Infection of chickens with low pathogenicity avian influenza (LPAI) virus results in mild clinical signs while infection with highly pathogenic avian influenza (HPAI) viruses causes death of the birds within 36-48 hours. Since natural killer (NK) cells have been shown to play an important role in influenza-specific immunity, we hypothesise that NK cells are involved in this difference in pathogenicity. To investigate this, the role of chicken NK-cells in LPAI virus infection was studied. Next activation of lung NK cells upon HPAI virus infection was analysed. Infection with a H9N2 LPAI virus resulted in the presence of viral RNA in the lungs which coincided with enhanced activation of lung NK cells. The presence of H5N1 viruses, measured by detection of viral RNA, did not induce activation of lung NK cells. This suggests that decreased NK-cell activation may be one of the mechanisms associated with the enhanced pathogenicity of H5N1 viruses.

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.

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.

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.

Infectious Bronchitis Virus, a member of the Coronaviridae, is a respiratory pathogen in poultry. We found that in vitro stimulation with IBV resulted in ChIFN-gamma production in splenocytes of both infected birds and uninfected birds. The non-specific stimulation did not occur when other avian viruses or other coronaviruses were used or when mammalian cells were stimulated with IBV. Inactivation of IBV reduced ChIFN-gamma production, but ChIFN-gamma remained elevated compared to unstimulated cells. An increase in ChIFN-gamma mRNA was detected in splenocytes from IBV-infected and uninfected chickens as early as 1 h after stimulation with IBV. These results indicate that IBV acts as a polyclonal stimulus, inducing a rapid production of IFN-gamma even without previous exposure to the virus. (c) 2008 Elsevier Inc. All rights reserved.

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 4 dpi, 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 4 dpi, 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 7 dpi 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-1β, IL-6, IL-8, IL-10, IL-18 and IFN-γ mRNA was found 1 dpi. 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.

The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-γ) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-γ production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-γ production by CD4+ and CD8+ cells.

Infection of chickens with low pathogenicity avian influenza (LPAI) virus results in mild clinical signs while infection with highly pathogenic avian influenza (HPAI) viruses causes death of the birds within 36–48 hours. Since natural killer (NK) cells have been shown to play an important role in influenza-specific immunity, we hypothesise that NK cells are involved in this difference in pathogenicity. To investigate this, the role of chicken NK-cells in LPAI virus infection was studied. Next activation of lung NK cells upon HPAI virus infection was analysed. Infection with a H9N2 LPAI virus resulted in the presence of viral RNA in the lungs which coincided with enhanced activation of lung NK cells. The presence of H5N1 viruses, measured by detection of viral RNA, did not induce activation of lung NK cells. This suggests that decreased NK-cell activation may be one of the mechanisms associated with the enhanced pathogenicity of H5N1 viruses.