Veterinary Microbiology, 26 ( 1991 ) 161-166 Elsevier Science Publishers B.V., Amsterdam
16l
Cytotoxic activity of Staphylococcus hyicus R.P. Allaker, M. Whitlock* and D.H. Lloyd Department of Veterinary Medicine and Animal Husbandry, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts AL9 7TA, UK (Accepted 26 April 1990)
ABSTRACT Allaker, R.P., Whitlock, M. and Lloyd, D.H., 1991. Cytotoxic activity of Staphylococcus hyicus. Vet. Microbiol., 26:161-166. Culture supernatants from a number of Staphylococcus hyicus strains caused toxic effects to both murine fibroblast and porcine keratinocyte cells in culture. The extent of cytotoxicity was shown to differ between strains and may provide an indication of strain virulence. Purification of cytotoxic activity produced by S. hyicus (strain P 119) using preparative isoelectric-focussing demonstrated it to be cytolytic, haemolytic and non-proteolytic. The cytotoxin demonstrates certain properties in common with the delta haemolysin of Staphylococcus aureus.
INTRODUCTION
Staphylococcus hyicus is the causative organism of porcine exudative epidermitis (Underdahl et al., 1965 ). The organism is a resident of healthy pig skin; however, the factors which enable it to survive on the skin and occasionally produce disease are poorly understood. Certain biologically active compounds, including proteolytic enzymes (Takeuchi et al., 1987) and an epidermolytic toxin (Amtsberg, 1979), have been implicated in the pathogenesis of the disease. Cytotoxic compounds may be of significance and differences in the amount of cytotoxin produced by different strains of S. hyicus may exist. A measure ofcytotoxin production may therefore provide an indication of potential virulence. Eukaryotic cells maintained in culture are useful indicators of cytotoxicity (Allaker et al., 1985 ) and this paper describes the testing of culture supernatants from S. hyicus strains for cytotoxic activity. *Present address: School of Natural Sciences, Hatfield Polytechnic, College Lane, Hatfield, Hefts A L l 0 9AB, Great Britain.
0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
162
R.P. ALLAKER ETAL,
MATERIALS AND METHODS
Bacterial cultures S. hyicus strains were isolated from the skin of healthy and diseased piglets from a number of sources in Europe (Table 1 ). All isolates were maintained as lyophilised cultures until required. Broth culture medium contained 1.7% w/v tryptone (Oxoid, L42), 1% w/v yeast extract (Oxoid, L21 ), 0.5% w/v NaC1 and 0.25% w/v K2HPO4. Two culture methods were used: (a) For cytotoxic assays bacterial strains were grown in 50 ml volumes of broth in 500 ml volume flasks. (b) Exoproteins for subsequent purification and identification were obtained by culturing S. hyicus on the surface of a dialysis bag (approx. 12 cm × 5 cm) containing broth ( 150 ml) enclosed in a glass flask (Ikigai et al., 1988). All cultures were incubated at 37°C in air for 24 h on a shaking platform ( 100 rev/min). Those from method (a) were grown to a standard optical density reading of 1.6, measured at 540 nm. All cultures were centrifuged and the supernatants filtered (Millipore filters; 0.45 ~m) to remove residual cells. Filtered supernatants were then dialysed against water at 4°C for 24 h and their pH adjusted to 7.2 with 2 M NaOH. Cytotoxic assay McCoy cells (mouse fibroblasts) and porcine keratinocytes were used to assay for cytotoxic activity. McCoy cells were grown in Minimal Essential Medium supplemented with Earle's salts, L-glutamine, antibiotics, non-essential amino acids and 10% fetal bovine serum. Cell monolayers were subcultured when confluent, by trypsinisation followed by reseeding into fresh medium. Keratinocyte cells were obtained and maintained as described by Sieber and Wells (1988). All reagents were supplied by Gibco. For the cytotoxic assay, wells of microtitre plates (Gibco) were seeded with 0.1 ml cell suspension containing 5 × 105 cells/ml. After 2 h incubation dilutions of test supernatants, other test samples or controls (uninoculated bacterial culture medium) were added. The cells were then incubated for 4 days at 37 °C in air with 5% CO2. The percentage of damaged cells, in comparison to healthy control cells, was tlaen visually determined. Six replicate wells of the McCoy cells and four replicate wells of the keratinocytes were examined for each determination. Cell viability was assessed by trypan blue dye (0.25%) exclusion. The proportion of damaged cells was recorded as follows: + + + + > 80%; + + + 60-80%; + + 40-60%; + 20-40%; - < 20%. Culture supernatant fractionation The culture supernatant constituents from a known virulent strain of S. hyicus, P119, (Lloyd et al., 1990) were separated in order to characterise
CYTOTOXIC ACTIVITY OF STAPHYLOCOCCUS HYICUS
163
the cytotoxin. Supernatants (culture method b) were subjected to preparative isoelectric-focussing in free solution using the BioRad Rotofor cell. The supernatant volume obtained from two dialysis bag cultures was adjusted to 50 ml with distilled water; 2% w/v ampholytes pH 5-8 (Sigma) and 0.5% v/v Tween 20 were added. Fractionation was carried out for 6 h at 12 W constant power; fractions 2 to 8 were then pooled, the volume adjusted to 50 ml with distilled water and refractionation carried out for a further 6 h. Ampholytes were removed from the focussed proteins in the resulting 20 fractions by adding an equal volume of 2 M NaC1 to each fraction. The fractions were then dialysed against water at 4°C for 24 h and the pH adjusted to 7.2.
Cytotoxin identification Alternate fractions obtained from isoelectric-focussing were assayed for cytotoxicity using McCoy ceils, proteolytic activity using azocasein (Millet, 1970) and haemolytic activity using bovine erythrocytes. Haemolysis was assessed using 5% v/v washed bovine erythrocytes in phosphate buffered saline (pH 7.3 ). Equal volumes of serial twofold dilutions of the fractions were added to 0.1 ml erythrocyte suspensions in round-bottomed microtitre plates (Gibco). The plates were then incubated at 37 °C and haemolysis recorded visually after 16 h. RESULTS
The dialysed culture supernatants of the S. hyicus strains caused toxic effects to McCoy and porcine keratinocyte cells. McCoy cells were more sensitive to the toxic effects than keratinocyte cells. Detachment from the culture plate, a decrease in cell size and the crenation of the cell membrane were characteristic effects, but cell membranes were not sufficiently damaged to allow an adequate uptake of trypan blue to stain the cellular contents. At the 1 in 2 supernatant dilution a high level ofcytotoxic activity ( + + + + ) to the McCoy and keratinocyte cells was apparent for all the strains tested, while at the 1 in 10 the extent of cytotoxicity was shown to differ between strains (Table 1 ), particularly with the McCoy cells. At the higher dilution all of the strains isolated from healthy pigs demonstrated a low level of cytotoxic activity to the McCoy cells ( + o r + + ), while 5 out of 8 from diseased pigs demonstrated a high activity ( + + + ÷ ). Three of the strains from diseased pigs had low cytotoxic activity ( - o r + ) to the McCoy cells. Purification of the exoproteins produced by S. hyicus (P 119 ) using isoelectric-focussing showed that cytotoxic activity was located in fractions 1 and 3 (Table 2). The McCoy cells were completely lysed and readily stained with trypan blue. Complete haemolysis of bovine erythrocytes was observed with a 1 in 4 dilution of fractions 1 and 3 (Table 2). None of the fractions exhib-
164
R.P. ALLAKER ET AL.
TABLE l
S. hyicus strain d e s i g n a t i o n s , origins a n d cytotoxic effects o f culture s u p e r n a t a n t s on M c C o y and k e r a t i n o c y t e cells Strain
PII9 P411 SKI70 310LS 970CE 28.17.86 28.8.86 133CE AB.3B AB.5A PG 1 102A
C o n d i t i o n o f pig ~
XE XE XE XE H H H H XE XE XE XE
C y t o t o x i c i t y score at I in 10 dilution M c C o y cells
R.eratinocyles
++++ ++++ ++++ + ++ + + + + + + + +
++ ++ +++ + ++ N.T. 2 N.T. N.T. N.T. N.T.
