lung γδ T cells, but this induction needs other signals from non γδ T cells. • CpG-ODN 2007 induced IFN-γ secretion by TCR co-stimulated γδ T cells. • Degranulation by chicken γδ T cells need other signals rather than those mediated by TLR3 and TLR21. • Phosphoantigen stimulation elicited neither IFN-γ production nor degranulation by chicken γδ T cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Toll-like receptor ligands reduced incidence of MD or delay onset of this disease. [•] Among the ligands, LPS was the most potent to induce immunity against MD. [•] CpG-ODN was also able to induce immunity against MD. [•] Immunity was associated with expression of IFNs in spleen and lungs. [Copyright &y& Elsevier]