The present study was undertaken to assess the antimicrobial activity of Lactobacillus spp. (L. salivarius, L. johnsonii, L. reuteri, L. crispatus, and L. gasseri) against Campylobacter jejuni as well as their immunomodulatory capabilities. The results demonstrated that lactobacilli exhibit differential antagonistic effects against C. jejuni and vary in their ability to elicit innate responses in chicken macrophages. All lactobacilli exerted inhibitory effects on C. jejuni growth, abrogated the production of the quorum sensing molecule autoinducer-2 (AI-2) by C. jejuni and inhibited the invasion of C. jejuni in human intestinal epithelial cells. Additionally, all lactobacilli, except L. reuteri, significantly reduced the expression of virulence-related genes in C. jejuni, including genes responsible for motility (flaA, flaB, and flhA), invasion (ciaB), and AI-2 production (luxS). All lactobacilli enhanced C. jejuni phagocytosis by macrophages and increased the expression of interferon (IFN)-γ, interleukin (IL)-1β, IL-12p40, IL-10, and chemokine (CXCLi2) in macrophages. Furthermore, L. salivarius, L. reuteri, L. crispatus, and a mixture of all lactobacilli significantly increased expression of the co-stimulatory molecules CD40, CD80, and CD86 in macrophages. In conclusion, these findings demonstrate that lactobacilli possess anti-Campylobacter and immunomodulatory activities. Further studies are needed to assess their protective efficacy against intestinal colonization by C. jejuni in broiler chickens.

Infectious laryngotracheitis (ILT) is an acute respiratory disease of poultry caused by infectious laryngotracheitis virus (ILTV). Control of the disease with live attenuated vaccines administered via eye drop build upon immune responses generated by the eye-associated lymphoid tissues. The aim of this study was to assess cytokine and lymphocyte changes in the conjunctiva-associated lymphoid tissues (CALT) and Harderian gland (HG) stimulated by the ocular inoculation of the ILTV chicken embryo origin (CEO) vaccine strain and virulent strain 63140. This study offers strong evidence to support the roles that the CALT and HG play in the development of protective ILTV immune responses. It supports the premise that ILTV-mediated immunomodulation favors the B cell response over those of T cells. Further, it provides evidence that expansions of CD8α+ cells, with the concomitant expression of the Granzyme A gene, are key to reducing viral genomes in the CALT and halting ILTV cytolytic replication in the conjunctiva. Ultimately, this study revealed that the early upregulation of interleukin (IL)-12p40 and Interferon (IFN)-γ cytokine genes, which shape the antigen-specific cell-mediated immune responses, retarded the decline of virus replication, and enhanced the development of lesions in the conjunctiva epithelium.

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.

The objective of this study was to determine how cytokine transcription profiles correlate with patterns of infectious laryngotracheitis virus (ILTV) replication in the trachea, Harderian gland, and trigeminal ganglia during the early and late stages of infection after intratracheal inoculation. Viral genomes and transcripts were detected in the trachea and Harderian gland but not in trigeminal ganglia. The onset of viral replication in the trachea was detected at day one post-infection and peaked by day three post-infection. The peak of pro-inflammatory (CXCLi2, IL-1β, IFN-γ) and anti-inflammatory (IL-13, IL-10) cytokine gene transcription, 5 days post-infection, coincided with the increased recruitment of inflammatory cells, extensive tissue damage, and limiting of virus replication in the trachea. In contrast, transcription of the IFN-β gene in the trachea remained unaffected suggesting that ILTV infection blocks type I interferon responses. In the Harderian gland, the most evident transcription change was the early and transient upregulation of the IFN-γ gene at 1 day post-infection, which suggests that the Harderian gland is prepared to rapidly respond to ILTV infection. Overall, results from this study suggest that regulation of Th1 effector cells and macrophage activity by Th1/2 cytokines was pertinent to maintain a balanced immune response capable of providing an adequate Th1-mediated protective immunity, while sustaining some immune homeostasis in preparation for the regeneration of the tracheal mucosa.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. Reducing Campylobacter numbers in the intestinal tract of chickens will minimize transmission to humans, thereby reducing the incidence of infection. We have previously shown that oral pre-treatment of chickens with C. jejuni lysate and Poly D, L-lactide-co-glycolide polymer nanoparticles (PLGA NPs) containing CpG oligodeoxynucleotide (ODN) can reduce the number of C. jejuni in infected chickens. In the current study, the effects of these pre-treatments on the composition and functional diversity of the cecal microbiota, in chickens experimentally infected with C. jejuni, were investigated using next-generation sequencing. The taxonomic composition analysis revealed a reduction in cecal microbial diversity and considerable changes in the taxonomic profiles of the microbial communities of C. jejuni-challenged chickens. On the other hand, irrespective of the dose, the microbiota of PLGA-encapsulated CpG ODN- and C. jejuni lysate-treated chickens exhibited higher microbial diversity associated with high abundance of members of Firmicutes and Bacteroidetes and lower numbers of Campylobacter than untreated-chickens. These findings suggest that oral administration of encapsulated CpG ODN and C. jejuni lysate can reduce colonization by C. jejuni by enhancing the proliferation of specific microbial groups. The mechanisms that mediate these changes remain, however, to be elucidated.

