Pure spirochetal isolates were analysed by PCR and subsequent sequencing of the 16S ribosomal RNA-tRNA-Ile intergenic spacer region (ISR2) and 16S ribosomal RNA genes [7, 10]. oral treponemes closely related to ulcer treponemes led us to hypothesize that treponemes are transferred to the skin by biting and licking behavior [4]. However, further studies are needed to reveal if treponemes are involved with the aetiology as the primary cause of the syndrome or if they are secondary invaders of the skin ulcers. The aim of this study was to test if inoculation with T A4 could induce lesions of ear necrosis in pigs, as evaluated by clinical and histopathological examination. The immune response was analysed?and gingival samples were continuously checked for treponemes to study the colonization of the gingiva and to enable comparison with the inoculation strain. Methods The study was performed at the department of Clinical Sciences at the Swedish University of Agricultural Sciences, Uppsala, in research animal facilities (see Declarations). The study included eight pigs (A-H) of Yorkshire/Hampshire crossbreed from two litters born by Yorkshire gilts. Two males and two females from each litter were randomly selected at four weeks, when weaned, and were allocated to the treatment group (After disinfection with ethanol, both ears were injected with 0.5?ml solution, with Mouse monoclonal to RUNX1 an estimate of 109 bacteria (challenge pigs, A-E) or isotonic saline (control pigs, F-H), evenly distributed at four sites, approximately ? in on the earlobe and 0.5?cm from the ventral margin, on both sides of the earlobes. On pigs C, E, and F a blunt trauma was made by shutting a forceps on five sites along the margin of the ear. The wet bandage was kept for one week after injection. Pig B was excluded from 12-O-tetradecanoyl phorbol-13-acetate the study from day 7 due to fainting and vomiting. At day 29 the inoculation was repeated. Thereafter the pigs were grouped two and two in the pens (A?+?C, D?+?E, F?+?G), except for pig H that was individually housed. The ears and the skin of the pigs were examined daily. Serum samples were collected from at days 0, 7, 11, 14, 21, 29, 35, 42, 49, 56. Gingival samples were taken with cottons swabs. At day 56 biopsies were collected from each ear from all pigs under anesthesia, and the pigs were euthanized by intravenous injection with 140?mg/kg pentobarbital sodium and phenytoin sodium (Euthasol? vet. Virbac Animal Health). After fixation in formalin the biopsies were embedded in paraffin and sections of 5C7?m were cut 12-O-tetradecanoyl phorbol-13-acetate and stained with hematoxyline and eosin (HE) and Warthin-Starry silver staining (W-S). Gingival samples were investigated by phase contrast microscopy and inoculated and cultured as previously described [8]. Pure spirochetal isolates were analysed by PCR and subsequent sequencing of the 16S ribosomal RNA-tRNA-Ile intergenic spacer region (ISR2) and 16S ribosomal RNA genes [7, 10]. Sequences were processed in CLC Main Workbench 12-O-tetradecanoyl phorbol-13-acetate 6.7 (CLC Bio) and the megablast algorithm BLAST was used for homology searches [11]. For the enzyme-linked immunosorbant assay (ELISA)T A4 lysate was prepared by washing bacterial pellets from eight 10?ml cultures three times with isotonic saline, suspended in 8?ml of isotonic saline and subjected to ultrasonic treatment using a horn-type sonicator (Vibra-cell VC-505; Sonics & Materials Inc., Newton, NJ, USA) at a frequency of 20?kHz. The cell lysate was sterile filtered using 0.2 m syringe filters (Sartorius, Goettingen, Germany) and quantified.