Veterinary Microbiology, 25 ( 1 9 9 0 ) 2 9 7 - 3 0 2 Elsevier Science Publishers B.V., A m s t e r d a m
297
Short Communication Assay of protein A in Staphylococcus hyicus subsp, hyicus by ELISA and immunoelectron microscopy S. Takeuchi, Y.
K o b a y a s h i a a n d Y. M o r i a
Department of Agriculture, Fukui Prefectural College, 97-21-30batake, Fukui 910, Japan ~National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan (Accepted 6 March 1990)
ABSTRACT Takeuchi, S., Kobayashi, Y. and Mori, Y., 1990. Assay of protein A in Staphylococcus hyicus subsp. hyicus by ELISA and immunoelectron microscopy. Vet. Microbiol., 25: 297-302. The presence and quantity of protein A in Staphylococcus hyicus subsp, hyicus isolates were examined by an enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. Cellbound protein A was demonstrated in 45 (94%) of 48 isolates from diseased pigs and in 113 (86%) of 132 isolates from healthy pigs by ELISA using peroxidase-conjugated rabbit antibody, but was not found in isolates from chickens and cows. Most of these swine isolates contained about 100 to 300 ng of cell-bound protein A/ml. Extracellular protein A was not detected in any isolates from pigs, chickens or cows. In the immunoelectron microscopy assay, swine isolates were labeled with goat antimouse IgG conjugated to colloidal gold particles, but chicken and cow isolates were not labeled.
INTRODUCTION
Staphylococcus hyicus subsp, hyicus is considered to be an etiological agent of exudative epidermitis in young pigs. The organism is also isolated from the skin and nares of healthy pigs, chickens and cattle (Sato et al., 1972; Devriese, 1977, 1980; Devriese and Derycke, 1979; Takeuchi et al., 1985 ). Some workers (Mfiller et al., 1981; Phillips and Kloos, 1981; Hoover et al., 1983; Takeuchi et al., 1988 ), using a hemagglutination test or agar gel immunodiffusion test, demonstrated that swine isolates of S. hyicus subsp, hyicus produced protein A, which possesses the capability of binding the Fc region of immunoglobulins from various mammalian species. In the present study, the authors examined the presence and quantity of cell-bound and extracellular protein A in S. hyicus subsp, hyicus isolates from pigs, chickens and cows by 0378-1135/90/$03.50
© 1 9 9 0 - - Elsevier Science Publishers B.V.
298
s. TAKEUCHI ET AL.
an enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. MATERIALS AND METHODS
Bacterial isolates A total of 257 isolates of S. hyicus subsp, hyicus were examined for the presence of protein A. Of these isolates, 48 were obtained from lesions of pigs affected with exudative epidermitis, 132 from nasal cavities or external ears of clinically healthy pigs, 71 from nasal cavities of healthy chickens and 6 from mastitic udders of cows (Takeuchi et al,, 1985 ). S. aureus strain Cowan I and strain Wood 46 were used as protein A positive and negative controls, respectively.
Samples for ELISA For cell-bound protein A analysis, the bacteria to be tested were inoculated on brain heart infusion (BHI) agar (Difco) and cultivated at a 37 °C for 18 h. The bacterial cells were suspended in sterile 0.9 M NaC1 solution containing 0.5% formalin and incubated at 37 °C for 1 h for inactivation. Cells were then washed three times with 0.9 M NaC1 solution and resuspended in the saline solution to a concentration of MacFarland No. 3. For extracellular protein A analysis, the bacteria to be tested were inoculated in BHI broth supplemented with 0.3% yeast extract (Difco) and 1% maltose and cultivated at 35 °C for 20 h as described by Cox et al. (1986). The culture fluids were centrifuged at 12 000 rpm for 10 min and the resulting supernatants were filtered through a 0.45/~m filter (Millipore). The bacterial cells and supernatants were stored at - 80 ° C until used for ELISA.
