Runting-Stunting poultry disease overview

Set of metagenomes to investigate the ethiology of the Runting-Stunting poultry disease. One day old specific pathogen-free broilers (USDA-ARS, SEPRL, Athens, GA) were orally infected with 1 ml of gut content from 12-day-old commercial broiler chickens which showed the typical signs of runting-stunting-syndrome (RSS) in chicken (growth retardation > 40%, cystic lesions in the small intestine). Before inoculation, the gut content of RSS affected chicken was centrifuged at 4°C for 30 min at 3000 x g. the obtained supernatant was filtered first through a 0.45 mkm filter followed by filtration through a 0.22 um filter. A second group of broilers was mock-infected with phosphate buffered saline. Five days, 8 d, and 12 d after infection, 10 birds of each group were euthanized and necropsy was performed. The duodenal loop was taken for histological examination. The analysis of the sections showed that cystic lesions were only present in the infected group. The highest number of lesions was observed at 8 d after infection. Based on this result the gut content harvested at 5 d after infection was used for subsequent experiments. The purification of the gut content was performed following a multi-step centrifugation protocol. In a first step, the samples were centrifuged at 16000 x g to remove cellular organelles and debris. The obtained supernatant was filtered twice as described above. Next the filtrate was centrifuged through a 10% sucrose cushion made in TEN buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.5) at 174899 x g for 3h. The obtained pellets (RSS+, RSS-) were resuspended in 400 mkl TEN buffer. To purify nucleic acids, the RNA and DNA localized outside of viral particles needed to be degraded. To this end, 40 mkl of 10x DNA I buffer (Roche), 20 units of DNase I (Roche) and 10 ug of RNase I (Roche) was added to 360 mkl of the viral suspension. The mixture was incubated for 1 h at 37C. Both samples (RSS+, Con) were then split and 200 mkl of each sample was used for purification of either the DNA (QIAamp DNA Blood Mini Kit, Qiagen) or RNA (High Pure RNA isolation Kit, Roche) following the manufacturers instructions. The resulting samples (RSS+ RNA, RSS- RNA, RSS+ DNA and RSS- DNA) were amplified separately using two different protocols to amplify the metagenome (called respectively ChickenRuntingStuntingPRnaVir2008, ChickenRuntingStuntingMRnaVir2008, ChickenRuntingStuntingPDnaVir2008 and ChickenRuntingStuntingMDnaVir2008). The RNA containing samples were amplified using the Transplex Whole Transcriptome Amplification Kit (Sigma). The amplification of the DNA library for both DNA samples was performed using GenomiPhi V2 DNA Amplification Kit (GE Healthcare). Both protocols were applied as recommended by the manufacturer. The resulting cDNA library was submitted to 454 Life Science for sequencing using the GS-FLX platform. Duplicate sequences were removed.