Application of chicken microarrays for gene expression analysis in other avian species

BACKGROUND: With the threat of emerging infectious diseases such as avian influenza, whose natural hosts are thought to be a variety of wild water birds including duck, we are armed with very few genomic resources to investigate large scale immunological gene expression studies in avian species. Multiple options exist for conducting large gene expression studies in chickens and in this study we explore the feasibility of using one of these tools to investigate gene expression in other avian species. RESULTS: In this study we utilised a whole genome long oligonucleotide chicken microarray to assess the utility of cross species hybridisation (CSH). We successfully hybridised a number of different avian species to this array, obtaining reliable signals. We were able to distinguish ducks that were infected with avian influenza from uninfected ducks using this microarray platform. In addition, we were able to detect known chicken immunological genes in all of the hybridised avian species. CONCLUSION: Cross species hybridisation using long oligonucleotide microarrays is a powerful tool to study the immune response in avian species with little available genomic information. The present study validated the use of the whole genome long oligonucleotide chicken microarray to investigate gene expression in a range of avian species

Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection

Characterization of the chicken and duck response to H5N1 avian influenza infection

© 2011 Dr. Simon BurggraafAvian influenza viruses are increasingly widespread in poultry and show varied disease severity depending on their hemagglutinin and neuraminidase structure. Whilst many influenza viruses, such as the H5N3 subtype, are of low-pathogenicity, H5N1 influenza viruses result in rapid mortality that in poultry occurs in a matter of hours. The mechanisms of disease pathogenesis are still somewhat poorly understood. Ducks often appear asymptomatic, with few strains of H5N1 causing severe pathogenesis. The gradual progression of infection in ducks contrasts the rapid nature of infection in chickens and it is not clear how this difference in virulence comes about. One possibility is that the innate immune response in chickens and ducks varies during infection and this may be critical to the clinical outcome. With this in mind, we investigated the expression of several key proinflammatory cytokines following infection of chickens and ducks with highly-pathogenic H5N1 and low-pathogenic H5N3 influenza virus. Two H5N1 strains, A/Muscovy duck/Vietnam/453/2004 (Vt453) and A/Duck/Indramayu/BBVW/109/2006 (Ind109) were compared, as well as the low pathogenic H5N3 A/Duck/Victoria/1462/2008 (Vc1462) strain. Intriguingly, in the chicken, H5N1 viruses caused fatal infections, a high viral load and increased production of proinflammatory molecules. Inflammatory molecules such as IL6, IFNγ, the acute phase reactant SAA and also NO inducing gene iNOS, were raised by up to 80 fold at 24 hours post infection. Meanwhile, infection with the Vc1462 H5N3 influenza strain induced a comparably low cytokine response in chickens. In contrast, ducks displayed only small changes in these cytokines and this only occurred later in the infection period. Only the Vt453 H5N1 strain caused mortality in ducks and this was associated with increased levels of cytokines, such as IFNγ and iNOS, as well as increased virus replication in the lung and heart. These observations support the belief that hypercytokinemia may contribute to pathogenesis in chickens, whilst the lower cytokine response in ducks may explain their resistance to disease and decreased mortality. Given the increased levels of inflammatory molecules and the observation that IL6, a pleitropic inflammatory gene, is highly upregulated in the sera of H5N1 infected patients, it appeared likely that IL6 had an impact on hypercytokinemia induced disease severity. Since IL6 signalling results in increased levels of downstream inflammatory molecules, potentially triggering hypercytokinemia, we aimed to suppress the levels of IL6 during H5N1 infection. To investigate whether a more moderated IL6 response may improve the severity of H5N1 infection in chickens, we aimed to inhibit the signalling of IL6 using Madindoline-A (Mad-A) and Galielallactone (Gal). Following H5N1 infection, chickens treated with Mad-A/Gal inhibitors had reduced levels of IL6 and IL6-stimulated genes, such as SAA and AGP as well as lower NO and iNOS levels. However, even though this decreased IL6 response was associated with reduced viral titres, chickens did not appear to have increased survival. Nevertheless, these studies provide insight into the role possible therapeutics could play to target inflammation and improve the immune response during H5N1 infection. Therefore, examining the key components of the inflammatory response during H5N1 induced hypercytokinemia in chickens and ducks may help us understand how to ameliorate the initial hyperinflammatory response and prolong survival so that an adaptive response which alleviates viral replication can commence

