INFECrION AND IMMUNITY, July 1993, p. 2973-29770019-9567/93/072973-05$02.00/0Copyright © 1993, American Society for Microbiology

Vol. 61, No. 7

Purification of Exfoliative Toxin Produced by Staphylococcushyicus and Its Antigenicity

TAISHI TANABE,* HISAAKI SATO, MASAHIKO KURAMOTO, AND HIROSHI SAITODepartment of Veterinary Microbiology, School of Veterinary Medicine and

Animal Sciences, Kitasato University, Towada, Aomori 034, JapanReceived 30 November 1992/Accepted 24 March 1993

We previously reported the isolation of an exfoliative toxin from culture filtrates of Staphylococcus hyicus(shET) and reproduction of exfoliation in piglets injected with partially purified shET. In this study, we purifiedshET and compared the biological and physicochemical properties of shET and Staphylococcus aureusexfoliative toxin (sETA and sETB). shET was purified by ammonium sulfate precipitation, DEAE-celulofineA-500 column chromatography, Sephadex G-75 gel filtration, and polyacrylamide gel electrophoresis (7.5%polyacrylamide). Purified shET (p-shET) did not cause exfoliation of the epidermis in suckling mice but didcause exfoliation in 1-day-old chickens, whereas sETA and sETB produced by S. aureus caused exfoliation insuckling mice but not in 1-day-old chickens. The molecular mass of p-shET was determined as 27 kDa bysodium dodecyl sulfate-polyacrylamide gel electrophoresis. p-shET did not show any cross-reactivity withsETA and sETB in Western immunoblotting analysis or the immunodiffusion test.

Staphylococcus hyicus is known to be an important causeof exudative epidermitis in pigs (EEP) (19). EEP is a skindisease that affects pigs younger than 1 month (5) and ischaracterized by exudation, exfoliation, and vesicle forma-tion with erosion of the skin (5, 10).

Staphylococcal scalded-skin syndrome in humans is asso-ciated with infection by phage group 2 Staphylococcusaureus (13). Exfoliation in this disease is caused by staphy-lococcal exfoliative toxin (sET). sET has been divided intotwo serotypes, sETA and sETB (8). sETA is a heat-stabletoxin, and its molecular weight is 26,950 (11). sETB is aheat-labile toxin, and its molecular weight is 27,274 (11).Amtsberg (1) showed that the culture filtrate of S. hyicus

contains an exotoxin which causes exfoliation in piglets. Healso showed that the molecular mass of this exotoxin is lessthan 30 kDa, that its exfoliative activity is abolished by heattreatment, and that it is serologically different from S. aureussET. We isolated this exotoxin from culture supernatants ofS. hyicus P-1 and designated it shET (16). Its molecular masswas estimated as 27 kDa by sodium dodecyl sulfate-polyac-rylamide gel electrophoresis (SDS-PAGE). Exfoliative activ-ity was not found in shET heated at 60°C for 15 min.Recently, we also reported that piglets and chickens weresusceptible to shET but that mice were not susceptible (15).

In the present paper we describe the antigenicity and someproperties of purified shET (p-shET).

MATERIALS AND METHODSBacterial strain. S. hyicus P-1 (17) isolated from a pig

affected with erythema and incrustation of the body surfacewas used in this study. This strain was lyophilized and storedat 4°C. Lyophilized bacteria were suspended in heart infu-sion broth (HIB [Difcol) and cultured on heart infusion agar(HIA [Difco]) at 37°C for 18 h. The bacteria were thensuspended in 15% glycerol and stored at -80°C.

S. aureus ZM and J-sETB-8 were kindly supplied by S.Sakurai, Jikeikai University School of Medicine. Bothstrains were lyophilized and stored at 4°C. The lyophilized

* Corresponding author.

organisms were suspended in HIB and cultured at 37°C for18 h on HIB before being used as the inoculum for toxinproduction.

