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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV that infects avian species and lead to huge economical losses in the poultry industry. The unique immunomodulatory properties of Retinoic acid (RA), an active component of vitamin A, highlights its potential to enhance chicken's resistance to infectious diseases and perhaps vaccine-induced immunity. Therefore, the present study evaluated the effects of in ovo supplementation of RA on the immunogenicity and protective efficacy of an inactivated avian influenza virus vaccine. On embryonic day 18, eggs were inoculated with either 90 μmol RA/200 μL/egg or diluent into the amniotic sac. On days 7 and 21 post-hatch, birds were vaccinated with 15 μg of β-propiolactone (BPL) inactivated H9N2 virus via the intramuscular route. One group received BPL in combination with an adjuvant, while the other group received saline solution and served as a non-vaccinated control group. Serum samples were collected on days 7, 14, 21, 28, 35, and 42 post-primary vaccination (ppv) for antibody analysis. On day 24 ppv, spleens were collected, and splenocytes were isolated to analyze cytokine expression, interferon gamma (IFN-γ) production, and cell population. On day 28 ppv, birds in all groups were infected with H9N2 virus and oral and cloacal swabs were collected for TCID 50 (50 % Tissue Culture Infectious Dose) assay up to day 7 post-infection. The results demonstrated that in ovo administration of RA did not significantly enhance the AIV vaccine-induced antibody response against H9N2 virus compared to the group that received the vaccine alone. However, RA supplementation enhanced the frequency of macrophages (KUL01 + ), expression of inflammatory cytokines and production of IFN-γ by splenocytes. In addition, RA administration reduced oral shedding of AIV on day 5 post-infection. In conclusion, these findings suggest that RA can be supplemented in ovo to enhance AIV vaccine efficacy against LPAIV., Competing Interests: Declaration of Competing Interest Bahram Shojadoost was employed by the company “CEVA Animal Health”. The remaining authors declare that the research was conducted in the absence of any commercial of financial relationship that could be construed as potential conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Over the past decades, avian influenza (AI) outbreaks have been reported across different parts of the globe, resulting in large-scale economic and livestock loss and, in some cases raising concerns about their zoonotic potential. The virulence and pathogenicity of H5Nx (e.g., H5N1, H5N2) AI strains for poultry could be inferred through various approaches, and it has been frequently performed by detecting certain pathogenicity markers in their haemagglutinin (HA) gene. The utilization of predictive modeling methods represents a possible approach to exploring this genotypic-phenotypic relationship for assisting experts in determining the pathogenicity of circulating AI viruses. Therefore, the main objective of this study was to evaluate the predictive performance of different machine learning (ML) techniques for in-silico prediction of pathogenicity of H5Nx viruses in poultry, using complete genetic sequences of the HA gene. We annotated 2137 H5Nx HA gene sequences based on the presence of the polybasic HA cleavage site (HACS) with 46.33% and 53.67% of sequences previously identified as highly pathogenic (HP) and low pathogenic (LP), respectively. We compared the performance of different ML classifiers (e.g., logistic regression (LR) with the lasso and ridge regularization, random forest (RF), K-nearest neighbor (KNN), Naïve Bayes (NB), support vector machine (SVM), and convolutional neural network (CNN)) for pathogenicity classification of raw H5Nx nucleotide and protein sequences using a 10-fold cross-validation technique. We found that different ML techniques can be successfully used for the pathogenicity classification of H5 sequences with ∼99% classification accuracy. Our results indicate that for pathogenicity classification of (1) aligned deoxyribonucleic acid (DNA) and protein sequences, with NB classifier had the lowest accuracies of 98.41% (+/-0.89) and 98.31% (+/-1.06), respectively; (2) aligned DNA and protein sequences, with LR (L1/L2), KNN, SVM (radial basis function (RBF)) and CNN classifiers had the highest accuracies of 99.20% (+/-0.54) and 99.20% (+/-0.38), respectively; (3) unaligned DNA and protein sequences, with CNN's achieved accuracies of 98.54% (+/-0.68) and 99.20% (+/-0.50), respectively. ML methods show potential for regular classification of H5Nx virus pathogenicity for poultry species, particularly when sequences containing regular markers were frequently present in the training dataset., (Copyright © 2023. Published by Elsevier B.V.)

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.

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.

Poultry infection with avian influenza viruses (AIV) is a continuous source of concern for poultry production and human health. Uncontrolled infection and transmission of AIV in poultry increase the potential for viral mutation and reassortment, possibly resulting in the emergence of zoonotic viruses. To this end, implementing strategies to disrupt the transmission of AIV in poultry, including a wide array of traditional and novel methods, is much needed. Vaccination of poultry is a targeted approach to reduce clinical signs and shedding in infected birds. Strategies aimed at enhancing the effectiveness of AIV vaccines are multi-pronged and include methods directed towards eliciting immune responses in poultry. Strategies include producing vaccines of greater immunogenicity via vaccine type and adjuvant application, and increasing bird responsiveness to vaccines by modification of the gastrointestinal tract (GIT) microbiome and dietary interventions. This review provides an in-depth discussion of recent findings surrounding novel AIV vaccines for poultry, including reverse genetics vaccines, vectors, protein vaccines and virus-like particles, highlighting their experimental efficacy among other factors such as safety and potential for use in the field. In addition to the type of vaccine employed, vaccine adjuvants also provide an effective way to enhance AIV vaccine efficacy; therefore, research on different types of vaccine adjuvants and vaccine adjuvant delivery strategies is discussed. Finally, the poultry gastrointestinal microbiome is emerging as an important factor in the effectiveness of prophylactic treatments. In this regard, current findings on the effects of the chicken GIT microbiome on AIV vaccine efficacy are summarized here.