+ + + +
N.T.
~XE = e x u d a t i v e e p i d e r m i t i s ; H = healthy. 2N.T. = not tested. TABLE 2 Cytotoxic, h a e m o l y t i c a n d proteolytic activities o f u n f r a c t i o n a t e d a n d f r a c t i o n e d (isoelectricfocussing ) culture s u p e r n a t a n t f r o m S. hyicus ( P 119 ) Unfractionated supernatant z
C y t o t o x i c i t y score ( 1 in 2 d i l u t i o n ) H a e m o l y t i c activity ( I in 4 dilution ) Proteolytic activity (units/ml)
+ + + + 0 700
Fractions 1 and 3
5 to 19
+ + + + ~
-
Complete
0
0
0
~Complete lysis o f cells. 2Undialysed.
ited any proteolytic activity; however, the unfractionated supernatant demonstrated considerable protease activity (Table 2). DISCUSSION
Kawano et al. ( 1983 ) using bacteriophage typing ofS. hyicusdemonstrated that two or more kinds of phage patterns were present in isolates from pigs with exudative epidermitis. However all isolates from healthy pigs showed a
CYTOTOXIC ACTIVITY OF STAPHYLOCOCCUS HYICUS
165
single-phage pattern. Hunter et al. (1970) found that avirulent strains were present both in diseased and healthy pigs, whilst virulent strains were recovered only from pigs affected with exudative epidermitis or those which had been in contact with the disease. Of the three strains with a low cytotoxic activity, isolated from diseased pigs in this study, SK170 is known to be avirulent (Lloyd et al., 1990) and the others may be similar. If cytotoxic activity is a good indicator of potential virulence our results concur with the findings of other workers and suggest that diseased pigs are likely to yield strains with both high and low cytotoxicity scores; healthy pigs from farms with no disease problem may yield only strains with low cytotoxicity scores. Further studies are required to confirm this hypothesis. Some bacterial proteases are known to be cytotoxic (Eke et al., 1989) and the unfractionated S. hyicus, P 119, culture supernatant demonstrated considerable protease activity. However, none of the purified fractions exhibited proteolysis. The removal of certain divalent cations important for S. hyicus protease stability (Takeuchi et al., 1985 ) during purification might account for this loss in enzyme activity, although addition of calcium, magnesium or zinc salts, after the purification procedures, failed to restore it (Allaker and Lloyd, unpublished observations). Purification of the cytotoxin from S. hyicus (P 119 ) revealed a highly cytolytic factor and haemolytic activity against bovine erythrocytes correlated with this. Thus the cytotoxin has certain properties in common with the delta haemolysin produced by S. aureus, which is also haemolytic and cytolytic (Molby, 1983). Studies using concentrated culture supernatants introduced into the skin of pigs are currently being undertaken to further characterise the toxic components produced by S. hyicus. ACKNOWLEDGMENTS
This study was supported by a grant from the Wellcome Trust.
REFERENCES Allaker, R.P., Greenman, J., Osborne, R.H. and Gowers, J.l., 1985. Cytotoxic activity of Propionibacterium acnes and other skin organisms. Br. J. Dermatol., 113: 229-235. Amtsberg, G., 1979. Nachweis von Exfoliation auslosenden Substanzen in Kulturen von Staphylococcus hyicus des Schweines und Staphylococcus epidermidis Biotyp 2 des Rindes. Zentralbl. VeterinS.rmed., 26: 257-272. Eke, P.I., Laughon, B.E. and Rotim, V.O., 1989. Cytotoxic activity of crude extracts of Bacteroides gingivalis. J. Med. Microbiol., 28: 5-8. Hunter, D., Todd, J.N. and Larkin, M., 1970. Exudative epidermitis of pigs: the serological identification and distribution of the associated Staphylococcus. Br. Vet. J., 126: 225-229. lkigai, H., Seki, K., Nishihara, S. and Masuda, S., 1988. Simplified method for preparation of
166
R.P. ALLAKER ET AL.
