, 1999). Serum opacification activity has been observed in other streptococci, such as Streptococcus pyogenes and S. suis (Ward & Rudd, 1938; Baums et al., 2006). Serum opacity factor (SOF) is a bifunctional protein consisting of highly

conserved C-terminal repetitive sequences, and the N-terminal serum opacification domain (Rakonjac et al., 1995; Courtney et al., 2002). SOF caused S. pyogenes to invade and adhere to cells, and was demonstrated to be a virulence determinant of S. pyogenes using a murine infection model (Timmer et al., 2006; Gillen et al., 2008). The repetitive ABT-199 research buy C-terminal fibronectin-binding domains and high similarity in part of the N-terminal domain between serum opacity genes were observed in FnBA of S. dysgalactiae strain S2 and several SOFs from S. pyogenes strains (Courtney et al., 1999; Katerov et al., 2000). Although many variable sequences of sof genes exist in S. pyogenes, only a few genes coding SOF were reported to exist in S. dysgalactiae isolates from mammals. GCSD isolated from fish also possesses serum opacification activity. However, the gene encoding activity has been not identified. The aim of this study was to identify the sof gene, named sof-FD, and to determine its distribution in GCSD isolated from farmed fish. The designed oligonucleotides targeting

sof-FD were applied to a PCR assay to discriminate between fish GCSD and mammalian S. dysgalactiae. A total of 316 GCSD strains, isolated from farmed fish (amberjack, 276; yellowtail, 40) between I-BET-762 datasheet 2002 and 2008 in Japan, were used to detect SOF. Streptococcus

dysgalactiae isolated from pigs (n = 17) diagnosed with endocarditis in the Kumamoto Prefectural Meat Inspection Office was used as the source of mammalian isolates. Lancefield streptococcal grouping (Lancefield, 1933) was performed for these isolates using a Pastorex Strep Ribonuclease T1 test (Bio-Rad, Marnes-la-Coquette, France). The fish and mammalian isolates were identified using a PCR assay targeting the 16S–23S rRNA spacer region (Forsman et al., 1997; Hassan et al., 2003). Streptococcus dysgalactiae ssp. dysgalactiae ATCC 43078 and S. dysgalactiae ssp. equisimilis ATCC 35666 were used as reference strains. All the isolates were cultured on Todd-Hewitt (TH) agar (Difco, Sparks, MD) at 37 °C for 24 h. Serum opacification activity was detected using the microtitre plate method (Johnson & Kaplan, 1988) with minor modifications. Bacterial strains were cultured in TH broth at 37 °C for 24 h. The supernatants or 0.5% (sodium dodecyl sulfate) SDS extracts of bacterial cells were filtered using a 0.45-μm filter (Sartorius Stedim Japan K. K., Japan). Fish serum was obtained from healthy amberjacks (average weight 1250 g, n = 15). Briefly, fish were anaesthetized using FA-100 (Tanabe Pharma, Osaka, Japan), then bled from the caudal peduncle. After clotting of blood, the serum was separated by centrifugation for 20 min at 5000 g.