Influenza viruses cause severe respiratory infections in humans and birds, triggering global health concerns and economic burden. Influenza infection is a dynamic process involving complex biological host responses. The objective of this study was to illustrate global biological processes in ileum and cecal tonsils at early time points after chickens were infected with low pathogenic avian influenza virus (LPAIV) H9N2 through transcriptome analysis. Total RNA isolated from ileum and cecal tonsils of non-infected and infected layers at 12-, 24- and 72-h post-infection (hpi) was used for mRNA sequencing analyses to characterize differentially expressed genes and overrepresented pathways. Statistical analysis highlighted transcriptomic signatures significantly occurring 24 and 72 hpi, but not earlier at 12 hpi. Interferon (IFN)-inducible and IFN-stimulated gene (ISG) expression was increased, followed by continued expression of various heat-shock proteins (HSP), including HSP60, HSP70, HSP90 and HSP110. Some upregulated genes involved in innate antiviral responses included DDX60, MX1, RSAD2 and CMPK2. The ISG15 antiviral mechanism pathway was highly enriched in ileum and cecal tonsils at 24 hpi. Overall, most affected pathways were related to interferon production and the heat-shock response. Research on these candidate genes and pathways is warranted to decipher underlying mechanisms of immunity against LPAIV in chickens., (© 2021. The Author(s).)

Avian influenza viruses (AIVs) infect a wide range of hosts, including humans and many avian species. Efforts have been made to control this pathogen in chickens using vaccination programs, but that has been met with varying degrees of success. Therefore, identification of more efficacious vaccination strategies is warranted. This study was undertaken to investigate the potential effects of probiotics on the immunogenicity of a beta-propiolactone-whole inactivated virus (WIV) vaccine of H9N2 subtype adjuvanted with the Toll-like receptor-21 ligand, CpG oligodeoxynucleotides 2007 (CpG). Eighty-four 1-day-old White Leghorn layers were allocated into six groups. Two out of six groups received a mixture of probiotic Lactobacillus spp. (PROB) biweekly from days 1 35 of age. Chickens were intramuscularly vaccinated with WIV either alone or adjuvanted with AddaVax™ (WIV+Add) or CpG (WIV+CpG), and one group received saline (phosphate-buffered saline). Primary and secondary vaccinations occurred at days 14 and 28 of age, respectively. The results revealed that the group that received probiotics and was vaccinated with CpG-adjuvanted WIV H9N2 vaccine had higher hemagglutination inhibition titers than the other treatment groups at days 14 and 21 postprimary vaccination. Probiotics did not induce higher IgM or IgY titers in chickens receiving the WIV vaccine only. Concerning their effect on cell-mediated immune responses, probiotics enhanced interferon-gamma (IFN-γ) gene expression and significantly increased secretion of IFN-γ protein by splenocytes in chickens vaccinated with CpG-adjuvanted WIV H9N2. Together, these findings suggest the use of probiotics to enhance the immunogenicity of CpG-adjuvanted WIV H9N2 vaccines. Additional studies are required to better understand the specific interactions between probiotics and the gut microbiota and different types of cells of the gastrointestinal tract to decipher the underlying mechanisms of how probiotics modulate immune responses to vaccines.

Avian influenza viruses can cause economically devastating diseases in poultry and have the potential for zoonotic transmission. To mitigate the consequences of avian influenza, disease prediction systems have become increasingly important. In this study, we have proposed a framework for the prediction of the occurrence and spread of avian influenza events in a geographical area. The application of the proposed framework was examined in an Indonesian case study. An extensive list of historical data sources containing disease predictors and target variables was used to build spatiotemporal and transactional datasets. To combine disparate sources, data rows were scaled to a temporal scale of 1-week and a spatial scale of 1-degree × 1-degree cells. Given the constructed datasets, underlying patterns in the form of rules explaining the risk of occurrence and spread of avian influenza were discovered. The created rules were combined and ordered based on their importance and then stored in a knowledge base. The results suggested that the proposed framework could act as a tool to gain a broad understanding of the drivers of avian influenza epidemics and may facilitate the prediction of future disease events., Competing Interests: The authors have declared that no competing interests exist.