concentrated exoproteins produced by Staphj,Iococcus aureus grown on surface of cellophane bag containing liquid medium. Microbiol. lmmunol., 32: 225-228. Kawano, J., Shimizu, A. and Kimura, S., 1983. Bacteriophage typing ofoClap]~.v[ococcus h),ic~ts subsp, h.vicus isolated from pigs. Am. J. Vet. Res., 44: 1476-1479. Lloyd, D.H., Allaker, R.P., Smith, I.M. and Mackie, A., 1990. Colonisation ofgnotobiotic piglets by Staphylococcus hyicus and the development of exudative epidermitis. Microb. Ecol. Health Dis., 3: 15-18. Millet, J., 1970. Characterisation of proteinases excreted by Bacillus subtilis Marburg strain during sporulation. J. Appl. Bacteriol., 33: 207-219. Molby, R., 1983. Isolation and properties of membrane damaging toxins. In: C.S. Easmon and C. Adlam (Editors), Staphylococci and Staphylococcal Infection, Vol. 2. Academic Press, London, pp. 619-669. Sieber, V.K. and Wells, J., 1988. The use of plucked hairs as a biological dosemeter. Br. J. Radiol. Suppl. 19: 92-95. Takeuchi, S., Kobayashi, Y. and Morozumi, T., 1985. Purification and so~ne properties of protease produced by Slaph),lococcus h)'icus subsp, h),icus strain No. 111. Jpn. J. Vet. Sci., 47: 769-775. Takeuchi, S., Kobayashi, Y. and Morozumi, T., 1987. Proteolytic zymograms of Slaph),lococct¢s h)'icus subsp, h),icus isolated from pigs, chickens and cows. Vet. Microbiol., 14: 47-52. Underdahl, N.R., Grace, O.D. and Twiehaus, M.J., 1965. Porcine exudative epidermitis: characterisation of bacterial agent. Am. J. Vet. Res., 26:617-624.
16l
Cytotoxic activity of Staphylococcus hyicus R.P. Allaker, M. Whitlock* and D.H. Lloyd Department of Veterinary Medicine and Animal Husbandry, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts AL9 7TA, UK (Accepted 26 April 1990)
ABSTRACT Allaker, R.P., Whitlock, M. and Lloyd, D.H., 1991. Cytotoxic activity of Staphylococcus hyicus. Vet. Microbiol., 26:161-166. Culture supernatants from a number of Staphylococcus hyicus strains caused toxic effects to both murine fibroblast and porcine keratinocyte cells in culture. The extent of cytotoxicity was shown to differ between strains and may provide an indication of strain virulence. Purification of cytotoxic activity produced by S. hyicus (strain P 119) using preparative isoelectric-focussing demonstrated it to be cytolytic, haemolytic and non-proteolytic. The cytotoxin demonstrates certain properties in common with the delta haemolysin of Staphylococcus aureus.
INTRODUCTION
Staphylococcus hyicus is the causative organism of porcine exudative epidermitis (Underdahl et al., 1965 ). The organism is a resident of healthy pig skin; however, the factors which enable it to survive on the skin and occasionally produce disease are poorly understood. Certain biologically active compounds, including proteolytic enzymes (Takeuchi et al., 1987) and an epidermolytic toxin (Amtsberg, 1979), have been implicated in the pathogenesis of the disease. Cytotoxic compounds may be of significance and differences in the amount of cytotoxin produced by different strains of S. hyicus may exist. A measure ofcytotoxin production may therefore provide an indication of potential virulence. Eukaryotic cells maintained in culture are useful indicators of cytotoxicity (Allaker et al., 1985 ) and this paper describes the testing of culture supernatants from S. hyicus strains for cytotoxic activity. *Present address: School of Natural Sciences, Hatfield Polytechnic, College Lane, Hatfield, Hefts A L l 0 9AB, Great Britain.
