P-FPMI-67 - P-FPMI-67
Briefly, a 2-3 m section of jejunum was transected and isolated from the functional digestive tract without compromising the mesenteric attachment or altering intestinal contents. The continuity of the remaining digestive tract was re-established by end-to-end anastomoses and the isolated section of jejunum was closed at both ends by inversion sutures. This section was sub-divided using silk ligatures into 10-15 cm long segments, designated as loops and each loop was separated by 15-70 cm long interspaces. These titrations were performed over a 20-fold range (50-1000 µL of stock virus) using field isolate BRV (3.5 X 107 pfu/mL) diluted to a total volume of 5 mL with calcium and magnesium-free phosphate-buffered saline, (PBS) [0.137 M NaCl (Sigma- Aldrich Canada Ltd., Oakville, ON, Canada), 2.7 mM KCl (Sigma-Aldrich), 8 mM Na2HPO4 (Sigma-Aldrich), 1.47 mM KH2PO4 (Sigma-Aldrich) ; pH 7.3] and injected into duplicate loops.
Duplicate control loops were mock-infected with 5 mL of PBS for a total of at least four infected loops and two control loops/animal. Four animals were used. Calves were treated post-surgery with enrofloxacin (Baytril, Bayer Inc. Animal Health, Toronto, ON, Canada) and flunixine meglumine (Cronyxin, Bioniche Animal Health Canada, Belleville, ON, Canada) and euthanized 18 h post-infection. Intestinal contents were collected from each intestinal loop at the time of tissue collection and the total volume of fluid/loop was recorded.
Tissue infected with 500 µL of BRV stock and their respective animal matched PBS control loops were used for microarray analysis and these tissues were collected immediately following euthanasia, rinsed in PBS, and stored in RNAlater (Ambion, Inc. Austin, TX, USA) for 24 hours at room temperature before being frozen at 20°C to preserve the RNA integrity.