For years, avian influenza has influenced economies and human health around the world. The emergence and spread of avian influenza virus have been uncertain and sudden. The virus is likely to spread through several pathways such as poultry transportation and wild bird migration. The complicated and global spread of avian influenza calls for surveillance tools for timely and reliable prediction of disease events. These tools can increase situational awareness and lead to faster reaction to events. Here, we aimed to design and evaluate a decision support framework that aids decision makers by answering their questions regarding the future risk of events at various geographical scales. Risk patterns were driven from pre-built components and combined in a knowledge base. Subsequently, questions were answered by direct queries on the knowledge base or through a built-in algorithm. The evaluation of the system in detecting events resulted in average sensitivity and specificity of 69.70% and 85.50%, respectively. The presented framework here can support health care authorities by providing them with an opportunity for early control of emergency situations.

Selenium is a trace mineral that has antioxidant activities and can influence the immune system. However, antiviral effects of selenium have not been well studies in chickens. Chickens were therefore fed diets supplemented with two levels of two different sources of selenium (organic: selenium enriched yeast; SEY or inorganic: sodium selenite; SS). Chickens in the control groups did not receive supplemental dietary selenium. At 14 and 21 days of age, chickens were vaccinated with an inactivated low pathogenicity avian influenza virus (AIV, subtype H9N2) vaccine and blood samples were collected to determine the level of antibodies using hemagglutination inhibition (HI) and ELISA. At 30 days of age, chickens were also challenged with the same virus and swab samples were collected to assess the amount of virus shedding. Antibody levels, as measured by HI, increased significantly in the chickens that received higher levels of SEY at 16 days post vaccination. ELISA titers for IgM and IgY were higher in selenium supplemented chickens. Comparing to challenged control, virus shedding was lower in organic as well as inorganic selenium treated groups. Therefore, it may be concluded that supplemental dietary selenium could enhance vaccine conferred immunity thereby impacting protection against viral challenge in chickens., (Copyright © 2020. Published by Elsevier B.V.)

Chickens infected with avian influenza virus (AIV) transmit the virus via respiratory and cloacal shedding. While previous mathematical models have shown that the innate immune response is necessary for the early suppression of virus production in infected respiratory cells, the different pathways by which the innate immune response can affect cloacal viral shedding have not been studied in chickens. The present study aims to evaluate the sensitivity of H9N2 low pathogenic AIV shedding in chicken gastrointestinal cells to different type-I interferon (IFN) response pathways, and to determine the impact of a cellular eclipse phase (latent period) on the time to peak virus shedding using a mathematical model describing within host viral kinetics. Our model results demonstrate that a mechanistic model that incorporates 1) the intracellular antiviral effects of type-I IFN on virus production, 2) destruction of infected cells by type-I IFN activated Natural Killer cells, and 3) an eclipse phase is most consistent with experimental cloacal virus shedding data. These results provide a potential mechanistic explanation for the delay to peak cloacal virus shedding observed in experimental studies conducted in chickens, as well as an improved understanding of the primary type-I IFN pathways involved in the control of cloacal virus shedding, which may lead to the development of more targeted vaccine candidates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Social media services such as Twitter are valuable sources of information for surveillance systems. A digital syndromic surveillance system has several advantages including its ability to overcome the problem of time delay in traditional surveillance systems. Despite the progress made with using digital syndromic surveillance systems, the possibility of tracking avian influenza (AI) using online sources has not been fully explored. In this study, a Twitter-based data analysis framework was developed to automatically monitor avian influenza outbreaks in a real-time manner. The framework was implemented to find worrisome posts and alerting news on Twitter, filter irrelevant ones, and detect the onset of outbreaks in several countries. The system collected and analyzed over 209,000 posts discussing avian influenza on Twitter from July 2017 to November 2018. We examined the potential of Twitter data to represent the date, severity and virus type of official reports. Furthermore, we investigated whether filtering irrelevant tweets can positively impact the performance of the system. The proposed approach was empirically evaluated using a real-world outbreak-reporting source. We found that 75% of real-world outbreak notifications of AI were identifiable from Twitter. This shows the capability of the system to serve as a complementary approach to official AI reporting methods. Moreover, we observed that one-third of outbreak notifications were reported on Twitter earlier than official reports. This feature could augment traditional surveillance systems and provide a possibility of early detection of outbreaks. This study could potentially provide a first stepping stone for building digital disease outbreak warning systems to assist epidemiologists and animal health professionals in making relevant decisions.