Isolation and purification of toxins. The exfoliative toxinswere isolated and purified by the method of Sato et al. (16)with slight modifications. TY medium (6), which contained10 g of yeast extract, 17 g of Trypticase, 5 g of NaCl, 2.5 g ofK2HPO4, and 1,000 ml of distilled water, was used as theliquid medium for toxin production. TY medium was trans-ferred to 2,000-ml Erlenmeyer flasks (300 ml per flask) andsterilized at 121°C for 15 min. S. hyicus P-1 cultured on HIAat 37°C for 18 h was suspended in 0.15 M phosphate-bufferedsaline (PBS [pH 7.2]) at a concentration of 109 CFU/ml.Then 3 ml of this suspension was inoculated into each flask,and the flasks were incubated at 37°C for 16 h in a shaker(Toyoshima, Tokyo, Japan) operated at 75 oscillations permin. The culture were centrifuged at 10,000 x g for 20 min at4°C, and the supernatant was passed through a 0.45-,um-pore-size membrane filter (Toyo Roshi, Tokyo, Japan). Solidammonium sulfate was added to the culture filtrate to 90%saturation, and the solution was allowed to stand for 60 minat 4°C and then centrifuged at 10,000 x g for 30 min at 4°C.The precipitate was dissolved in 10 ml of Tris-hydrochloride(TH) buffer (pH 7.5) and dialyzed against the same buffer for24 h at 4°C. The dialyzed solution was layered on a DEAE-cellulofine A-500 column (1 by 20 cm; Chisso Corp., Tokyo,Japan) previous equilibrated with TH buffer and eluted withthe same buffer. Fractions of the effluent yielding a highconcentration of protein were pooled and concentrated to 5ml by ultrafiltration through a UK-10 ultrafilter (ToyoRoshi). This concentrated solution was loaded on a Sepha-dex G-75 column (2.2 by 35 cm; Pharmacia, Uppsala,Sweden) equilibrated with TH buffer and eluted with thesame buffer. Fractions of the effluent yielding a high concen-tration of protein were pooled and concentrated to 2 ml byultrafiltration through a UK-10 ultrafilter. sETA and sETBwere isolated from the supernatants of S. aureus ZM andJ-sETB-8 cultures and purified by the method describedabove.

Protein determination. The protein concentration was de-termined by bicinchoninic acid protein assay (18, 20) with

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bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.)as the standard. The bicinchoninic acid protein assay kit waspurchased from Pierce Chemical Co., Rockford, Ill.

Native PAGE. Samples of eluates obtained from theSephadex G-75 gel filtration were layered on a polyacryl-amide gel slab, consisting of a 4.5% stacking gel and 7.5%separation gels with Tris-glycine buffer (pH 8.3), by themethod of Davis (3) and run at 15 mA per gel on the stackinggels and at 30 mA per gel on the separation gels. Afterelectrophoresis, a portion of each gel was transferred to a

polyvinylidene difluoride membrane (Atto Corp., Tokyo,Japan), stained with 1% amido black 10B (E. Merck AG,Darmstadt, Germany), and destained with 7% acetic acid.The remaining gel, corresponding to the protein bands, was

eluted with Maxyield GP (Atto) and concentrated through aUK-10 ultrafilter at 4°C. The concentrated samples wereexamined for exfoliative activity, and the samples causingthe typical Nikolsky sign (peeling off of the skin surfaceeasily caused by slight rubbing with the fingertip) were

designated purified shET (p-shET).SDS-PAGE. SDS-PAGE was performed at room temper-

ature by the method of Laemmli (9). A mixture of 0.05 ml of0.5 M TH buffer (pH 6.8), 0.08 ml of 10% SDS, 0.02 ml of2-mercaptoethanol (Bio-Rad Laboratories, Richmond, Ca-lif.), and 0.05 ml of 0.02% bromophenol blue in 80% glycerolwas added to 0.2 ml of protein solution, and the mixture wasallowed to stand overnight at room temperature. This samplesolution was layered on linear-gradient (10 to 20%) SDS-polyacrylamide gel slabs and run at 30 mA per gel. Theproteins in the slabs were transferred to polyvinylidenedifluoride membranes, stained with 0.25% Coomassie bril-liant blue R-250 (E. Merck), and destained with 7% aceticacid by the method of Fairbanks et al. (4).

