Magmax lysis binding solution

Fecal samples were maintained frozen at −80 °C until processed. A 300-μg sample of feces was placed into 1 mL of PBS and mixed thoroughly by vortexing for ~3 min. Solids were removed by centrifugation for 30 s at 100× g. Avoiding solid material, 175 mL of the supernatant was transferred to a tube containing 232 µL of MagMax lysis/binding solution (Thermo Fisher) prepared in accordance with the manufacturer’s instructions. These tubes also contained ~125 µL of 1.0 mm glass beads (Biospec Products), and ~1258 µL of 0.1 mm glass beads (Biospec Products). Bacilli were disrupted by shaking in a bead beater (Biospec Products) for 5 min. The sample was clarified by centrifugation at 16,000× g for 3 min. Using 20 µL of the clarified preparation DNA was isolated using the MagMAX total nucleic acid isolation kit; (Thermo Fisher), as instructed by the manufacturer. The IS900 element was detected by PCR, which was performed as described above.

Oral, nasal, and rectal swabs were collected prior to euthanasia and placed immediately into 2 mL viral transport medium (PBS containing 1% BSA with double strength antibiotic/antimycotic solution [Thermo Fisher Scientific]) and urine was collected by manual expression of the bladder; all specimens were stored at -80°C. At post mortem examination, blood, lymph node, salivary gland, lung, heart, liver, kidney, spleen, jejunum, ileum, large intestine, gonad and brain were collected for viral RNA detection, virus isolation, host gene detection, histology, immunohistology and proteomics. Tissue samples were collected into either 800 μL Magmax lysis/binding solution (Thermo Fisher Scientific) containing 1 mm stainless steel beads for RNA extraction or 10% neutral buffered formalin (Australian Biostain Pty. Ltd. Traralgon VIC) for 48 h prior to routine processing for histology. For proteomic analysis, tissues were homogenised in a ratio of 1:10 sample to SDT buffer (4% (w/v) SDS, 100 mM DTT, 100 mM Tris-HCl pH 7.6). Samples were then boiled at 95°C for 5 min and lysates were cleared by centrifugation at 13000 x g for 10 min.