Evolutionarily conserved pattern recognition receptors, including Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) that are present in microbes. PAMPs induce several pathways downstream of TLRs that lead to induction of antiviral responses. The objective of this study was to investigate the stimulatory effect of various PAMPs (in the form of TLR ligands) in reducing Marek's disease virus (MDV) infection in chicken embryo fibroblast cells (CEFs). To this end, CEFs were pretreated with Pam3CSK4, Poly(IC), lipopolysaccharide (LPS), and CpG ODN as TLR2, TLR3, TLR4, and TLR21 ligands, respectively for 24 h followed by infection with MDV. The results indicated that pretreatment with Poly(IC) resulted in a robust reduction (by about 81%) of MDV infection in CEFs at 96 h postinfection while a moderate reduction was observed with treatment of Pam3CSK4 (35%), LPS (26%), and CpG ODN (23%) PAMPs. Transcriptional analysis of gene expression in CEFs demonstrated that all TLR ligand treatments and MDV infection significantly increased the expression of type I interferons, interleukin (IL)-1β, interferon regulatory factor 7 (IRF7), interferon induced protein with tetratricopeptide repeats 5 (IFIT5), and myxoma-resistance protein (Mx). Further studies are needed to explore the mechanism by which PAMPs, particularly the TLR3 ligands could reduce MDV infection in CEFs, which may play an important role in controlling the replication of MDV in chicken.

Immunity against Marek's disease (MD), caused by Gallid herpesvirus 2 (GaHV-2), in chickens is mediated by both innate and adaptive responses. The present study evaluated the effects of GaHV-2 infection on distribution and frequency of γδ T cells in tissues, as well as their expression of cytokines. We found that the infected chickens had significantly higher number of γδ T cells in their spleens by 10 and 21 days post-infection (d.p.i.) and nearly 100% of these γδ T cells were CD8+ at 21 d.p.i. Conversely, the number of γδ T cells in the cecal tonsils of GaHV-2-infected birds decreased compared to uninfected birds. Splenic γδ T cells had up-regulated expression of interferon-γ early in infection followed by simultaneous gene expression of interleukin-10 during the later phases. In conclusion, these results suggest a potential role for γδ T cells in host response to GaHV-2 and further elucidate the underlying immunological mechanisms of interactions between this virus and its host.