Compounding with the problem of frequent antigenic shift and occasional drift of the segmented genome of Avian Influenza Virus (AIV), vaccines based on major surface glycoproteins such as haemagglutinin (HA) to counter heterosubtypic AIV infection in chickens remain unsuccessful. In contrast, neuraminidase (NA), the second most abundant surface glycoprotein present in viral capsid is less mutable and, in some instances, successful in eliciting inter-species cross-reactive antibody responses. However, without selective activation of B-cells and T-cells, the ability of NA to induce strong cell mediated immune responses is limited, thus NA based vaccines cannot singularly address the risk of virus escape from host defence. To this end, the highly conserved ectodomain of influenza matrix protein-2 (M2e) has emerged as an attractive cross-protective vaccine target. The present study describes the potential of recombinant Lactococcus lactis (rL. lactis) in expressing functional influenza NA or M2e proteins and conferring effective mucosal and systemic immune responses in the intestine as well as in the upper respiratory airways (trachea) of chickens. In addition, lavages collected from trachea and intestine of birds administered with rL. lactis expressing influenza NA or M2e protein were found to protect MDCK cells against avian influenza type A/PR/8/34 (H1N1) virus challenge. Although minor, the differences in the expression of pro-inflammatory cytokines gene transcripts targeted in this study among the birds administered with either empty or rL. lactis could be attributed to the activation of innate response by L. lactis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Macrophages are an important cell type of the innate immune system that upon activation, can exert antiviral functions and also can induce virus-specific adaptive immune responses. Macrophage interaction with certain probiotic bacteria such as lactobacilli can enhance antiviral functions of these cells. We have previously shown that administration of lactobacilli to chickens can effectively augment immune response to vaccine antigens. Here, we investigated the effects of representative strains of three Lactobacillus species, L. acidophilus, L. reuteri and L. salivarius used alone or in combination, in enhancing antiviral activity of chicken macrophages against avian influenza virus in an in vitro model using MQ-NCSU cells. Treatment of macrophages with probiotic lactobacilli significantly enhanced the antiviral functions, as determined by the virus titration assay. We also found that lactobacilli stimulation of macrophages induced significantly higher expression of interleukin (IL)-1β, interferon (IFN)- γ and IFN-α cytokine genes as well as interferon regulatory factor-7 (IRF7), 2',5'-oligoadenylate synthetase (OAS) and interferon-inducible transmembrane protein M3 (IFITM3) genes. Furthermore, macrophages that were treated with lactobacilli had significantly enhanced production of nitric oxide (NO) and IFN-γ protein as well as surface expression of the costimulatory molecule CD40. However, the antiviral and immunostimulatory effects of probiotic lactobacilli largely depended on the Lactobacillus species studied. Collectively, the results from our study using an in vitro model showed that certain Lactobacillus species can effectively augment antiviral responses in chicken macrophages., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The chicken upper respiratory tract is the portal of entry for respiratory pathogens including avian influenza virus (AIV). There is a paucity of information about the role of airway epithelial cells in the induction of antiviral responses in the chicken trachea. A better understanding of the role of these cells in the initiation of innate responses may improve prophylactic or therapeutic strategies for control of viral infections. The present study aimed to characterize antiviral innate responses in chicken tracheal epithelial cells (cTECs) induced by TLR ligands. The results demonstrated that stimulation of cTECs with TLR ligands induced antiviral responses, and subsequently reduced the replication of AIV in cTECs. Additionally, stimulated cTECs were able to influence the function of other cells such as macrophages. Overall, these results provided evidence that cTECs mount antiviral responses after stimulation with TLR ligands through IRF7 and NF-κB signaling pathways, leading to activation of other cells, such as macrophages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the last several decades, avian influenza virus has caused numerous outbreaks around the world. These outbreaks pose a significant threat to the poultry industry and also to public health. When an avian influenza (AI) outbreak occurs, it is critical to make informed decisions about the potential risks, impact, and control measures. To this end, many modeling approaches have been proposed to acquire knowledge from different sources of data and perspectives to enhance decision making. Although some of these approaches have shown to be effective, they do not follow the process of knowledge discovery in databases (KDD). KDD is an iterative process, consisting of five steps, that aims at extracting unknown and useful information from the data. The present review attempts to survey AI modeling methods in the context of KDD process. We first divide the modeling techniques used in AI into two main categories: data-intensive modeling and small-data modeling. We then investigate the existing gaps in the literature and suggest several potential directions and techniques for future studies. Overall, this review provides insights into the control of AI in terms of the risk of introduction and spread of the virus.

Selenium supplementation in poultry feeds has been known to have beneficial effects on the bird health and performance; however antiviral effects of selenium have remained largely unknown. In this study, we have evaluated the effects of supplementation of chicken diets with organic (Selenium Enriched Yeast; SEY) and inorganic selenium (Sodium Selenite; SS) on low pathogenicity avian influenza virus (H9N2) shedding in the cloacal and oropharyngeal swab samples as well as examined the expression of immune related genes. Chickens were fed two doses (High- 0.30 mg/kg of feed; Low- 0.15 mg/kg of feed) of selenium supplementation for 2 weeks followed by low pathogenicity avian influenza virus challenge. Our results showed that the cloacal shedding of virus in all the selenium supplemented groups was significantly lower when compared to the non-supplemented control groups. In addition, the oropharyngeal shedding of virus in chickens fed with organic selenium supplementation was significantly lower than that in the chickens that received either inorganic selenium supplemented feed or controls. Furthermore, the expression of interferon stimulated genes (Viperin, OAS: 2'-5' oligoadenylate synthetase and MDA5: melanoma differentiation-associated gene) in the cecal tonsils was significantly elevated in the selenium treated groups when compared to controls. Additionally, a significantly higher transcription of interferon (IFN)-α, IFN-β and IFN-γ genes in the cecal tonsils and spleens of chickens receiving SEY-L and SS-H supplemented feed was also observed at post virus challenge time points compared to untreated controls. The results of this study demonstrated that supplementation of chicken diets with selenium, can enhance antiviral defense and thus, may have a beneficial effect in controlling viral infections in poultry., (Copyright © 2018. Published by Elsevier B.V.)

