coli TOP10. The recombinant E. coli TOP10 lysates

showed opacification activity in the fish serum. Figure 3 shows the results obtained by Western blotting using the His antibody and serum agar overlay method for purified rSOF-OFD. An immune-stained band at c. 70 kDa was observed. Selleck Lumacaftor Meanwhile, the serum overlay agar with a native PAGE gel showed an opaque band at c. 150 kDa. When an SDS-PAGE gel was used on agarose containing fish serum, the opaque band was not observed. To discriminate between the mammalian and fish isolates, a primer set for PCR targeting the sof-FD gene was determined. Although bands of c. 400 bp were observed in the 16 fish isolates with different genotypes, no bands were observed in the mammalian isolates (Fig. 4). One of the two fish isolates

lacking SOF activity was PCR-positive. This could be due to a putative insertion sequence into the sof-FD gene (data Proteasome inhibitor not shown). However, another SOF-negative strain did not harbour the sof-FD gene when other primers targeting other regions of the sof-FD gene were used. Beall et al. (2000) and Goodfellow et al. (2000) reported that about half of clinical isolates of S. pyogenes possessed the sof gene and opacification activity. In the present study, almost all of the fish isolates showed serum opacification activity in both culture supernatants and SDS extracts. Moreover, the PCR assay targeting the sof-FD gene showed high sequence identity. This study also determined sequences of the entire sof-FD gene from fish isolates with varying degrees of opacification activity (OD660 = 0.1–0.6). The determined sequences included entire SOF-FD amino acid sequences with 100% identity to each other. These results suggested the clonal expansion and homogeneity of S. dysgalactiae isolated from farmed fish in Japan (Nishiki et al., 2010). Further studies are in progress to

reveal the mechanism of variations in the SOF activity in fish GCSD isolates. Recently, GCSD was isolated HSP90 from blood culture of a patient who had handled raw fish, and the characteristics of the GCSD were the same as those of isolates from farmed fish in Japan (Koh et al., 2008). To discriminate between fish and mammalian isolates is important to protect the public from the potential threat of zoonosis. The primer set targeting the sof-FD gene discriminated between mammalian and fish isolates. However, at least one PCR-negative strain was determined in this study and such PCR-negative strains could increase in future. A previous study demonstrated that PCR targeting the sodA gene was able to discriminate between mammalian and fish isolates (Nomoto et al., 2008). Because there were only a few nucleotide differences in the sodA gene between mammalian and fish isolates, the PCR assay could be used to discriminate between fish and mammalian isolates under strict annealing conditions. Therefore, it is possible that nonspecific reactions occurred.