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Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P ؊ ) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P ؊ substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P ؊ ) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.Staphylococcus hyicus is known to be a causative agent of exudative epidermitis (EE) in pigs (28). EE is a generalized infection of the skin characterized by greasy exudation, exfoliation, and vesicle formation (7, 13).Amtsberg (1) has shown that the culture filtrate of S. hyicus contains an exotoxin that causes exfoliation in piglets. He has also suggested that the exfoliative activity of this exotoxin is similar to the exfoliative toxin (ET) produced by Staphylococcus aureus. We isolated this exotoxin from the culture supernatant of S. hyicus P-1 and designated it SHET (26). In 1993, we described some of the characteristics of purified SHET from the culture supernatant of S. hyicus (29). The molecular mass of SHET was estimated to be 27 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and it was determined that SHET differs from ETA and ETB in antigenicity and animal susceptibility (29).We have recently reported that the SHET-producing strain of S. hyicus causes EE in pigs while the non-SHET-producing strain does not and that SHET can be divided into more than two serotypes (30; H. Sato, T. Tanabe, T. Watanabe, K. Teruya, A. Ohtake, H. Saito, and N. Maehara, Proc. 14th IPVS Cong. Italy, p. 339, 1996). Andresen et al. (2, 3) have also reported that SHET has many serotypes. In this study, we detected the large plasmid in several strains of S. hyicus. We then confirmed the antigenic differences between SHETs from the plasmid-carrying strain and the plasmidless strain and between the SHET-producing ability of the plasmid-carrying strain and its plasmid-cured substrains. MATERI...

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The Staphylococcus hyicus exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in Escherichia coli. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.Staphylococcus hyicus is the causative agent of exudative epidermitis (EE) in pigs. Amtsberg (1) suggested that the culture filtrate of S. hyicus contains an exotoxin involved in exfoliative activity. Sato et al. (17) isolated such an exotoxin from the culture filtrate of S. hyicus and designated it SHET.Exfoliative toxin (ET)-producing strains of Staphylococcus aureus cause staphylococcal scalded skin syndrome in humans (11). The ET has been divided into two serotypes, ETA and ETB (9).The ET and SHET have some similarities, including their target site (5,17,20) and their molecular weight (7,8,17,18), but they only react with homologous antibodies. The production of ETA is controlled by chromosomal DNA, while that of ETB is controlled by plasmid DNA (12,13,15). S. hyicus P-23, the SHETB-producing strain, harbors the large plasmid (pKUH-1). The plasmid-eliminated substrain of S. hyicus P-23 has lost its toxic activity. From these findings, it appears that SHETB production is controlled by plasmid DNA (H. Sato, T. Tanabe, T. Watanabe, K. Teruya, A. Ohtake, H. Saito, and N. Maehara, Proc. 14th IPVS Cong. Italy, p. 339, 1996).In this study, we cloned the SHETB gene (shetb) on plasmid DNA of S. hyicus P-23 in Escherichia coli and determined the nucleotide sequence and the predicted amino acid sequence.S. hyicus P-23 is a SHETB-producing strain and was isolated from a pig affected with EE (19). E. coli DH5␣ was used as the host strain in cloning experiments. Bacteria were grown in TY broth (6) for S. hyicus and in Luria-Bertani broth (16) for E. coli. Both bacteria were cultured in a Bio-shaker BR-160LF (Taitec Co., Ltd., Tokyo, Japan) at 37°C with shaking operated at 75 oscillations per min for 18 h. The vector plasmid pUC118 (Takara Shuzo Co. Ltd., Tokyo, Japan) was used in the cloning experiments.Isolation of plasmid DNA from S. hyicus and E. coli was carried out by a modification of the method described by O'Reilly et al. (12) and Birnboim and Doly (3), respectively. To purify plasmid DNA from S. hyicus and E. coli, the dye-buoyant density centrifugation was performed in a P65AT rotor (Hitachi Koki Co., Ltd., Tokyo) at 45,000 rpm, and supercoiled DNA was separated from residual chromosomal DNA and nicked plasmid DNA.The large plasmid (pKUH-1) of S. hyicus P-23 was digested to completion with restriction endonucleases EcoRI, BamHI, and HindIII (Nippon Gene Inc., Toyama, Japan), and the resulting fragments were separated by electrophoresis with a 1.0% agarose gel. The transfer of the DNA onto a nylon membrane (Hybond N ϩ ; Amersham Pharmacia Biotech Co. Ltd., Uppsala, Sweden) was performed by alkaline blotting with fixation by a UV cross-linker. The nylon membrane was hybridized to a biotin-labelled ET probe (conserv...