ELISA One hundred/tl of the bacterial cells or supernatants, diluted 5- or 10-fold, were coated to wells in microtiter plates (Dynatech Laboratories Inc., Germany) and incubated at 37°C for 16 h. After washing, 100/~1 of peroxidaseconjugated rabbit antibody (Kirkegaard & Perry Laboratories Inc., MD ), diluted 1000 X, were added to each well of the plates and incubated at 25 ° C for 1 h. After washing, 100/tl of substrate solution (o-phenylenediamine dihydrochloride) was added to each well. After 30 min incubation at room temperature, the reaction was stopped and the optical density (OD) was measured. Recombinant protein A (Repligen Corporation Massachusetts) was used as a standard sample in the ELISA.
Immunoelectron microscopy The bacteria, freshly grown on BHI agar, were suspended in PBS containing fetal calf serum (PBS-FCS), washed by centrifugation, and resuspended
299
PROTEIN A ASSAY OF STAPHYLOCOCCUS HYICUS
to a density of approximately 109 C F U / m l in PBS-FCS. The bacterial suspensions were applied to carbon-coated grids (Oken shoji, Japan), which were pasted on a glass microscope slide with a piece of double-sided adhesive tape. After 5 min, excess suspension was removed with a wedge of filter paper, and goat anti-mouse IgG conjugated to colloidal gold particles (E.Y. Laboratories Inc., California) was applied to each grid. The grids were incubated at room temperature for 1 h and excess complex was removed with a piece of filter paper. After washing with PBS, the grids were stained with 0.5% ammonium molybdate solution and examined under an electron microscope (Hitachi, Japan) at 60 kV accelerating voltage. RESULTS
ELISA The presence and quantity of protein A in isolates ofS. hyicus subsp, hyicus were examined by the ELISA using peroxidase-conjugated rabbit antibody. In the ELISA, OD values of >0.201 were considered to be positive for protein A because the OD value of S. aureus strain Wood 46 was < 0.150. Cellbound protein A was demonstrated in 45 (94%) of 48 isolates from diseased pigs and in 113 (86%) of 132 isolates from healthy pigs. However, all isolates from chickens and cows were negative for cell-bound protein A. On the other hand, extracellular protein A was not detected in any isolate from diseased pigs, healthy pigs, chickens or cows. When purified recombinant protein A was examined by ELISA, a linear relationship was demonstrated between the OD values and the concentra-
E C~
r"
o
=.
I
1 0 1 - 130 1 3 1 - 160 1 6 1 - 190
II
1 9 1 - 220 221 - 250 2 5 1 - 280 281-310
oc o
311 - 3 4 0 3 4 1 - 370
~: .E
371 - 4 0 0 401 - 1,30
o n
431 - 4 6 0 461 - &90
I
I
] Healthy p=gs
>491 0
Diseased pigs
I
I
10
20
No. of
0
I
t
10
20
isolates
Fig. 1. Distribution of concentrations of cell-bound protein A in bacterial cell suspensions of MacFarland No. 3 of swine isolates.
300
S. T A K E U C H I ET AL.
•
A
G
-°*'~°"
,
B
~i¸ i
¸~¸
ii~i~!i
Fig. 2. Immunoelectron micrograph of strain Cowan I of S. aureus (A), swine isolates (B, C) and chicken isolates (D) of S. hyicus subsp, hyicus incubated with goat anti-mouse IgG conj ugated to gold colloidal particles.
tions of 20 to 100 ng of protein A/ml. Therefore, the quantitiy of cell-bound protein A in the S. hyicus subsp, hyicus isolates from diseased and healthy pigs was estimated by reference to this standard curve. As shown in Fig. 1, most of the swine isolates contained about 100 to 300 ng of cell-bound protein A / m l in nondiluted bacterial cell suspensions of a concentration of MacFarland No. 3. The average quantity of cell-bound protein A produced by them was about 200 ng/ml. In particular, strain T 19, which was isolated from a pig with exudative epidermitis, produced about 1000 ng of cell-bound protein A/ml. Strain Cowan I of S. aureus, used as positive control, produced > 1200 ng of cell-bound protein A/ml.