0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
162
R.P. ALLAKER ETAL,
MATERIALS AND METHODS
Bacterial cultures S. hyicus strains were isolated from the skin of healthy and diseased piglets from a number of sources in Europe (Table 1 ). All isolates were maintained as lyophilised cultures until required. Broth culture medium contained 1.7% w/v tryptone (Oxoid, L42), 1% w/v yeast extract (Oxoid, L21 ), 0.5% w/v NaC1 and 0.25% w/v K2HPO4. Two culture methods were used: (a) For cytotoxic assays bacterial strains were grown in 50 ml volumes of broth in 500 ml volume flasks. (b) Exoproteins for subsequent purification and identification were obtained by culturing S. hyicus on the surface of a dialysis bag (approx. 12 cm × 5 cm) containing broth ( 150 ml) enclosed in a glass flask (Ikigai et al., 1988). All cultures were incubated at 37°C in air for 24 h on a shaking platform ( 100 rev/min). Those from method (a) were grown to a standard optical density reading of 1.6, measured at 540 nm. All cultures were centrifuged and the supernatants filtered (Millipore filters; 0.45 ~m) to remove residual cells. Filtered supernatants were then dialysed against water at 4°C for 24 h and their pH adjusted to 7.2 with 2 M NaOH. Cytotoxic assay McCoy cells (mouse fibroblasts) and porcine keratinocytes were used to assay for cytotoxic activity. McCoy cells were grown in Minimal Essential Medium supplemented with Earle's salts, L-glutamine, antibiotics, non-essential amino acids and 10% fetal bovine serum. Cell monolayers were subcultured when confluent, by trypsinisation followed by reseeding into fresh medium. Keratinocyte cells were obtained and maintained as described by Sieber and Wells (1988). All reagents were supplied by Gibco. For the cytotoxic assay, wells of microtitre plates (Gibco) were seeded with 0.1 ml cell suspension containing 5 × 105 cells/ml. After 2 h incubation dilutions of test supernatants, other test samples or controls (uninoculated bacterial culture medium) were added. The cells were then incubated for 4 days at 37 °C in air with 5% CO2. The percentage of damaged cells, in comparison to healthy control cells, was tlaen visually determined. Six replicate wells of the McCoy cells and four replicate wells of the keratinocytes were examined for each determination. Cell viability was assessed by trypan blue dye (0.25%) exclusion. The proportion of damaged cells was recorded as follows: + + + + > 80%; + + + 60-80%; + + 40-60%; + 20-40%; - < 20%. Culture supernatant fractionation The culture supernatant constituents from a known virulent strain of S. hyicus, P119, (Lloyd et al., 1990) were separated in order to characterise
CYTOTOXIC ACTIVITY OF STAPHYLOCOCCUS HYICUS
163
the cytotoxin. Supernatants (culture method b) were subjected to preparative isoelectric-focussing in free solution using the BioRad Rotofor cell. The supernatant volume obtained from two dialysis bag cultures was adjusted to 50 ml with distilled water; 2% w/v ampholytes pH 5-8 (Sigma) and 0.5% v/v Tween 20 were added. Fractionation was carried out for 6 h at 12 W constant power; fractions 2 to 8 were then pooled, the volume adjusted to 50 ml with distilled water and refractionation carried out for a further 6 h. Ampholytes were removed from the focussed proteins in the resulting 20 fractions by adding an equal volume of 2 M NaC1 to each fraction. The fractions were then dialysed against water at 4°C for 24 h and the pH adjusted to 7.2.