Low pathogenic avian influenza virus (AIV) infection in chickens can result in economic losses and has impacts on human health. Poultry vaccination is a tool that can be used to decrease infection and transmission of AIVs. Prior research has demonstrated that Toll-like receptor (TLR) ligands can act as vaccine adjuvants and their addition to inactivated AIV vaccines can enhance immune responses elicited in chickens. The objective of this study was to compare the adjuvant capabilities of TLR5 ligand (flagellin) and TLR21 ligand (CpG ODN 2007) administered either alone or in combination with an intramuscular formaldehyde inactivated H9N2 whole virus vaccine in chickens. Along with the inactivated virus, chickens were administered either a single dose of CpG ODN 2007 (2 or 10 μg), flagellin (0.4 or 2 μg), or a combination of both ligands. An additional group received AddaVax™, an oil emulsion style adjuvant. Chickens were vaccinated twice and serum and lachrymal samples were collected weekly following the primary vaccination, and antibody-mediated immune responses were quantified. Results showed that vaccines containing CpG ODN 2007 induce significantly greater systemic and lachrymal antibody responses than vaccines containing flagellin or AddaVax. Combinations of flagellin and CpG ODN 2007 did not demonstrate inhibitory, additive, or synergistic effects on systemic or lachrymal antibody-mediated immune responses. Additionally, for both flagellin and CpG ODN 2007, a fivefold higher dose of each did not induce significantly higher antibody-mediated immune responses compared with the lesser dose. Future studies should examine the induction of cell-mediated immune responses when flagellin, CpG ODN 2007, or other TLR ligands are administered either alone or combined as adjuvants for inactivated H9N2 AIV vaccines.

Mucosal vaccine delivery systems have paramount importance for the induction of mucosal antibody responses. Two studies were conducted to evaluate immunogenicity of inactivated AIV antigens encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). In the first study, seven groups of specific pathogen free (SPF) layer-type chickens were immunized subcutaneously at 7-days of age with different vaccine formulations followed by booster vaccinations two weeks later. Immune responses were profiled by measuring antibody (Ab) responses in sera and lachrymal secretions of vaccinated chickens. The results indicated that inactivated AIV and CpG ODN co-encapsulated in PLGA NPs (2x NanoAI+CpG) produced higher amounts of hemagglutination inhibiting antibodies compared to a group vaccinated with non-adjuvanted AIV encapsulated in PLGA NPs (NanoAI). The tested adjuvanted NPs-based vaccine (2x NanoAI+CpG) resulted in higher IgG responses in the sera and lachrymal secretions at weeks 3, 4 and 5 post-vaccination when immunized subcutaneously. The incorporation of CpG ODN led to an increase in Ab-mediated responses and was found useful to be included both in the prime and booster vaccinations. In the second study, the ability of chitosan and mannan coated PLGA NPs that encapsulated AIV and CpG ODN was evaluated for inducing antibody responses when delivered via nasal and ocular routes in one-week-old SPF layer-type chickens. These PLGA NPs-based and surface modified formulations induced robust AIV-specific antibody responses in sera and lachrymal secretions. Chitosan coated PLGA NPs resulted in the production of large quantities of lachrymal IgA and IgG compared to mannan coated NPs, which also induced detectable amounts of IgA in addition to the induction of IgG in lachrymal secretions. In both mucosal and subcutaneous vaccination approaches, although NPs delivery enhanced Ab-mediated immunity, one booster vaccination was required to generate significant amount of Abs. These results highlight the potential of NPs-based AIV antigens for promoting the induction of both systemic and mucosal immune responses against respiratory pathogens., Competing Interests: The authors have declared that no competing interests exist.

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.

Objective: Infection of chickens with low pathogenic avian influenza virus, such as H9N2 virus, culminates in decreased egg production and increased mortality and morbidity if co-infection with other respiratory pathogens occurs. We have previously observed the induction of antibody- and cell-mediated immune responses after intramuscular administration of an H9N2 beta-propiolactone inactivated virus vaccine to chickens. Given the fact that in ovo vaccination represents a practical option for vaccination against H9N2 AIV in chickens, in the current study, we set out to characterize immune responses in chickens against a beta-propiolactone inactivated H9N2 virus vaccine after primary vaccination in ovo on embryonic day 18, and secondary intramuscular vaccination on day 14 post-hatch. We also included the Toll-like receptor 21 ligand, CpG ODN 2007, and an oil emulsion adjuvant, AddaVax™, as adjuvants for the vaccines., Results: Antibody-mediated immune responses were observed after administering the secondary intramuscular vaccine. Cell-mediated immune responses were observed in chickens that received the beta-propiolactone inactivated H9N2 virus combined with AddaVax™. Our results demonstrate that adaptive immune responses can be induced in chickens after a primary in ovo vaccination and secondary intramuscular vaccination.

