All procedures with animals were approved by the Committee for Animal Protection and Use of the Institute of Microbiology. PR4 is a commensal strain of B. choerinum isolated from fecal flora of 8-week-old pigs of (LW × L) × Pn breed using modified trypticase–phytone–yearst (MTPY) agar [30]. The isolate was identified using the random amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) procedure according to Sakata

et al. [31] and compared with porcine bifidobacteria strains from the German Resource Centre Selisistat ic50 for Biological Material. EcN is E. coli Nissle 1917 (EcN, serovar O6:K5:H1). This serum-sensitive non-virulent E. coli strain is used as a human and veterinary probiotic [16]. LT2 is a serum-resistant LT2 strain of S. enterica serovar Typhimurium causing lethal sepsis in germ-free piglets [26]. Fresh cultures of bacteria were prepared for each experiment by cultivation at 37°C overnight. PR4 was cultivated in an anaerobic chamber in 10 ml TPY

broth (Scharlau, Barcelona, Spain). The cells were harvested by centrifugation at 4000 g for 10 min. The pellet was washed twice with 0·05 M phosphate buffer, pH 6·5 containing 500 mg/l cysteine see more (PBC). EcN and LT2 were cultivated on meat-peptone agar slopes (blood agar base; Oxoid, Basingstoke, UK). Bacteria were resuspended to the density of 5 × 108 colony-forming units (CFU)/ml and given to gnotobiotic pigs in milk diet. The number of CFU estimated by spectrophotometry at 600 nm was verified by a cultivation method. One-week-old germ-free pigs were orally associated/infected

with 1 × 108 CFU of bacteria in 5 ml of milk diet. Di-associated pigs were infected with S. Typhimurium 24 h after the association with PR4 or EcN, respectively. All experimental pigs were euthanized 24 h after the last bacteria treatment by exsanguination Diflunisal under halothane anaesthesia. The germ-free control group was euthanized at the same age. Six experimental groups of 1-week-old gnotobiotic pigs (five pigs in each group) from five hysterectomies of miniature sows were investigated: (i) germ-free piglets, (ii) pigs mono-associated with LT2 (LT2 strain of S. enterica serovar Typhimurium), (iii) pigs mono-associated with PR4 (B. choerinum strain PR4), (iv) pigs mono-associated with EcN (E. coli strain Nissle 1917), (v) pigs di-associated with PR4+LT2 and (vi) LT2 pigs di-associated with EcN+ LT2. Experimental animals were euthanized and samples of peripheral blood, intestinal lavages and homogenized tissues (from the spleen, mesenteric lymph nodes and liver) were serially diluted in PBC. Appropriate dilutions were transferred to sterile 60 mm Petri dishes, which were immediately filled with the media for bifidobacteria (TPY agar; Scharlau) supplemented with 100 mg/l mupirocin and 1 ml/l of concentrated glacial acetic acid [30]. Bifidobacteria were incubated in an anaerobic jar (Anaerobic Plus System; Oxoid) in CO2/H2 (90/10%) atmosphere at 37°C for 3 days. E.