Immunoelectron microscopy Immunoelectron microscopy was used to confirm the presence of cell-bound protein A in swine isolates and its absence in chicken and cow isolates. Strain Cowan I of S. aureus was strongly labeled with the electron-dense gold particles (Fig. 2A). Swine isolates of S. hyicus subsp, hyicus, which were positive for cell-bound protein A in the ELISA, were also labeled with the gold parti-
PROTEIN A ASSAY OF STAPHYLOCOCCUS HYICUS
301
cles (Fig. 2B, C), but the n u m b e r o f b o u n d gold particles was m u c h smaller than that o f S. aureus strain Cowan I. However, chicken (Fig. 2D) or cow isolates were not labeled with the gold particles. DISCUSSION Although almost all o f the swine isolates o f S. hyicus subsp, hyicus were positive for cell-bound protein A, all o f the chicken and bovine isolates were negative for it in the ELISA. Also, in the immunoelectron microscopy assay, swine isolates were labeled with goat anti-mouse IgG conjugated to colloidal gold particles, but no chicken and bovine isolates were labeled. These results of protein A assay were similar to those reported by Phillips and Kloos ( 1981 ), H o o v e r et al. ( 1983 ) and Takeuchi et al. ( 1988 ). This difference between the S. hyicus subsp, hyicus isolates o f different host origins was also recognized in proteolytic zymogram patterns described previously (Takeuchi et al., 1987). Protein A is also produced by most h u m a n and bovine isolates of Staphylococcus aureus (Forsgren, 1970; Kronvall et al., 1971, 1972). It activates serum complement, induces chemotaxis and hypersensitivity and inhibits phagocytosis (Forsgren et al., 1983). LeFevre and Jensen (1987) suggested that protein A, when combined with other factors, may contribute to the pathogenicity o f S. aureus. On the other hand, Teranishi et al. (1988) reported that protein A positive isolates of S. hyicus subsp, hyicus adhered to Vero cells more effectively than did protein A negative isolates. There is a paucity o f data on the biological activities o f protein A among S. hyicus subsp. hyicus. Therefore, it is necessary to examine the biological activities of protein A in order to clear the contribution of protein A to the pathogenicity o f S. hyicus subsp, hyicus.
REFERENCES Cox, H.U., Schmeer, N.S. and Newman, S.S., 1986. Protein A in Staphylococcus intermedius isolates from dogs and cats. Am. J. Vet. Res., 47: 1881-1884. Devriese, L.A., 1977. Isolation and identification of Staphylococcus hyicus. Am. J. Vet. Res., 38: 787-792. Devriese, L.A., 1980. Pathogenic staphylococci in poultry. World's Poult. Sci. J., 36: 227-236. Devriese, L.A. and Derycke, J., 1979. Staphylococcus hyicus in cattle. Res. Vet. Sci., 26: 356358. Forsgren, A., 1970. Significanceof protein A production by staphylococci. Infect. Immun., 2: 672-673. Forsgren, A, Ghetie, V., Lindmark, R. and Sjoquist, J., 1983. Protein A and its exploitation, pp. 429-480. In: C.S.F. Easmon and C. Adlam (Editors), Staphylococciand staphylococcalinfections, Vol. 2. Academic Press Inc., New York, NY. Hoover, D.G., Tatini, S.R. and Maltais, J.B., 1983. Characterization of staphylococci. Appl. Environ. Microbiol., 46: 649-660.