Toll-like receptor (TLR)3 signaling pathway is known to induce type 1 interferons (IFNs) and proinflammatory mediators leading to antiviral response against many viral infections. Double-stranded ribonucleic acid (dsRNA) has been shown to act as a ligand for TLR3 and, as such, has been a focus as a potential antiviral agent in many host-viral infection models. Yet, its effectiveness and involved mechanisms as a mediator against low pathogenic avian influenza virus (LPAIV) have not been investigated adequately. In this study, we used avian fibroblasts to verify whether dsRNA induces antiviral response against H 4 N 6 LPAIV and clarify whether type 1 IFNs and proinflammatory mediators such as interleukin (IL)-1β are contributing to the dsRNA-mediated antiviral response against H 4 N 6 LPAIV. We found that dsRNA induces antiviral response in avian fibroblasts against H 4 N 6 LPAIV infection. The treatment of avian fibroblasts with dsRNA increases the expressions of TLR3, IFN-α, IFN-β, and IL-1β. We also confirmed that this antiviral response elicited against H 4 N 6 LPAIV infection correlates, but is not attributable to type 1 IFNs or IL-1β. Our findings imply that the TLR3 ligand, dsRNA, can elicit antiviral response in avian fibroblasts against LPAIV infection, highlighting potential value of dsRNA as an antiviral agent against LPAIV infections. However, further investigations are required to determine the potential role of other innate immune mediators or combination of the tested cytokines in the dsRNA-mediated antiviral response against H 4 N 6 LPAIV infection.

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Toll like receptor (TLR) 3 is a critically important innate pattern recognizing receptor that senses many viral infections. Although, it has been shown that double stranded (ds) RNA can be used for the stimulation of TLR3 signaling pathway in a number of host-viral infection models, it's effectiveness as an antiviral agent against low pathogenic avian influenza virus (LPAIV) needs further investigation., Methods: In this study, first, we delivered TLR3 ligand, dsRNA, in ovo at embryo day (ED)18 since in ovo route is routinely used for vaccination against poultry viral and parasitic infections and infected with H4N6 LPAIV 24-h post-treatment. A subset of in ovo dsRNA treated and control groups were observed for the expressions of TLR3 and type I interferon (IFN)s, mRNA expression of interleukin (IL)-1β and macrophage recruitment coinciding with the time of H4N6 LPAIV infection (24 h post-treatment). Additionally, Day 1 chickens were given dsRNA intra-tracheally along with a control group and a subset of chickens were infected with H4N6 LPAIV 24-h post-treatment whereas the rest of the animals were observed for macrophage and type 1 IFN responses coinciding with the time of viral infection., Results: Our results demonstrate that the pre-hatch treatment of eggs with dsRNA reduces H4N6 replication in lungs. Further studies revealed that in ovo delivery of dsRNA increases TLR3 expression, type I IFN production and number of macrophages in addition to mRNA expression of IL-1β in lung 24-h post-treatment. The same level of induction of innate response was not evident in the spleen. Moreover, we discovered that dsRNA elicits antiviral response against LPAIV correlating with type I IFN activity in macrophages in vitro. Post-hatch, we found no difference in H4N6 LPAIV genome loads between dsRNA treated and control chickens although we observed higher macrophage recruitment and IFN-β response coinciding with the time of viral infection., Conclusions: Our findings imply that the TLR3 ligand, dsRNA has antiviral activity in ovo and in vitro but not in chickens post-hatch and dsRNA-mediated innate host response is characterized by macrophage recruitment and expressions of TLR3 and type 1 IFNs.

The impact of low pathogenic influenza viruses such as subtype H9N2, which infect the respiratory and the gastrointestinal tracts of chickens, on microbial composition are not known. Twenty-day-old specific pathogen-free chickens were assigned to two treatment groups, control (uninfected) and H9N2-infected (challenged via the oral-nasal route). Fecal genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were sequenced using the Illumina Miseq® platform. Sequences were curated using Mothur as described in the MiSeq SOP. Infection of chickens with H9N2 resulted in an increase in phylum Proteobacteria, and differential enrichment with the genera Vampirovibrio, Pseudoflavonifractor, Ruminococcus, Clostridium cluster XIVb and Isobaculum while control chickens were differentially enriched with genera Novosphingobium, Sphingomonas, Bradyrhizobium and Bifidobacterium. Analysis of pre- and post-H9N2 infection of the same chickens showed that, before infection, the fecal microbiota was characterized by Lachnospiracea and Ruminococcaceae family and the genera Clostridium sensu stricto, Roseburia and Lachnospiraceae incertae sedis. However, post-H9N2 infection, class Deltaproteobacteria, orders Clostridiales and Bacteroidiales and the genus Alistipes were differentially enriched. Findings from the current study show that influenza virus infection in chickens results in the shift of the gut microbiota, and the disruption of the host-microbial homeostasis in the gut might be one of the mechanisms by which influenza virus infection is established in chickens., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytosine-guanosinedeoxynucleotide (CpG) DNA can be used for the stimulation of the toll-like receptor (TLR)21 signalling pathway in avian species which ultimately leads to up-regulation of gene transcription for pro-inflammatory molecules including nitric oxide and recruitment of innate immune cells. The objective of this study was to determine the antiviral effect of NO, produced in response to in ovo delivery of CpG DNA, against avian influenza virus (AIV) infection. We found that when CpG DNA is delivered at embryo day (ED)18 in ovo and subsequently challenged with H4N6 AIV at ED19 pre-hatch and day 1 post-hatching, CpG DNA reduces H4N6 AIV replication associated with enhanced NO production and macrophage recruitment in lungs. In vitro, we showed that NO originating from macrophages is capable of eliciting an antiviral response against H4N6 AIV infection. This study provides insights into the mechanisms of CpG DNA-mediated antiviral response, particularly against AIV infection in avian species.