Antisera. p-shET, p-sETA, and p-sETB were converted totoxoid by treatment with 0.8% formaldehyde solution at37°C for 50 h. The samples were inoculated subcutaneouslyinto young adult mice (6 to 8 weeks old) once a week for 4weeks. The inoculum was a mixture of 50 ,ug of toxoid andthe same volume of Freund incomplete adjuvant (Difco). At4 days after the fourth injection, sarcoma 180 cells (106 cellsper 0.5 ml) were inoculated intraperitoneally. After 3 days,toxoid was inoculated in the same manner into the mice.Most mice had distended abdomens within 10 to 15 daysafter inoculation of sarcoma cells. At that time, the asciticfluid was withdrawn by paracentesis through an 18-gaugeneedle into a 10-ml syringe. Fluids were pooled and centri-fuged for 20 min at 1,500 x g. The supernatant was drawn offand stored at -20°C. Sera were obtained by cardiac punc-

ture from all immunized mice and were treated in the same

way as the ascitic fluid.Animals. Six inbred specific-pathogen-free BALB/c

mice, 1 to 3 days old (Japan SLC Co. Ltd., Hamamatsu,Japan) were used for the neonatal-mouse inoculation testof the exfoliative toxins. Two mice were used for eachtoxin sample. Nine 1-day-old specific-pathogen-free WhiteLeghorn chickens, bred on a Kitasato University farm, were

used for detection of shET during the purification process.

Six 1-day-old specific-pathogen-free White Leghorn chick-ens were used for the 1-day-old chicken inoculation test ofsET. Two chickens were used for each sET sample.

Inoculation tests. (i) Neonatal-mouse inoculation test.

p-shET, p-sETA, and p-sETB (10 pg each) were inoculatedsubcutaneously into 1- to 3-day-old BALB/c mice. Mice inwhich a positive Nikolsky sign was observed within 3 h wereconsidered sensitive to the exfoliative toxin inoculated.

(ii) One-day-old-chicken inoculation test. p-sETs (50 p,g

each) were inoculated subcutaneously into 1-day-old specif-ic-pathogen-free White Leghorn chickens. Chickens inwhich a positive Nikolsky sign was observed within 3 h wereconsidered sensitive to the toxin inoculated.ELISA. The toxins were diluted to 0.5 ,ug/ml in 0.05 M

carbonate-bicarbonate buffer (pH 9.6). Diluted toxins in100-,I quantities were dispensed into a 96-well microplate(Toyoshima) and absorbed by overnight incubation at 4°C.After the wells were washed four times with 0.15 M PBS (pH7.2) containing 0.05% Tween 20, 25% block ace (SnowBrand Milk Products Co. Ltd., Tokyo, Japan) was dispensedinto the plate, which was allowed to stand overnight at 4°C.The plate was washed, the test sera were dispensed into thewells, and the plate was allowed to stand at 37°C for 1 h.After another washing, 100 pl of peroxidase-conjugatedanti-mouse immunoglobulin G (1:20,000; lot 44999; Seika-gaku Kogyo Co., Ltd., Tokyo, Japan) was dispensed into thewells, and the plate was held at 37°C for 1 h. Then substratesolution (0.04% o-phenylenediamine plus 0.03% H202 in 0.2M Na2HPO4-0.1 M citric acid buffer [pH 4.6]) was dispensedinto each well, and the plate was allowed to stand at 25°C for1 h. Then 100 p1 of 3 N H2SO4 was dispensed into each well.The optical density at 490 nm was read with an enzymeimmunoassay reader (model 3544; Bio-Rad). The cutoffpoint was set as the sum of the average optical densities at490 nm of sera from nonimmunized mice and three times thestandard deviation. The ELISA antibody titer of the serumwas taken as the reciprocal of the highest serum dilution forwhich the optical density at 490 nm was above the cutoffpoint. The ELISA titers of anti-shET, anti-sETA, and anti-sETB sera or ascitic fluids were 1:25,600, 1:204,800, and1:12,800, respectively.Immunodiffusion test. The immunodiffusion test was car-

ried out by the Ouchterlony method (14a). The surroundingwells contained the three toxins (p-shET, p-sETA, andp-sETB). The center wells contained anti-shET, anti-sETA,and anti-sETB sera or ascitic fluids. After the immunodiffu-sion test, the gel was washed in PBS for 1 day and stainedwith 1% amido black 10B.Western blotting. Western immunoblotting was carried out

by the method of Towbin et al. (22). The antigens (p-shET,p-sETA, and p-sETB) were prepared by SDS-PAGE andtransferred to a polyvinylidene difluoride membrane. Theantiserum was anti-shET, anti-sETA, or anti-sETB serum orascitic fluid at a 1:1,000 dilution in 10% block ace. Thesecond antibody was peroxidase-conjugated anti-mouse im-munoglobulin G (lot F44989; Seikagaku Kogyo) diluted1:2,000 with 10% block ace. The color reaction of thesubstrate was developed with 0.05 M TH buffer (pH 7.6)containing 0.05% (wt/vol) 3,3'-diaminobenzidine and 0.01%H202.