302
S. T A K E U ( HI ET AL.
Kronvall, G., Dossett, J.H., Quie, P.G. and Williams, R.C. Jr., 1971. Occurrence of protein A in staphylococcal strains: Quantitative aspects and correlation to antigenic and bacteriophages. Infect. lmmun., 3: 10-15. Kronvall, G., Holmberg, O. and Ripa, T., 1972. Protein A in Staphylococcus aureus strains of human and bovine origin. Acta Pathol. Microbiol. Scand., B, 80: 735-742. LcFcvre, S.D. and Jensen, M.M., 1987. Staphylococcosis of turkeys. 2. Assay of protein A levels of staphylococci isolated from turkeys. Avian Dis., 31: 70-73. Mfiller, H., Schaeg, W. and Blobel, H., 1981. Protein A activity of Staphylococcus hyicus in comparison to protein A of Staphylococcus aureus. Zentralbl. Bakt. Hyg., A, 249:443-451. Phillips, W.E. Jr. and Kloos, W.E., 1981. Identification of coagulase-positive Staphylococcus intermedius and Staphylococcus hyicus subsp, hyicus isolates from veterinary clinical specimens. J. Clin, Microbiol., 14:671-673. Sato, G., Miura, S. and Terakado, N., 1972. Classification of chicken coagulase-positive staphylococci into 4 biological types and relation of the types to additional characteristics including coagulase antigenic type. Jpn. J. Vet. Res., 20:90-110. Takeuchi, S., Kobayashi, Y., Morozumi, T. and Niibori, S., 1985. Isolation and some properties of Staphylococcus hyicus subsp, hyicus from pigs, chickens and cows. Jpn. J. Vet. Sci., 47: 841-843. Takeuchi, S., Kobayashi, Y. and Morozumi, T., 1987. Proteolytic zymograms of Staphylococcus hyicus subsp, hyicus isolated from pigs, chickens and cows. Vet. Microbiol., 14: 47-52. Takeuchi, S., Kobayashi, Y. and Morozumi, T. and Mort, Y., 1988. Protein A in Staphylococcu.s hyicus subsp, hyicus isolates from pigs, chickens and cows. J pn. J. Vet. Sci., 50:153-157. Tcranishi, H., Shimizu, A., Kawano, J. and Kimura, S., 1988. Comparative adhesion of protein A - positive and protein A - negative strains of porcine Staphylococcus hyicus subsp, hvicus to Vero cells. Jpn. J. Vet. Sci., 50: 825-827.
297
Short Communication Assay of protein A in Staphylococcus hyicus subsp, hyicus by ELISA and immunoelectron microscopy S. Takeuchi, Y.
K o b a y a s h i a a n d Y. M o r i a
Department of Agriculture, Fukui Prefectural College, 97-21-30batake, Fukui 910, Japan ~National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan (Accepted 6 March 1990)
ABSTRACT Takeuchi, S., Kobayashi, Y. and Mori, Y., 1990. Assay of protein A in Staphylococcus hyicus subsp. hyicus by ELISA and immunoelectron microscopy. Vet. Microbiol., 25: 297-302. The presence and quantity of protein A in Staphylococcus hyicus subsp, hyicus isolates were examined by an enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. Cellbound protein A was demonstrated in 45 (94%) of 48 isolates from diseased pigs and in 113 (86%) of 132 isolates from healthy pigs by ELISA using peroxidase-conjugated rabbit antibody, but was not found in isolates from chickens and cows. Most of these swine isolates contained about 100 to 300 ng of cell-bound protein A/ml. Extracellular protein A was not detected in any isolates from pigs, chickens or cows. In the immunoelectron microscopy assay, swine isolates were labeled with goat antimouse IgG conjugated to colloidal gold particles, but chicken and cow isolates were not labeled.
INTRODUCTION
Staphylococcus hyicus subsp, hyicus is considered to be an etiological agent of exudative epidermitis in young pigs. The organism is also isolated from the skin and nares of healthy pigs, chickens and cattle (Sato et al., 1972; Devriese, 1977, 1980; Devriese and Derycke, 1979; Takeuchi et al., 1985 ). Some workers (Mfiller et al., 1981; Phillips and Kloos, 1981; Hoover et al., 1983; Takeuchi et al., 1988 ), using a hemagglutination test or agar gel immunodiffusion test, demonstrated that swine isolates of S. hyicus subsp, hyicus produced protein A, which possesses the capability of binding the Fc region of immunoglobulins from various mammalian species. In the present study, the authors examined the presence and quantity of cell-bound and extracellular protein A in S. hyicus subsp, hyicus isolates from pigs, chickens and cows by 0378-1135/90/$03.50
© 1 9 9 0 - - Elsevier Science Publishers B.V.
