agalactiae PAGU 330T (=ATCC 13813T), Streptococcus suis PAGU 580T (=ATCC 43765T), S. dysgalactiae ssp. equisimilis PAGU 375T (=NCFB 1356T) and Streptococcus marimammalium PAGU 780T (=CCUG 48494T). All strains were grown on 5% defibrinated sheep blood agar plates at 37 °C and 5% CO2. Antigens were extracted using the Lancefield procedure (Slotved et al., 2002) and serologically grouped by a capillary precipitation test. Briefly, 0.1 mL of 0.2 N HCl was added to the bacteria pellet, and the acid suspension was placed in a water bath (100 °C) for 15 min. pH was adjusted to 7 by the addition of drops of 0.2 N NaOH. The suspension was centrifuged for 10 min at 1000 g

and the supernatant was transferred (acid antigen extract) to a test tube. When acid antigen extracts were mixed with equal amounts of the antiserum (Statens Serum Institut, Copenhagen, Denmark), they formed insoluble antigen–antibody Selleckchem Forskolin complexes GSK2118436 purchase visible as a precipitate in positive reactions. The organisms were biochemically characterized using the Streptogram (Wako Pure Chemical, Osaka, Japan) and Rapid ID 32 Strep (bioMérieux, Tokyo, Japan) systems, according to the manufacturers’ instructions.

Morphology and hemolysis of the colonies were determined after 24-h incubation on sheep blood agar at 37 °C and 5% CO2. PCR amplification of the 16S rRNA gene sequencing of the purified PCR products was carried out (Kawamura et al., 1999). After confirming amplicons of 16S rRNA gene on 1% agarose gels, the sequence was determined using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan). 16S rRNA gene (>1300 bases) sequences of both strands of the gene were determined using the 3130 Genetic Analyzer (Applied

Biosystems). The sequences of the other streptococci used for alignment and for calculating levels of homology were obtained from GenBank. Multiple Thalidomide sequence alignments of DNA sequences were performed using clustal x software (Thompson et al., 1997). Phylogenetic distances were calculated using the neighbor-joining method (Saitou & Nei, 1987). The phylogenetic tree was constructed using treeview software (Page, 1996). DNA–DNA hybridization was performed, as described by Ezaki et al. (1989). Briefly, purified DNA (100 μg mL−1) of each strain was heat denatured and then diluted to 10 μg mL−1 with phosphate-buffered saline (PBS) containing 0.1 M MgCl2. The diluted DNA solution was distributed onto a microplate (Nunc-Immunoplate, Roskilde, Denmark) at 100 μL per well, and the plate was incubated at 30 °C for 12 h. The solution was discarded and the plate was dried. DNA from group M strains and S. marimammalium CCUG 48494T were labeled with photobiotin (Vector Laboratories, CA). The plate was prehybridized for 30 min and then hybridized for 2 h at 30 °C (optimal conditions) and 40 °C (stringent conditions) using 2 × SSC containing 50% formamide.