In poultry, systemic administration of commercial vaccines consisting of inactivated avian influenza virus (AIV) requires the simultaneous delivery of an adjuvant (water-in-oil emulsion). These vaccines are often limited in their ability to induce quantitatively better local (mucosal) antibody responses capable of curtailing virus shedding. Therefore, more efficacious adjuvants with the ability to provide enhanced immunogenicity and protective anti-AIV immunity in chickens are needed. While the Toll-like receptor (TLR) 21 agonist, CpG oligodeoxynucleotides (ODNs) has been recognized as a potential vaccine adjuvant in chickens, poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, successfully tested as vaccine delivery systems in other species, have not been extensively explored. The present study, therefore, assessed both systemic and mucosal antibody-mediated responses following intramuscular vaccination (administered at 7 and 21days post-hatch) of chickens with PLGA encapsulated H9N2 AIV plus encapsulated CpG ODN 2007 (CpG 2007), and nonencapsulated AIV plus PLGA encapsulated CpG 2007 vaccine formulations. Virus challenge was performed at 2weeks post-secondary vaccination using the oculo-nasal route. Our results showed that chickens vaccinated with the nonencapsulated AIV vaccine plus PLGA encapsulated CpG 2007 developed significantly higher systemic IgY and local (mucosal) IgY antibodies as well as haemagglutination inhibition antibody titres compared to PLGA encapsulated AIV plus encapsulated CpG 2007 vaccinated chickens. Furthermore, chickens that received CpG 2007 as an adjuvant in the vaccine formulation had antibodies exhibiting higher avidity indicating that the TLR21-mediated pathway may enhance antibody affinity maturation qualitatively. Collectively, our data indicate that vaccination of chickens with nonencapsulated AIV plus PLGA encapsulated CpG 2007 results in qualitatively and quantitatively augmented antibody responses leading to a reduction in virus shedding compared to the encapsulated AIV plus PLGA encapsulated CpG 2007 formulation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Avian influenza virus (AIV), a mucosal pathogen, gains entry into host chickens through respiratory and gastrointestinal routes. Most commercial AIV vaccines for poultry consist of inactivated, whole virus with adjuvant, delivered by parenteral administration. Recent advances in vaccine development have led to the application of nanoparticle emulsion delivery systems, such as poly (d,l-lactic-co-glycolic acid) (PLGA) nanoparticles to enhance antigen-specific immune responses. In chickens, the Toll-like receptor 21 ligand, CpG oligodeoxynucleotides (ODNs), have been demonstrated to be immunostimulatory. The objective of this study was to compare the adjuvant potential of CpG ODN 2007 encapsulated in PLGA nanoparticles with nonencapsulated CpG ODN 2007 when combined with a formalin-inactivated H9N2 virus, through intramuscular and aerosol delivery routes. Chickens were vaccinated at days 7 and 21 posthatch for the intramuscular route and at days 7, 21, and 35 for the aerosol route. Antibody-mediated responses were evaluated weekly in sera and lacrimal secretions in specific pathogen-free chickens. The results indicate that nonencapsulated CpG ODN 2007 in inactivated AIV vaccines administered by the intramuscular route generated higher antibody responses compared to the encapsulated CpG ODN 2007 formulation by the same route. Additionally, encapsulated CpG ODN 2007 in AIV vaccines administered by the aerosol route elicited higher mucosal responses compared to nonencapsulated CpG ODN 2007. Future studies may be aimed at evaluating protective immune responses induced with PLGA encapsulation of AIV and adjuvants.