298
s. TAKEUCHI ET AL.
an enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. MATERIALS AND METHODS
Bacterial isolates A total of 257 isolates of S. hyicus subsp, hyicus were examined for the presence of protein A. Of these isolates, 48 were obtained from lesions of pigs affected with exudative epidermitis, 132 from nasal cavities or external ears of clinically healthy pigs, 71 from nasal cavities of healthy chickens and 6 from mastitic udders of cows (Takeuchi et al,, 1985 ). S. aureus strain Cowan I and strain Wood 46 were used as protein A positive and negative controls, respectively.
Samples for ELISA For cell-bound protein A analysis, the bacteria to be tested were inoculated on brain heart infusion (BHI) agar (Difco) and cultivated at a 37 °C for 18 h. The bacterial cells were suspended in sterile 0.9 M NaC1 solution containing 0.5% formalin and incubated at 37 °C for 1 h for inactivation. Cells were then washed three times with 0.9 M NaC1 solution and resuspended in the saline solution to a concentration of MacFarland No. 3. For extracellular protein A analysis, the bacteria to be tested were inoculated in BHI broth supplemented with 0.3% yeast extract (Difco) and 1% maltose and cultivated at 35 °C for 20 h as described by Cox et al. (1986). The culture fluids were centrifuged at 12 000 rpm for 10 min and the resulting supernatants were filtered through a 0.45/~m filter (Millipore). The bacterial cells and supernatants were stored at - 80 ° C until used for ELISA.
ELISA One hundred/tl of the bacterial cells or supernatants, diluted 5- or 10-fold, were coated to wells in microtiter plates (Dynatech Laboratories Inc., Germany) and incubated at 37°C for 16 h. After washing, 100/~1 of peroxidaseconjugated rabbit antibody (Kirkegaard & Perry Laboratories Inc., MD ), diluted 1000 X, were added to each well of the plates and incubated at 25 ° C for 1 h. After washing, 100/tl of substrate solution (o-phenylenediamine dihydrochloride) was added to each well. After 30 min incubation at room temperature, the reaction was stopped and the optical density (OD) was measured. Recombinant protein A (Repligen Corporation Massachusetts) was used as a standard sample in the ELISA.
Immunoelectron microscopy The bacteria, freshly grown on BHI agar, were suspended in PBS containing fetal calf serum (PBS-FCS), washed by centrifugation, and resuspended
299
PROTEIN A ASSAY OF STAPHYLOCOCCUS HYICUS
to a density of approximately 109 C F U / m l in PBS-FCS. The bacterial suspensions were applied to carbon-coated grids (Oken shoji, Japan), which were pasted on a glass microscope slide with a piece of double-sided adhesive tape. After 5 min, excess suspension was removed with a wedge of filter paper, and goat anti-mouse IgG conjugated to colloidal gold particles (E.Y. Laboratories Inc., California) was applied to each grid. The grids were incubated at room temperature for 1 h and excess complex was removed with a piece of filter paper. After washing with PBS, the grids were stained with 0.5% ammonium molybdate solution and examined under an electron microscope (Hitachi, Japan) at 60 kV accelerating voltage. RESULTS
ELISA The presence and quantity of protein A in isolates ofS. hyicus subsp, hyicus were examined by the ELISA using peroxidase-conjugated rabbit antibody. In the ELISA, OD values of >0.201 were considered to be positive for protein A because the OD value of S. aureus strain Wood 46 was < 0.150. Cellbound protein A was demonstrated in 45 (94%) of 48 isolates from diseased pigs and in 113 (86%) of 132 isolates from healthy pigs. However, all isolates from chickens and cows were negative for cell-bound protein A. On the other hand, extracellular protein A was not detected in any isolate from diseased pigs, healthy pigs, chickens or cows. When purified recombinant protein A was examined by ELISA, a linear relationship was demonstrated between the OD values and the concentra-
E C~
r"
o
=.