In vitro assay for exfoliative activity. The assay for exfoli-ative activity in cultured cells was carried out by the methodof Sato et al. (15) with slight modifications. A monolayerculture of NCTC 2544 cells (human epithelial cells) wasused. NCTC 2544 cells were grown in NCTC 135 medium(Flow Laboratories, Irvine, United Kingdom) supplementedwith 10% fetal calf serum (GM) and maintained in GMwithout fetal calf serum (MM). NCTC 2544 cell suspensions(100 VI containing 2 x 105 cells per ml) were added to thewells of a 96-well microculture plate (Corning Glass Works,Corning, N.Y.), which was then incubated overnight at 37°Cunder a 5% C02-95% air atmosphere. After incubation,shET, sETA, and sETB serially diluted with MM wereadded to the cell monolayers and the plate was incubated at37°C for 48 h. The negative control consisted of 0.01 M TH

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.0&6

D-1 D-2I- -

Fraction

FIG. 1. DEAE-celluloflne A-500 chromatography of the prepa-ration of S. hyicus P-1 precipitated by 90% saturation with ammo-nium sulfate. Column size, 1.0 by 20 cm; elution buffer, 0.01 M THbuffer (pH 7.5); flow rate, 15 ml/h.

buffer (pH 7.5) diluted with MM. After 48 h of incubation,toxin activity was determined by observing rounding of thecells. One exfoliative unit (EU) was calculated as the recip-rocal titer of the maximum dilution causing a rounding effecton NCTC 2544 cells after 48 h of injection.

RESULTS

Purification of exfoliative toxin. Figure 1 shows the elutionprofile on the DEAE-cellulofine A-500 column of the frac-tion precipitated from the culture filtrate of S. hyicus P-1 by90% saturated ammonium sulfate. Two major protein peaks(D-1 and D-2) were observed. When the peak samples wereinoculated into 1-day-old chickens, exfoliation was seen onlyin the chickens inoculated with D-1. Subsequently, 5.46 mgof the D-1 preparation was loaded onto a Sephadex G-75column. The elution proffle, as shown in Fig. 2, consisted ofone minor peak (S-1) and one major peak (S-2). When thepeak samples were inoculated into 1-day-old chickens, ex-foliation was seen only in the chickens inoculated with S-2.Therefore, the S-2 preparation was regarded as partiallypurified exfoliative toxin (pp-shET).

Figure 3 shows the native PAGE patterns of the partiallypurified toxins. pp-shET gave five major protein bands (B-1through B-5) on the polyacrylamide gel. The motility of B-2corresponded to the toxic protein band of pp-sETA andpp-sETB. When the extract of each protein band of pp-shETwas inoculated into 1-day-old chickens, exfoliation was seenonly in the chickens inoculated with B-2 extract. Therefore,the B-2 extract was regarded as p-shET. Figure 4 shows theSDS-PAGE pattern of each purified toxin. p-shET gave one

8 1

B 2

83

B 4

B~ ~5

shET sETA sETB

FIG. 3. Native PAGE patterns of S-2 preparations of partiallypurified toxins.

protein band on the gel, and its molecular mass was 27 kDa.p-sETA and p-sETB also gave one band (27 kDa) on the gel.Table 1 shows details of the purification of shET. The totalamount of protein was reduced during the purification pro-cess; however, the exfoliative activity (EU per milligram ofprotein) was increased from 8.8 to 400.0. Therefore, it wasconfirmed that shET was increasingly purified at each puri-fication step.

