Abstract: Infection with highly pathogenic avian influenza (HPAI) in birds and mammals is associated with severe pathology and increased mortality. We hypothesize that in contrast to low pathogenicity avian influenza (LPAI) infection, HPAI infection of chicken dendritic cells (DC) induces a cytokine deregulation which may contribute to their highly pathogenic nature. Infection of DC with LPAI H7N1 and H5N2 resulted in viral RNA and NP expression without increase in time, in contrast to HPAI H7N1 and H5N2 mRNA expression. No increase in IFN mRNA was detected after infection with LPAI, but after LPAI H5N2, and not LPAI H7N1, infection the level of bioactive IFNα/β significantly increased. After HPAI H7N1 and H5N2 infection, significant increases in IL-8, IFN-α, IFN-γ mRNA expression and in TLR1, 3, and 21 mRNA were observed. This enhanced activation of DC after HPAI infection may trigger deregulation of the immune response as seen during HPAI infection in chickens.
Abstract: Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds.
Abstract: Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.
Abstract: In the poultry industry, infections with avian influenza virus (AIV) can result in significant economic losses. The risk and the size of an outbreak might be restricted by vaccination of poultry. A vaccine that would be used for rapid intervention during an outbreak should be safe to use, highly effective after a single administration and be suitable for mass application. A vaccine that could be applied by spray or aerosol would be suitable for mass application, but respiratory applied inactivated influenza is poorly immunogenic and needs to be adjuvanted. We chose aluminum OH, chitosan, cholera toxin B subunit (CT-B), and Stimune as adjuvant for an aerosolized vaccine with inactivated H9N2. Each adjuvant was tested in two doses. None of the adjuvanted vaccines induced AIV-specific antibodies after single vaccination, measured 1 and 3 weeks after vaccination by aerosol, in contrast to the intramuscularly applied vaccine. The aerosolized vaccine did enter the chickens' respiratory tract as CT-B-specific serum antibodies were detected after 1 week in chickens vaccinated with the CT-B-adjuvanted vaccine. Chickens showed no adverse effects after the aerosol vaccination based on weight gain and clinical signs. The failure to detect AIV-specific antibodies might be due to the concentration of the inactivated virus.
Abstract: Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells. Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8alpha+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers. Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function. In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens.
Abstract: Mammalian collectins have been found to play an important role in the defense against influenza A virus H9N2 inoculation, but for chicken collectins this has not yet been clarified. The aim of this study was to determine the effect of avian influenza A virus (AIV) inoculation on collectin gene expression in the respiratory tract of chickens and whether this was affected by age. For this purpose 1- and 4-week-old chickens were inoculated intratracheally with PBS or H9N2 AIV. Chickens were killed at 0, 8, 16 and 24h post-inoculation and trachea and lung were harvested for analysis. Viral RNA expression and mRNA expression of chicken collectins 1 and 2 (cCL-1 and cCL-2), chicken lung lectin (cLL) and chicken surfactant protein A (cSP-A) were determined using real-time quantitative RT-PCR. In lung, a decrease in mRNA expression of cCL-2, cLL and cSP-A after inoculation with H9N2 was seen in both 1- and 4-week-old birds, although at different time points, while in trachea changes were only seen in 4-week-old birds and expression was increased. Moreover, collectin expression correlated with viral RNA expression in lung of 1-week-old birds. These results suggest that both age and location in the respiratory tract affect changes in collectin mRNA expression after inoculation with H9N2 and indicate a possible role for collectins in the host response to AIV in the respiratory tract of chickens.