I
1 0 1 - 130 1 3 1 - 160 1 6 1 - 190
II
1 9 1 - 220 221 - 250 2 5 1 - 280 281-310
oc o
311 - 3 4 0 3 4 1 - 370
~: .E
371 - 4 0 0 401 - 1,30
o n
431 - 4 6 0 461 - &90
I
I
] Healthy p=gs
>491 0
Diseased pigs
I
I
10
20
No. of
0
I
t
10
20
isolates
Fig. 1. Distribution of concentrations of cell-bound protein A in bacterial cell suspensions of MacFarland No. 3 of swine isolates.
300
S. T A K E U C H I ET AL.
•
A
G
-°*'~°"
,
B
~i¸ i
¸~¸
ii~i~!i
Fig. 2. Immunoelectron micrograph of strain Cowan I of S. aureus (A), swine isolates (B, C) and chicken isolates (D) of S. hyicus subsp, hyicus incubated with goat anti-mouse IgG conj ugated to gold colloidal particles.
tions of 20 to 100 ng of protein A/ml. Therefore, the quantitiy of cell-bound protein A in the S. hyicus subsp, hyicus isolates from diseased and healthy pigs was estimated by reference to this standard curve. As shown in Fig. 1, most of the swine isolates contained about 100 to 300 ng of cell-bound protein A / m l in nondiluted bacterial cell suspensions of a concentration of MacFarland No. 3. The average quantity of cell-bound protein A produced by them was about 200 ng/ml. In particular, strain T 19, which was isolated from a pig with exudative epidermitis, produced about 1000 ng of cell-bound protein A/ml. Strain Cowan I of S. aureus, used as positive control, produced > 1200 ng of cell-bound protein A/ml.
Immunoelectron microscopy Immunoelectron microscopy was used to confirm the presence of cell-bound protein A in swine isolates and its absence in chicken and cow isolates. Strain Cowan I of S. aureus was strongly labeled with the electron-dense gold particles (Fig. 2A). Swine isolates of S. hyicus subsp, hyicus, which were positive for cell-bound protein A in the ELISA, were also labeled with the gold parti-
PROTEIN A ASSAY OF STAPHYLOCOCCUS HYICUS
301
cles (Fig. 2B, C), but the n u m b e r o f b o u n d gold particles was m u c h smaller than that o f S. aureus strain Cowan I. However, chicken (Fig. 2D) or cow isolates were not labeled with the gold particles. DISCUSSION Although almost all o f the swine isolates o f S. hyicus subsp, hyicus were positive for cell-bound protein A, all o f the chicken and bovine isolates were negative for it in the ELISA. Also, in the immunoelectron microscopy assay, swine isolates were labeled with goat anti-mouse IgG conjugated to colloidal gold particles, but no chicken and bovine isolates were labeled. These results of protein A assay were similar to those reported by Phillips and Kloos ( 1981 ), H o o v e r et al. ( 1983 ) and Takeuchi et al. ( 1988 ). This difference between the S. hyicus subsp, hyicus isolates o f different host origins was also recognized in proteolytic zymogram patterns described previously (Takeuchi et al., 1987). Protein A is also produced by most h u m a n and bovine isolates of Staphylococcus aureus (Forsgren, 1970; Kronvall et al., 1971, 1972). It activates serum complement, induces chemotaxis and hypersensitivity and inhibits phagocytosis (Forsgren et al., 1983). LeFevre and Jensen (1987) suggested that protein A, when combined with other factors, may contribute to the pathogenicity o f S. aureus. On the other hand, Teranishi et al. (1988) reported that protein A positive isolates of S. hyicus subsp, hyicus adhered to Vero cells more effectively than did protein A negative isolates. There is a paucity o f data on the biological activities o f protein A among S. hyicus subsp. hyicus. Therefore, it is necessary to examine the biological activities of protein A in order to clear the contribution of protein A to the pathogenicity o f S. hyicus subsp, hyicus.