Exfoliative activity of shET. Figure 5 shows the results ofthe 1-day-old-chicken inoculation test. When 50 ,ug of thepurified toxins was inoculated into the chickens, the Nikol-sky sign was observed in the chickens 30 min after inocula-tion of shET. However, in the chickens injected with sETAand sETB, the sign was not seen within 24 h after inocula-tion. Figure 6 shows the results of the neonatal-mouseinoculation test. When 10 ,ug of the purified toxins wasinoculated into neonatal mice, the Nikolsky sign was ob-served in the mice inoculated with sETA and sETB 30 and 60min after inoculation, respectively. However, in the miceinoculated with shET, the sign was not seen within 24 h.

Serological properties of shET. Figure 7 shows the resultsof the immunodiffusion test of the exfoliative toxins. shETformed a precipitin line only with anti-shET (Fig. 7A).Similarly, sETA and sETB formed precipitin lines only withtheir homologous antibodies (Fig. 7B and C, respectively).Figure 8 shows the results of the Western blotting analysis of

c-a

L S-1 , S-22r T~

Fraction Number

FIG. 2. Sephadex G-75 gel filtration of the D-1 fraction of S.hyicus P-1 obtained by DEAEcellofine A-500 chromatography.Column size, 2.2 by 35 cm; elution buffer, 0.01 M TH buffer (pH7.5); flow rate, 24 ml/h.

78K66K

43K *

30K ^

17K_12K_

M P shEr sETA sETB

FIG. 4. SDS-PAGE of purified exfoliative toxins. Lane MPcontained molecular mass markers (78, 66, 43, 30, 17, and 12 kDa).

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TABLE 1. Purification of shET

Total Total Total Sp act YieldPurification step vol activity protein ( g) ()

(ml) (EU)a (mg) Emg

Ammonium sulfate- 17.0 1,940.4 220.5 3.5 100precipitated extract'

DEAE-cellulofine (D-1)C 4.2 1,354.1 5.5 246.2 69.8Sephadex G-75 (S-2)C 1.3 842.0 2.5 336.8 43.4PAGE ,7.5% acrylamide) 1.0 120.0 0.3 400.0 6.2

(B-2)

a EU was calculated as the reciprocal titer of the maximum dilution causinga rounding effect on NCTC 2544 cells 48 h after injection.

b This extract was prepared from a 1,500-ml culture of S. hyicus.c Preparations possessed exfoliative activity.d B-2 preparation is p-shET.

the toxins. Anti-shET antibody reacted with the 27-kDaprotein band of shET alone. Similarly, anti-sETA and anti-sETB antibodies reacted with the 27-kDa protein bands ofonly their homologous toxins.

DISCUSSIONS. hyicus is a causative agent of EEP (19). Amtsberg

suggested that shET was one of the virulence factors of S.hyicus (1). Takeuchi et al. reported that the activity ofprotease isolated from the culture filtrates of S. hyicus isenhanced by Ca2+ ions and that its molecular mass is 32 kDa(21). They hypothesized that protease is one of the virulencefactors of S. hyicus. However, further investigation showedthat protease did not influence the virulence of this organ-ism. Recently, we reported the isolation of shET fromculture filtrates of S. hyicus and production of exfoliation inpiglets injected with pp-shET (16). We also stated that themolecular mass of shET seems to be 27 kDa. From theseobservations, it was suggested that shET is different fromprotease and is the causative agent of exfoliation.Amtsberg reported that concentrated culture filtrates from

S. hyicus caused exfoliation in piglets but not in mice andguinea pigs (1). Recently, we showed that only piglets andchickens are sensitive to shET (15). In the previous study(14), sETA and sETB caused exfoliation in humans and miceonly. In our study, shET caused exfoliation in 1-day-old butnot 15-day-old chickens. Similarly, Melish and Glasgowreported that sensitivity to sETA was lost in mice aged 5days and older (13). Jones reported that EEP occurs mainlyin piglets aged 1 month or less (5). These findings suggestedthat susceptibility to shET is age dependent, like that to

FIG. 6. Mouse inoculation test. Left, mouse inoculated withshET; center, mouse inoculated with sETA; right, mouse inoculatedwith sETB.

sETA. It seems that age-dependent occurrence of EEP iscorrelated with age-dependent susceptibility to shET.Kondo et al. reported that sETA and sETB did not show

cross-reactivity in the gel diffusion precipitin test (7). In thepresent study, shET did not show cross-reactivity withsETA or sETB. From these findings, it seems that thesethree toxins are antigenically different. The molecularmasses of sETA and sETB are approximately 26 to 27 kDa.Recently, we estimated that the molecular mass of shETproduced by S. hyicus is 27 kDa (16). In the present study,SDS-PAGE of shET yielded one protein band whose molec-ular mass was approximately 27 kDa. These findings sug-gested that the three types of toxin have similar molecularmasses.