Abstract: Newly hatched chickens are more susceptible to infectious diseases than older birds because of an immature immune system. The aim of this study was to determine to what extent host responses to avian influenza virus (AIV) inoculation are affected by age. Therefore, 1- and 4-week (wk) old birds were inoculated with H9N2 AIV or saline. The trachea and lung were sampled at 0, 8, 16 and 24h post-inoculation (h.p.i.) and gene expression profiles determined using microarray analysis. Firstly, saline controls of both groups were compared to analyse the changes in gene profiles related to development. In 1-wk-old birds, higher expression of genes related to development of the respiratory immune system and innate responses were found, whereas in 4-wk-old birds genes were up regulated that relate to the presence of higher numbers of leukocytes in the respiratory tract. After inoculation with H9N2, gene expression was most affected at 16 h.p.i. in 1-wk-old birds and at 16 and 24h.p.i. in 4-wk-old birds in the trachea and especially in the lung. In 1-wk-old birds less immune related genes including innate related genes were induced which might be due to age-dependent reduced functionality of antigen presenting cells (APC), T cells and NK cells. In contrast cytokine and chemokines gene expression was related to viral load in 1-wk-old birds and less in 4-wk-old birds. Expression of cellular host factors that block virus replication by interacting with viral factors was independent of age or tissue for most host factors. These data show that differences in development are reflected in gene expression and suggest that the strength of host responses at transcriptional level may be a key factor in age-dependent susceptibility to infection, and the cellular host factors involved in virus replication are not.
Abstract: To gain more insight in underlying mechanisms correlating to protection against avian influenza virus (AIV) infection, we investigated correlates of protection after AIV H9N2 infection and studied the contribution of different adjuvants to a protective response at host transcriptional level. One-day-old chickens were immunised with inactivated H9N2 supplemented with w/o, Al(OH)(3), CpG or without adjuvant. Two weeks later, birds were homologously challenged and at 1-4 days post challenge (d.p.c.) trachea and lung were collected. Birds immunised with H9N2+w/o or H9N2+Al(OH)(3) were protected against challenge infection and had lower viral RNA expression, less immune related genes induced after challenge, a lower amplitude of change of gene expression and smaller cellular influxes compared to the higher and prolonged gene expression in unprotected birds. We show that a limited number of differentially expressed genes correlates with reduced immune activation and subsequently reduced immunopathology after challenge with AIV.
Abstract: Interleukin-7 (IL-7) is a central regulator of T cell survival and homeostasis and its expression is indicative for naïve and memory T cells. We cloned chicken IL-7Ralpha (CHIL-7Ralpha) and determined its expression profile in chicken lymphocyte subpopulations. The predicted protein sequence contained 460 amino acids. The extracellular domain exhibited features typical of a type I cytokine receptor; a fibronectin type III domain and the GXWSXWS motif were conserved. ChIL-7Ralpha mRNA is highly expressed in lymphoid organs and in CD4+, CD8alpha+ and CD8beta+ cells. A monoclonal antibody was generated and expression of the protein investigated. ChIL-7Ralpha was expressed on CD4+ and CD8alpha+, but not CD8beta+, T cells, in contrast to the high mRNA expression levels in all of these cells. Upon polyclonal stimulation with ConA, IL-7Ralpha was rapidly down-regulated on T cells, suggesting that in the chicken expression of this receptor might also be correlated to the T cell activation status.
Abstract: Sampling the complete organ instead of defined parts might affect analysis at both the cellular and transcriptional levels. We defined host responses to H9N2 avian influenza virus (AIV) in trachea and different parts of the lung. Chickens were spray-inoculated with either saline or H9N2 AIV. Trachea and lung were sampled at 1 and 3 days post-inoculation (p.i.) for immunocytochemistry, real-time quantitative RT-PCR and gene-expression profiling. The trachea was divided into upper and lower parts and the lung into four segments, according to anatomy and airflow. Two segments contained the primary and secondary bronchi, cranial versus caudal (parts L1 and L3), and two segments contained the tertiary bronchi, cranial versus caudal (parts L2 and L4). Between the upper and lower trachea in both control and infected birds, minor differences in gene expression and host responses were found. In the lung of control birds, differences in anatomy were reflected in gene expression, and in the lung of infected birds, virus deposition enhanced the differences in gene expression. Differential gene expression in trachea and lung suggested common responses to a wide range of agents and site-specific responses. In trachea, site-specific responses were related to heat shock and lysozyme activity. In lung L1, which contained most virus, site-specific responses were related to genes involved in innate responses, interleukin activity and endocytosis. Our study indicates that the anatomy of the chicken lung must be taken into account when investigating in vivo responses to respiratory virus infections.
Abstract: Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4dpi, the number of KUL-01(+) PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1beta, IL-6, IL-8, IL-10, IL-18 and IFN-gamma mRNA was found 1dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.
