As described below, repeated measures of spleen volume and cell content were made https://www.selleckchem.com/products/AZD6244.html in four inoculated calves whereas change in regional distribution of phenotyped cells was determined by sequential euthanasia of six inoculated calves in comparison with two un-inoculated calves. Magnetic resonance imagery was performed with a 1·0 Tesla machine (Philips Intera, Andover, MA, USA). Sequences were acquired in a dorsal plane. The area imaged was from the spine to the ventral abdominal wall. A 40 cm field-of-view ensured that the entire spleen could be visualized. One-centimetre-thick

slices with a 2 mm gap were acquired using a short tau inversion recovery (STIR) sequence. This sequence resulted in a hyperintense spleen on a low intense background. The volume was calculated by tracing the outline of the spleen for the area on each slice and multiplying by the number of slices plus gap thickness

(3D-DOCTOR; Able Software Corporation, Lexington, MA, USA). Each calf’s spleen volume was calculated on the day prior to infection and then at 11 or 12 dpi, 2 calves each. Immediately following each MRI procedure, a 1 cm3 biopsy of marsupialized spleen was removed under local check details lidocaine anaesthesia for determining differential cell counts. Each biopsy was immediately processed into a single cell suspension using a tissue grinder (Tenbroek; Bellco Glass, Inc., NJ, USA), suspended in 50 mL of PBS and enumerated for differential cell counts by standard methods used for whole blood (28). Six inoculated calves were euthanized by captive bolt and jugular exsanguination Dimethyl sulfoxide for collection

of spleen tissue: one calf each on dpi 7, 8, 9 (fever day 1) and 14 (fever day 5), and two calves at 13 dpi (fever days 4 and 5). In this way, the spleens from three calves each were examined from two periods: a period just prior to, or including, the initiation of fever (7, 8 and 9 dpi) and a period several days after fever initiation (13 and 14 dpi). Spleen tissue from two uninfected calves was similarly collected. Multiple 15 × 15 × 5 mm sections of spleen were collected from each calf immediately posteuthanasia. Each section was placed into a cryostat mould containing Tissue-Tek® O.C.T.™ Compound (Sakura Fineteck USA, Inc., Torrance, CA, USA), snap frozen by floating on liquid nitrogen, and stored at −80°C. Cryostat sections (15 μm) were mounted on standard SuperFrost™ Plus slides (Electron Microscopy Services, Hatfield, PA, USA), fixed in 95% EtOH for 10 min and allowed to air dry overnight at room temperature. Formalin-fixed, paraffin-embedded samples of spleen were also collected from each calf and routinely stained in haematoxylin and eosin (H&E). Immunolabelling was carried out at room temperature in a humidified chamber. A Super PAP Pen HT™ (Research Products International Corp., Mt. Prospect, IL, USA) was used to create a hydrophobic margin to retain fluid reagents on slides.