Abnormal facial appearance, body asymmetry, limb deformities, and internal malformations

We describe a newborn girl with multiple congenital anomalies and abnormal phenotype comprising underdeveloped corpus callosum with ventriculomegaly, chorioretinal atrophy, pulmonary arterial hypertension, annular pancreas, horseshoe kidney, asymmetric limb and chest anomalies, spinal segmentation defects, hypertrichosis, and unusual face with large anterior fontanel, high anterior hairline, broad forehead, mildly underdeveloped midface, hypertelorism, depressed nasal bridge, short and upturned nose, large mouth, retrognathia, and large and malformed ears. Work-up included cytogenetic studies of lymphocytes and skin fibroblasts, subtelomere Multiplex Ligation-dependent Probe Amplification (MLPA), whole-genome oligo-array, and molecular analysis of SETBP1 and NSDHL: no abnormalities were found. Mucopolysaccharide urinary excretion was elevated. Results of metabolic studies for sterol and peroxisomal abnormalities in fibroblasts were normal. Additional electronic microscopy studies in skin fibroblasts did not show evidence for storage in fibroblasts or lysosomal changes. Nosologic considerations allowed exclusion of SchinzelGiedion and Urioste syndrome. This condition seems not to have been described before; a segregating Mendelian mutation is assumed. (C) 2011 Wiley Periodicals, In

H5N1 infection appears more widespread in chicken tissues in comparison to duck tissues.

<p>IHC for H5N1 antigen, left-hand panel shows staining in chicken tissues, right-hand panel shows staining in duck tissues (<b>A</b>) Chicken lung 24 hours post infection (h.p.i), with IHC stain showing H5N1 viral antigen as red/brown colour. (<b>B</b>) Duck lung 72 h.p.i., H5N1 antigen was less prevalent in duck lung tissue than in chicken. H5N1 antigen was detected in single cells scattered within the lung parenchyma and in the hyaline cartilage. (<b>C</b>) Chicken caecal tonsil 24 h.p.i., with H5N1 in caecal lymphoid follicles and submucosa. (<b>D</b>) Duck caecal tonsil 72 h.p.i., with similar H5N1 antigen staining. (<b>E</b>) Chicken liver tissue 24 h.p.i., with severe H5N1 antigen staining. (<b>F</b>) Duck liver 72 h.p.i., showed no signs of viral antigen. (<b>G</b>) Chicken heart tissue 24 h.p.i. showed H5N1 staining in the myocardium and typically near blood vessels. (<b>H</b>) Duck heart tissue 72 h.p.i., with IHC H5N1 antigen staining the myocardium. All scale bars  = 50 µm. (<b>I</b>) The graph shows viral replication efficiency between chickens and ducks across a range of organs following H5N1 Vt453 infection. In chickens 24 h.p.i, lung, caecal tonsil, liver and heart tissue showed between 5.5 and 7 log₁₀ TCID₅₀. Similarly, ducks had 7 log₁₀ TCID₅₀ of virus in heart tissue, but moderately less virus in other tissues, between 2.5 and 4 log₁₀ TCID₅₀.</p

Comparison of basal iNOS levels in chicken and duck tissues measured by QRT-PCR.

<p>iNOS mRNA was measured by QRT-PCR in chicken and duck organs. GAPDH was used as a housekeeping gene to standardize results. Expression is shown as the basal fold change increase of iNOS as relative to muscle tissue. Experiments were performed in triplicate with the data representative of 3 independent experiments. An asterisk indicates statistically significant differences of means with p<0.05.</p

H5N1 influenza infection results in increased serum NO levels in both chickens and ducks.

<p>Serum collected from chickens and ducks infected with the H5N1 strain Muscovy/duck/Vietnam/453, as assayed for NO production during the peak of influenza infection (chickens at 24 hours, ducks at 24 and 72 hours post infection). Displayed values are the mean of 2 experiments with 4 birds in each group. A single asterisk indicates statistically significant differences of means with p<0.05, double asterisk indicates statistically significant differences of means with p<0.01.</p