Commensal gut microbiota plays an important role in health and disease. The current study was designed to assess the role of gut microbiota of chickens in the initiation of antiviral responses against avian influenza virus. Day-old layer chickens received a cocktail of antibiotics for 12 (ABX-D12) or 16 (ABX-D16) days to deplete their gut microbiota, followed by treatment of chickens from ABX-12 with five Lactobacillus species combination (PROB), fecal microbial transplant suspension (FMT) or sham treatment daily for four days. At day 17 of age, chickens were challenged with H9N2 virus. Cloacal virus shedding, and interferon (IFN)-α, IFN-β and interleukin (IL)-22 expression in the trachea, lung, ileum and cecal tonsils was assessed. Higher virus shedding, and compromised type I IFNs and IL-22 expression was observed in ABX-D16 chickens compared to control, while PROB and FMT showed reduced virus shedding and restored IL-22 expression to levels comparable with undepleted chickens. In conclusion, commensal gut microbiota of chickens can modulate innate responses to influenza virus subtype H9N2 infection in chickens, and modulating the composition of the microbiome using probiotics- and/or FMT-based interventions might serve to promote a healthy community that confers protection against influenza virus infection in chickens.

Invariant natural killer T (iNKT) cells play important roles in antimicrobial defense and immune-regulation. We have previously shown that iNKT cells express certain toll-like receptors (TLR), and that TLR co-stimulation of iNKT cells in the presence of suboptimal concentrations of T cell receptor (TCR) agonists enhances cellular activation. In the present study, we investigated the regulatory effects of CpG oligonucleotides in mouse primary hepatic and splenic iNKT cells and in DN32.D3 iNKT cells. We show that CpG treatment of iNKT cells in the presence of higher concentrations of TCR agonists (α-GalCer or anti-CD3 mAb) results in the up-regulation of TLR9 in iNKT cells with a concurrent reduction in their cellular activation, as assessed by their production of IL-2, IL-4 and IFN-γ compared with controls. CpG-mediated down-regulation of iNKT cell activation has been found to depend, at least in part, on signaling by MyD88, a critical adapter moiety downstream of TLR9 signaling. Mechanistically, iNKT cells treated with CpG in the presence of TCR agonists show inhibition of MAPK signaling as determined by the levels of ERK1/2 and p38 MAPKs. Furthermore, CpG treatment leads to an increased induction of phosphatases, DUSP1 and SHP-1, that seem to impede MAPK and TCR signaling, resulting in the negative regulation of iNKT cell activation. Our findings therefore suggest a novel regulatory role for CpG in iNKT cells in the mediation of a negative feedback mechanism to control overactive iNKT cell responses and hence to avoid undesirable excessive immunopathology.

Dengue virus (DENV) is a mosquito-borne Flavivirus classified into four serotypes (DENV-1-4) that causes Dengue fever (DF), Dengue hemorrhagic Fever (DHF) or Dengue shock syndrome (DSS). An estimated 390 million people are at risk for infection with DENV and there are no effective vaccines or therapeutics. We utilized RNA chromatography coupled with quantitative mass spectrometry (qMS) to identify host RNA binding proteins (RBPs) that interact with DENV-2 RNA. We identified ERI3 (also PRNPIP and PINT1), a putative 3'-5' RNA exonuclease, which preferentially associates with DENV-2 genomic RNA via interactions with dumbbell structures in the 3' UTR. ERI3 is required for accumulation of DENV-2 genomic RNA and production of infectious particles. Furthermore, the mosquito homologue of ERI3 is required for DENV-2 replication in adult Aedes aegypti mosquitos implying that the requirement for ERI3 is conserved in both DENV hosts. In human cells ERI3 localizes to the Golgi in uninfected cells, but relocalizes near sites of DENV-2 replication in infected cells. ERI3 is not required for maintaining DENV-2 RNA stability or translation of the viral polyprotein, but is required for viral RNA synthesis. Our results define a specific role for ERI3 and highlight the importance of Golgi proteins in DENV-2 replication.