Avian influenza viruses (AIV) are of concern to the poultry industry. Outbreaks of AIV highlight the urgent need for effective control measures. Prophylactic strategies should be explored that rapidly elicit immunity against the virus. Toll-like receptors (TLRs) are innate immune molecules that can induce anti-viral responses, therefore the application of TLR ligands as prophylactic agents in chickens is gaining more attention. We hypothesized that treatment of chickens with TLR ligands reduces the shedding of AIV from infected birds. In addition, the effects of TLR ligand dose and route of administration on the efficiency of TLR ligands to reduce AIV shedding were examined. Chickens were treated with TLR2, 4, 7 and 21 ligands using different doses and routes of administration, 18h before AIV infection. Moreover, the expression of several candidate genes, such as type I interferons, PKR, OAS, viperin and IFITM3 was quantified at 3, 8 and 18h post-treatment with TLR ligands. The results revealed that route of administration and dosage affect the efficacy of TLR ligands to reduce virus shedding. Furthermore, varying effects were observed when different ligands were applied. Our results demonstrated that all TLR ligand treatments reduced AIV shedding, with the CpG-ODN 1826 being the most efficacious to reduce oral virus shedding, whereas LPS from Escherichia coli 026:B6 resulted in the largest reduction in cloacal virus shedding. Moreover, TLR ligands induced the expression of genes involved in antiviral responses such as type I interferons and interferon-stimulated genes in chicken trachea and cecal tonsils. These results raise the possibility of treatment of chickens with TLR ligands as anti-viral agents., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Commercial vaccines against avian influenza viruses (AIV) in chickens consist mainly of inactivated AIV, requiring parenteral administration and co-delivery of an adjuvant. Limitations in T helper 1 or T helper 2 biased responses generated by these vaccines emphasize the need for alternative, more efficacious adjuvants. The Toll-like receptor (TLR) 21 ligand, CpG oligodeoxynucleotides (ODN), has been established as immunomodulatory in chickens. Therefore, the objective of this study was to investigate the adjuvant potential of high (20μg) and low (2μg) doses of CpG ODN 2007 (CpG 2007) and CpG ODN 1826 (CpG 1826) when administered to chickens with a formalin-inactivated H9N2 AIV. Antibody responses in sera were evaluated in 90 specific pathogen free (SPF) chickens after intramuscular administration of vaccine formulations at 7 and 21 days post-hatch. Antibody responses were assessed based on haemagglutination inhibition (HI) and virus neutralization (VN) assays; virus-specific IgM and IgY antibody responses were evaluated by ELISA. The results suggest that the vaccine formulation containing low dose CpG 2007 was significantly more effective at generating neutralizing (both HI and VN) responses than formulations with high or low doses of CpG 1826 or high dose CpG 2007. Neutralizing responses elicited by low dose CpG 2007 significantly exceeded those generated by a squalene-based adjuvanted vaccine formulation during peak responses. A significantly higher IgM response was elicited by the formulation containing low dose CpG 2007 compared to high and low doses of 1826. Although the low dose of CpG 2007 elicited a higher IgY response than CpG 1826, the difference was not statistically significant. In conclusion, 2μg of CpG 2007 is potentially promising as a vaccine adjuvant when delivered intramuscularly with inactivated H9N2 virus to chickens. Future studies may be directed at determining the mucosal antibody responses to the same vaccine formulations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study related the replication of an H9N2 avian influenza virus in chickens to the induction of host acute immune response after aerosol or intranasal inoculation with the virus. On 1, 2, 4, and 7 days postinoculation (dpi), oropharyngeal swabs and tissue specimens of trachea, lungs, spleen, and cecal tonsils were collected for quantification of viral RNA. Expression of cytokine genes in lungs, spleen, and cecal tonsils was quantified by reverse transcriptase-PCR. Virus was detected in all oropharyngeal swabs up to 4 dpi in chickens from both inoculation groups. However, virus was detected more frequently (P<0.05) and in higher titers (1-4 log difference) in specimens of trachea and lungs from the group exposed to aerosols than from the group given intranasal drops. In accord with viral replication findings, expressions of cytokine genes interleukin (IL)-1β (on 2 and 7 dpi), IL-6 (on 2 dpi), and interferon (IFN)-γ (on 2 and 4 dpi) were up-regulated to a significantly higher level (P<0.05) in lung tissue specimens from the group exposed to virus aerosol than from controls that were given saline intranasally. Only IFN-γ on 1 dpi was up-regulated (P<0.05) above that of controls in lung tissue specimens from the group given intranasal drops of virus. In comparison, replication of the virus and induction of IL-1β and IL-6 genes were limited in spleen and cecal tonsil tissue specimens from both groups of chickens inoculated with the virus. These findings indicate that virus administered in aerosols was more efficient than virus administered as intranasal drops, in infecting the lower respiratory tract and in inducing the activity of the cytokine genes. The intense respiratory infection caused by virus aerosols might increase the shedding and transmission of the H9N2 virus in chickens.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Avian influenza viruses (AIV) of the H5N1 subtype have caused morbidity and mortality in humans. Although some migratory birds constitute the natural reservoir for this virus, chickens may play a role in transmission of the virus to humans. Despite the importance of avian species in transmission of AIV H5N1 to humans, very little is known about host immune system interactions with this virus in these species. The objective of the present study was to identify putative T cell epitopes of the hemagglutinin (HA) antigen of an H5 AIV in chickens. Using an overlapping peptide library covering the HA protein, we identified a 15-mer peptide, H5(246-260,) within the HA1 domain which induced activation of T cells in chickens immunized against the HA antigen of an H5 virus. Furthermore, H5(246-260) epitope was found to be presented by both major histocompatibility complex (MHC) class I and II molecules, leading to activation of CD4+ and CD8+ T cell subsets, marked by proliferation and expression of interferon (IFN)-gamma by both of these cell subsets as well as the expression of granzyme A by CD8+ T cells. This is the first report of a T cell epitope of AIV recognized by chicken T cells. Furthermore, this study extends the previous finding of the existence of dual-specific epitopes in other species to chickens. Taken together, these results elucidate some of the mechanisms of immune response to AIV in chickens and provide a platform for creation of rational vaccines against AIV in this species.