The animals were sedated (Nielsen et al., 2009b) and a catheter (22 G) was inserted into the left ear vein for inoculation (1 mL BW−1) of a saline suspension (108 CFU mL−1) of S. aureus once (group I) at the beginning of the experiment (0 h) or twice (groups II and III) at 0 h and at 12 h (Table 1) after the first inoculation (PI). Sham-infected animals were administered sterile saline. The S. aureus isolate S54F9 was obtained from a chronic embolic pulmonary abscess in a Danish slaughter pig. By staphylococcal protein Selleck RAD001 A (spa) typing and multilocus sequence typing (MLST), the isolate was found to belong to spa type t1333, MLST sequence type ST433 and clonal complex CC30 (Hasman et al., 2010), one of the

three predominant lineages of S. aureus demonstrated in Danish pigs. During the experiment, the animals were monitored clinically for signs of severe pain, which would have prompted immediate euthanasia as stated in the protocol that was approved by the Danish Animal Experimental Act (licence no. 2008/561-1462). Blood was sampled for bacteriology, haematology and clinical chemistry at different time points, and the various groups of pigs were euthanized with an intravenous

injection of 20% pentobarbital as indicated (Table 1). Following euthanasia, the animals underwent a thorough postmortem examination and tissues were sampled for histopathology and microbiology from predetermined sites and gross lesions. Tissues were processed using routine techniques and 4–5 μm sections were cut and stained with haematoxylin and eosin, and in selected cases, GDC0449 with phosphotungstic acid

haematoxylin for the demonstration of fibrin (Stevens & Wilson, 1996). Heparin-stabilized blood (10 mL) was collected aseptically for a quantitative microbiological examination. Three millilitres of the blood and 1 mL of decimal dilutions were added to empty Petri dishes and mixed with melted Luria–Bertani agar medium. Viable counts were determined after incubation for 48 h at 37 °C and presented as counts per millilitre blood. A quantitative bacteriological examination was performed on the lung tissue (left diaphragmatic lobe), spleen (dorsal half), liver (left lateral lobe) and bone tissue upon euthanasia (Jensen et al., 2010). Dapagliflozin Colony morphology was evaluated and representative colonies were subcultured on blood agar containing 5% sterile bovine blood and characterized phenotypically (Api ID 32 Staph, Biomerieux Inc., Marcy-l’Etoile, France). An automated complete blood cell count including a leucocyte differential count was conducted using EDTA-stabilized whole blood (ADVIA 120 analyzer, Bayer Healthcare Diagnostics, Berlin, Germany). The following parameters were recorded: white blood cells (WBC), the total neutrophil count, lymphocytes, monocytes, eosinophils, basophils, red blood cells, haematocrit, haemoglobin and platelet count.