Cytotoxin identification Alternate fractions obtained from isoelectric-focussing were assayed for cytotoxicity using McCoy ceils, proteolytic activity using azocasein (Millet, 1970) and haemolytic activity using bovine erythrocytes. Haemolysis was assessed using 5% v/v washed bovine erythrocytes in phosphate buffered saline (pH 7.3 ). Equal volumes of serial twofold dilutions of the fractions were added to 0.1 ml erythrocyte suspensions in round-bottomed microtitre plates (Gibco). The plates were then incubated at 37 °C and haemolysis recorded visually after 16 h. RESULTS
The dialysed culture supernatants of the S. hyicus strains caused toxic effects to McCoy and porcine keratinocyte cells. McCoy cells were more sensitive to the toxic effects than keratinocyte cells. Detachment from the culture plate, a decrease in cell size and the crenation of the cell membrane were characteristic effects, but cell membranes were not sufficiently damaged to allow an adequate uptake of trypan blue to stain the cellular contents. At the 1 in 2 supernatant dilution a high level ofcytotoxic activity ( + + + + ) to the McCoy and keratinocyte cells was apparent for all the strains tested, while at the 1 in 10 the extent of cytotoxicity was shown to differ between strains (Table 1 ), particularly with the McCoy cells. At the higher dilution all of the strains isolated from healthy pigs demonstrated a low level of cytotoxic activity to the McCoy cells ( + o r + + ), while 5 out of 8 from diseased pigs demonstrated a high activity ( + + + ÷ ). Three of the strains from diseased pigs had low cytotoxic activity ( - o r + ) to the McCoy cells. Purification of the exoproteins produced by S. hyicus (P 119 ) using isoelectric-focussing showed that cytotoxic activity was located in fractions 1 and 3 (Table 2). The McCoy cells were completely lysed and readily stained with trypan blue. Complete haemolysis of bovine erythrocytes was observed with a 1 in 4 dilution of fractions 1 and 3 (Table 2). None of the fractions exhib-
164
R.P. ALLAKER ET AL.
TABLE l
S. hyicus strain d e s i g n a t i o n s , origins a n d cytotoxic effects o f culture s u p e r n a t a n t s on M c C o y and k e r a t i n o c y t e cells Strain
PII9 P411 SKI70 310LS 970CE 28.17.86 28.8.86 133CE AB.3B AB.5A PG 1 102A
C o n d i t i o n o f pig ~
XE XE XE XE H H H H XE XE XE XE
C y t o t o x i c i t y score at I in 10 dilution M c C o y cells
R.eratinocyles
++++ ++++ ++++ + ++ + + + + + + + +
++ ++ +++ + ++ N.T. 2 N.T. N.T. N.T. N.T.
+ + + +
N.T.
~XE = e x u d a t i v e e p i d e r m i t i s ; H = healthy. 2N.T. = not tested. TABLE 2 Cytotoxic, h a e m o l y t i c a n d proteolytic activities o f u n f r a c t i o n a t e d a n d f r a c t i o n e d (isoelectricfocussing ) culture s u p e r n a t a n t f r o m S. hyicus ( P 119 ) Unfractionated supernatant z
C y t o t o x i c i t y score ( 1 in 2 d i l u t i o n ) H a e m o l y t i c activity ( I in 4 dilution ) Proteolytic activity (units/ml)
+ + + + 0 700
Fractions 1 and 3
5 to 19
+ + + + ~
-
Complete
0
0
0
~Complete lysis o f cells. 2Undialysed.
ited any proteolytic activity; however, the unfractionated supernatant demonstrated considerable protease activity (Table 2). DISCUSSION
Kawano et al. ( 1983 ) using bacteriophage typing ofS. hyicusdemonstrated that two or more kinds of phage patterns were present in isolates from pigs with exudative epidermitis. However all isolates from healthy pigs showed a
CYTOTOXIC ACTIVITY OF STAPHYLOCOCCUS HYICUS
165
single-phage pattern. Hunter et al. (1970) found that avirulent strains were present both in diseased and healthy pigs, whilst virulent strains were recovered only from pigs affected with exudative epidermitis or those which had been in contact with the disease. Of the three strains with a low cytotoxic activity, isolated from diseased pigs in this study, SK170 is known to be avirulent (Lloyd et al., 1990) and the others may be similar. If cytotoxic activity is a good indicator of potential virulence our results concur with the findings of other workers and suggest that diseased pigs are likely to yield strains with both high and low cytotoxicity scores; healthy pigs from farms with no disease problem may yield only strains with low cytotoxicity scores. Further studies are required to confirm this hypothesis. Some bacterial proteases are known to be cytotoxic (Eke et al., 1989) and the unfractionated S. hyicus, P 119, culture supernatant demonstrated considerable protease activity. However, none of the purified fractions exhibited proteolysis. The removal of certain divalent cations important for S. hyicus protease stability (Takeuchi et al., 1985 ) during purification might account for this loss in enzyme activity, although addition of calcium, magnesium or zinc salts, after the purification procedures, failed to restore it (Allaker and Lloyd, unpublished observations). Purification of the cytotoxin from S. hyicus (P 119 ) revealed a highly cytolytic factor and haemolytic activity against bovine erythrocytes correlated with this. Thus the cytotoxin has certain properties in common with the delta haemolysin produced by S. aureus, which is also haemolytic and cytolytic (Molby, 1983). Studies using concentrated culture supernatants introduced into the skin of pigs are currently being undertaken to further characterise the toxic components produced by S. hyicus. ACKNOWLEDGMENTS
This study was supported by a grant from the Wellcome Trust.
