The second group consisted of 26 fish isolates of GCSD, and one strain of pig GCSD that had PCR products above 1 kb, which was markedly larger than expected. The third group included two fish isolates (PF880 and PP1398) of GCSD that had PCR products above 2 kb, which was also larger than expected. On the basis of the nucleotide

sequences selleck chemicals llc of spegg genes extracted from fish and pig isolates, the size variation was confirmed to be due to the presence of IS in the spegg locus of fish isolates. The spegg locus obtained from GCSD fish strains (94414 and KNH07901) was 2059 bp long due to the presence of a 1224 bp IS. This IS was found to have 99% similarity to IS981SC of Streptococcus iniae (AY904444). The spegg locus sequence was interrupted 604 bp downstream from its start codon by IS981SC, resulting in a 3-bp (5′-ATA-3′) duplication at the insertion site. IS981SC contained two ORFs, designated ORF1 and ORF2, which

encode 86 and 279 amino acids, respectively. These ORFs were oriented in the direction opposite to that of the spegg (Fig. 2c). The spegg locus obtained from GCSD fish strain PP1398 was 3236 bp in length due HIF inhibitor to the presence of two IS: IS981SC and IS1161 (Fig. 3). The IS981SC sequence was interrupted after 50 bp from its noncodon part of the 3′ end by the IS1161-like element, resulting in a 13-bp (5′-ATTTTAATCTATT-3′) duplication at the insertion site. The IS1161-like element sequence has 98% similarity Axenfeld syndrome to IS1161 (NC_011375) of the S. pyogenes strain (NZ131). The IS1161-like element was 1164 bp in length. IS1161 has one ORF that encodes 342 amino acids, and this ORF was oriented in the direction opposite

to that of spegg, but in the same direction as that of the two ORFs in IS981SC (Fig. 2d). The hybrid IS981SC–IS1161-like element was found to be inserted into the same location as that of IS981SC in the spegg locus of fish strain KNH07901, resulting in an insertion mutation in ORF of spegg (Fig. 3). The spegg locus obtained from the pig GCSE (PAGU657) strain showed a five-nucleotide deletion mutation, from nucleotides 604 to nucleotides 608 (5′-AAGCT-3′), in the ORF of spegg (Fig. 2a). The spegg locus obtained from the pig strain of GCSE (PAGU656) yielded the expected product size and had 100% similarity to spegg variant 4 (AB105080), which has one ORF that encodes 234 amino acids (Fig. 2b). As expected from the sequence similarity results, phylogenetic analysis revealed that spegg of fish isolates (without IS) was related to that of β-hemolytic S. dysgalactiae ssp. equisimilis. Moreover, spegg of fish isolates could be distinguished from that of S. pyogenes (Fig. 4). The presence of the IS981SC–IS1161 hybrid IS-like element in various isolates of GCSD and GCSE was screened by PCR analysis using specific primers Seg8 and Seg9. All fish isolates of GCSD and one isolate of pig GCSD (dNo.