2

3

FIG. 5. One-day-old-chicken inoculation test. Left, chicken in-

oculated with shET; center, chicken inoculated with sETA; right,chicken inoculated with sETB.

2:.

FIG. 7. Immunodiffusion reaction between the purified toxinsand their antibodies. (A) Anti-p-shET serum (B) anti-p-sETA serum;(C) anti-p-sETB serum. Toxins: 1, p-sETA; 2, p-shET; 3, p-sETB.

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A.9 S

B c

78k s66k

30k -.

17k 4i12k_dS

MP 1 2 3 1 2 3 1 2 3

FIG. 8. Western blotting analysis of purified exfoliative toxins.(A) Anti-p-shET serum; (B) anti-p-sETA serum; (C) anti-p-sETBserum. Lanes: 1, p-sETA; 2, p-shET; 3, p-sETB.

In this study, we purified the 27-kDa protein from theculture supematant of S. hyicus and used it to produceexfoliation in chickens. It seems that the molecular mass andinduced exfoliative activity of shET are simialr to those ofsETA and sETB but that shET is different antigenically andin virulence for animals. We speculate that shET is one ofthe important pathogenic factors in EEP. Therefore, furtherinvestigation is needed to determine the toxicity and geneticcontrol of shET.

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3. Davis, B. J. 1964. Disc electrophoresis. II. Method and applica-tion to human serum proteins. Ann. N.Y. Acad. Sci. 12:404-427.

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5. Jones, L. D. 1961. Observation of exudative epidermitis. Vet.Med. 56:95-103.

6. Kapral, F. A., and M. M. Miller. 1971. Product of Staphylococ-cus aureus responsible for the scalded-skin syndrome. Infect.Immun. 4:541-545.

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8. Kondo, I., S. Sakurai, Y. Sarai, and S. Futaki. 1975. Two

serotypes of exfoliatin and their distribution in staphylococcalstrains isolated from patients with scalded skin syndrome. J.Clin. Microbiol. 1:397-400.

9. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

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12. Machida, K., S. Sakurai, I. Kondo, and S. Ikawa. 1988. Rela-tionship between susceptibility and immune response to staph-ylococcal exfoliative toxin A in mammalian species. Microbiol.Immunol. 32:1079-1084.

13. Melish, M. E., and L. A. Glasgow. 1970. The staphylococcalscalded skin syndrome: development of an experiment model.N. Engl. J. Med. 282:1114-1119.

14. Melish, M. E., L. A. Glasgow, and M. D. Turner. 1972. Thestaphylococcal scalded-skin syndrome: isolation and partialcharacterization of the exfoliative toxin. J. Infect. Dis. 125:129-140.

14a.Ouchterlony, 0. 1949. Antigen-antibody reactions in gels. ActaPathol. Microbiol. Scand. 26:753-759.

15. Sato, H., M. Kuramoto, T. Tanabe, and H. Saito. 1991. Suscep-tibility of various animals and cultured cells to exfoliative toxinproduced by Staphylococcus hyicus subsp. hyicus. Vet. Micro-biol. 28:157-169.

16. Sato, H., T. Tanabe, M. Kuramoto, K. Tanaka, T. Hashimoto,and H. Saito. 1991. Isolation of exfoliative toxin from Staphy-lococcus hyicus subsp. hyicus and its exfoliative activity in thepiglet. Vet. Microbiol. 27:263-275.

17. Sato, H., T. Tanabe, M. Nakanowatari, J. Oyama, N. Ymazaki,H. Yoshikawa, T. Yoshikawa, H. Koyama, and H. Saito. 1990.Isolation of Staphylococcus hyicus subsp. hyicus from the pigsaffected with exudative epidermitis and experimental infectionin the piglet. Kitasato Arch. Exp. Med. 63:119-130.

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20. Sorensen, K., and U. BrodbecL 1986. A sensitive protein assaymethod using Micro-titer plates. Experientia 42:161-162.

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