REFERENCES Allaker, R.P., Greenman, J., Osborne, R.H. and Gowers, J.l., 1985. Cytotoxic activity of Propionibacterium acnes and other skin organisms. Br. J. Dermatol., 113: 229-235. Amtsberg, G., 1979. Nachweis von Exfoliation auslosenden Substanzen in Kulturen von Staphylococcus hyicus des Schweines und Staphylococcus epidermidis Biotyp 2 des Rindes. Zentralbl. VeterinS.rmed., 26: 257-272. Eke, P.I., Laughon, B.E. and Rotim, V.O., 1989. Cytotoxic activity of crude extracts of Bacteroides gingivalis. J. Med. Microbiol., 28: 5-8. Hunter, D., Todd, J.N. and Larkin, M., 1970. Exudative epidermitis of pigs: the serological identification and distribution of the associated Staphylococcus. Br. Vet. J., 126: 225-229. lkigai, H., Seki, K., Nishihara, S. and Masuda, S., 1988. Simplified method for preparation of
166
R.P. ALLAKER ET AL.
concentrated exoproteins produced by Staphj,Iococcus aureus grown on surface of cellophane bag containing liquid medium. Microbiol. lmmunol., 32: 225-228. Kawano, J., Shimizu, A. and Kimura, S., 1983. Bacteriophage typing ofoClap]~.v[ococcus h),ic~ts subsp, h.vicus isolated from pigs. Am. J. Vet. Res., 44: 1476-1479. Lloyd, D.H., Allaker, R.P., Smith, I.M. and Mackie, A., 1990. Colonisation ofgnotobiotic piglets by Staphylococcus hyicus and the development of exudative epidermitis. Microb. Ecol. Health Dis., 3: 15-18. Millet, J., 1970. Characterisation of proteinases excreted by Bacillus subtilis Marburg strain during sporulation. J. Appl. Bacteriol., 33: 207-219. Molby, R., 1983. Isolation and properties of membrane damaging toxins. In: C.S. Easmon and C. Adlam (Editors), Staphylococci and Staphylococcal Infection, Vol. 2. Academic Press, London, pp. 619-669. Sieber, V.K. and Wells, J., 1988. The use of plucked hairs as a biological dosemeter. Br. J. Radiol. Suppl. 19: 92-95. Takeuchi, S., Kobayashi, Y. and Morozumi, T., 1985. Purification and so~ne properties of protease produced by Slaph),lococcus h)'icus subsp, h),icus strain No. 111. Jpn. J. Vet. Sci., 47: 769-775. Takeuchi, S., Kobayashi, Y. and Morozumi, T., 1987. Proteolytic zymograms of Slaph),lococct¢s h)'icus subsp, h),icus isolated from pigs, chickens and cows. Vet. Microbiol., 14: 47-52. Underdahl, N.R., Grace, O.D. and Twiehaus, M.J., 1965. Porcine exudative epidermitis: characterisation of bacterial agent. Am. J. Vet. Res., 26:617-624.