REFERENCES Cox, H.U., Schmeer, N.S. and Newman, S.S., 1986. Protein A in Staphylococcus intermedius isolates from dogs and cats. Am. J. Vet. Res., 47: 1881-1884. Devriese, L.A., 1977. Isolation and identification of Staphylococcus hyicus. Am. J. Vet. Res., 38: 787-792. Devriese, L.A., 1980. Pathogenic staphylococci in poultry. World's Poult. Sci. J., 36: 227-236. Devriese, L.A. and Derycke, J., 1979. Staphylococcus hyicus in cattle. Res. Vet. Sci., 26: 356358. Forsgren, A., 1970. Significanceof protein A production by staphylococci. Infect. Immun., 2: 672-673. Forsgren, A, Ghetie, V., Lindmark, R. and Sjoquist, J., 1983. Protein A and its exploitation, pp. 429-480. In: C.S.F. Easmon and C. Adlam (Editors), Staphylococciand staphylococcalinfections, Vol. 2. Academic Press Inc., New York, NY. Hoover, D.G., Tatini, S.R. and Maltais, J.B., 1983. Characterization of staphylococci. Appl. Environ. Microbiol., 46: 649-660.
302
S. T A K E U ( HI ET AL.
Kronvall, G., Dossett, J.H., Quie, P.G. and Williams, R.C. Jr., 1971. Occurrence of protein A in staphylococcal strains: Quantitative aspects and correlation to antigenic and bacteriophages. Infect. lmmun., 3: 10-15. Kronvall, G., Holmberg, O. and Ripa, T., 1972. Protein A in Staphylococcus aureus strains of human and bovine origin. Acta Pathol. Microbiol. Scand., B, 80: 735-742. LcFcvre, S.D. and Jensen, M.M., 1987. Staphylococcosis of turkeys. 2. Assay of protein A levels of staphylococci isolated from turkeys. Avian Dis., 31: 70-73. Mfiller, H., Schaeg, W. and Blobel, H., 1981. Protein A activity of Staphylococcus hyicus in comparison to protein A of Staphylococcus aureus. Zentralbl. Bakt. Hyg., A, 249:443-451. Phillips, W.E. Jr. and Kloos, W.E., 1981. Identification of coagulase-positive Staphylococcus intermedius and Staphylococcus hyicus subsp, hyicus isolates from veterinary clinical specimens. J. Clin, Microbiol., 14:671-673. Sato, G., Miura, S. and Terakado, N., 1972. Classification of chicken coagulase-positive staphylococci into 4 biological types and relation of the types to additional characteristics including coagulase antigenic type. Jpn. J. Vet. Res., 20:90-110. Takeuchi, S., Kobayashi, Y., Morozumi, T. and Niibori, S., 1985. Isolation and some properties of Staphylococcus hyicus subsp, hyicus from pigs, chickens and cows. Jpn. J. Vet. Sci., 47: 841-843. Takeuchi, S., Kobayashi, Y. and Morozumi, T., 1987. Proteolytic zymograms of Staphylococcus hyicus subsp, hyicus isolated from pigs, chickens and cows. Vet. Microbiol., 14: 47-52. Takeuchi, S., Kobayashi, Y. and Morozumi, T. and Mort, Y., 1988. Protein A in Staphylococcu.s hyicus subsp, hyicus isolates from pigs, chickens and cows. J pn. J. Vet. Sci., 50:153-157. Tcranishi, H., Shimizu, A., Kawano, J. and Kimura, S., 1988. Comparative adhesion of protein A - positive and protein A - negative strains of porcine Staphylococcus hyicus subsp, hvicus to Vero cells. Jpn. J. Vet. Sci., 50: 825-827.