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 viruses (AIV) are of great concern to the worldwide community as well as the poultry industry. Although existing vaccines are successful in limiting the spread of the virus, these vaccines do not eliminate virus shedding into the environment. As a result, it is of great importance to enhance the efficacy of existing AIV vaccines. Therefore, the objective of the present study was to utilize the immunostimulatory Toll-like receptor ligands poly I:C, lipopolysaccharide (LPS), and CpG DNA motifs, either alone or in combination with each other, as adjuvants to enhance the immunogenicity of an inactivated AIV vaccine. Chickens were vaccinated twice, 14 days apart. Antibody-mediated responses were assessed by collected sera and lacrimal secretions, while cell-mediated immunity was assessed by stimulating splenocytes from vaccinated chickens in vitro with the vaccine antigen. The results suggest that CpG alone served as the best single-ligand adjuvant compared to poly I:C or LPS, as it significantly enhanced antibody-mediated responses, as determined by enzyme-linked immunosorbant assay. Furthermore, upon combining CpG with poly I:C, a robust antibody-mediated and cell-mediated immune response was elicited, resulting in an enhanced hemagglutination inhibition titer and splenocyte proliferation respectively. Future studies may be aimed at assessing the efficacy of the poly I:C and CpG combination adjuvant in protecting against AIV infection.

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.

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.

Invariant NKT (iNKT) cells are glycolipid-reactive lymphocytes with anti-microbial properties. Toll-like receptor (TLR)-primed antigen-presenting cells are known to activate iNKT cells, however, the expression and function of TLRs in iNKT cells remain largely unknown. Here, we show that TCR-activation of murine iNKT cells by α-GalactosylCeramide (α-GalCer) or anti-CD3 antibodies can result in increased expression of TLR genes. TLR3, 5 and 9-mediated costimulation of TCR-preactivated iNKT cells resulted in enhancement of iNKT cell activation, as determined by their cytokine production. Expression of TLR3 and 9 at protein level was also confirmed in TCR-activated iNKT cells. Furthermore, TCR-preactivation followed by TLR9-costimulation of iNKT cells increased their ability to induce maturation of dendritic cells. Thus, our findings show that iNKT cells can up-regulate their TLR expression upon TCR activation and a subsequent TLR-signaling in these cells can lead to their enhanced activation, suggesting a new possible mode of iNKT cell activation.

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.

Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens. Several TLRs have been identified in chickens and their expression has been detected in many immune cell subsets including in B cells. However, the mechanisms through which TLRs modulate B cell responses have not been well characterized in chickens. The aim of the present study was to elucidate the responses mounted by cells of the bursa of Fabricius to treatment with the TLR 3, 4 and 21 ligands, poly I:C, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODN), respectively. The relative expression of several immune system genes was quantified at 1, 3, 8 and 18 h post-treatment. The results show that all three ligands induced the up-regulation of interferon (IFN)-γ and interleukin (IL)-10 transcripts and promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. Furthermore, the results indicated that LPS and poly I:C induced the greatest IFN-γ and IL-10 responses, respectively, while CpG ODN was the most efficacious inducer of CD80 and MHC-II expression. Future studies may be aimed at elucidating the mechanisms of TLR-mediated activation of chicken B cells. These mechanisms may be then exploited for the development of adjuvants with enhanced ability to stimulate B cell responses.

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.

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.

Chickens infected with Marek's disease virus (MDV) become lifelong carriers regardless of their susceptibility to clinical disease. Therefore various viral immune-evasive mechanisms must play a role in MDV-host interactions. MDV has previously been shown to influence the expression of major histocompatibility complex (MHC) class II molecules. However, little is known about the underlying mechanisms of this phenomenon. In the present study, we studied the effect of MDV infection on the expression of several genes associated with IFN-gamma-inducible MHC class II expression at 4, 7, 14, and 21 days post-infection (dpi). There was a significant (p <or= 0.05) downregulation of MHC class II beta chain expression throughout the experiment, while other components of the MHC class II heterotrimer (i.e., alpha chain and the invariant chain) were significantly downregulated only at 4 and 21 dpi. Furthermore, the expression of components of the IFN-gamma-receptor complex was significantly downregulated at 4 dpi. In contrast, a number of other IFN-gamma-signaling molecules, including signal transducer and activator of transcription 1 (STAT1), interferon-responsive factor 1 (IRF-1), and class II transactivator (CIITA) were significantly upregulated at most time points. The results of this study shed light on the possible mechanisms by which MDV may evade host immunosurveillance.