Veterinary Microbiology, 30 ( 1992 ) 73-85 Elsevier Science Publishers B.V., Amsterdam
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Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix
rhusiopathiae Shigeki Kobayashi, Hisaaki Sato, Kazuhiko Hirose and Hiroshi Saito Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan (Accepted 20 June 1991 ) ABSTRACT Kobayashi, S., Sato, H., Hirose, K. and Saito, H., 1992. Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae. Vet. Microbiol., 30: 73-85. Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar la) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5 ). Anti-NaOH P-2 and antiCF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and lhe antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.
INTRODUCTION
Swine erysipelas is widespread through the world and constitutes a serious problem to the swine-raising industry. A live-cell vaccine of an acriflavin-fast attenuated strain (Koganei) of Erysipelothrix rhusiopathiae has been commonly used for prophylaxis in Japan (Seto et al., 1971 ). Recently, Sawada et al. (1987b) and Sawada and Takahashi (1987) reported that protective antigens were present in the culture filtrate of strain Koganei. Traub (1947), White and Verwey (1970a) and Rothe (1982b) reported that immunization against erysipelas with a killed vaccine required antigens in both the bacterial cells and the culture filtrates. Dedi6 (1949), Erler ( 1973 ) and Rothe (1982b) demonstrated that the cell antigens extracted by alkaline treatment have moderate immunizing potency. These antigenic substances have been detected in protein fractions, but they are more difficult to separate from those 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
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fractions. The protective antigens from the culture filtrate and from cell components extracted by alkaline treatment were compared in this study. MATERIALS AND METHODS
Bacterial strains. Strains Koganei 65-0.15 (serovar 1a) of acriflavin-fast attenuated E. rhusiopathiae was used for the preparation of antigens. Virulent strains Fujisawa (serovar la) and Agata (serovar 5 ) were used for challenge. The Marienfelde strain was used in the growth agglutination (GA) test.
Mice. Four-week-old ddY SPF mice were purchased from the Japan SLC Co. Medium. Tryptic soy agar (Difco) containing 0.3% Tris aminomethane and 0.1% Tween 80 (TT-TS broth) was used for maintenance of the strains. The test m e d i u m was HI broth (Difco) containing 0.1% Tween 80, 0.25% glucose, 0.3088% NaRHPO4 and 0.442% K H 2 P O 4 with the pH adjusted to 7.6. Bacterial suspension (0.1 ml; 1 X 108 C F U / m l ) was added to 1000 ml of the test m e d i u m and incubated, without shaking, at 37 °C for 24 hr.
Preparation of crude antigen Crude alkaline-extracted antigen. After incubation, the bacteria were centrifuged at 10 000 g for 20 min and the pellets were washed in Dulbecco's phosphate buffered saline (PBS, pH 7.4). The bacterial cell components were extracted with gentle stirring at a ratio of 10 g per pellet to 100 ml of 0.01 N NaOH solution at 4 °C for 16 h. After extraction, the suspension was centrifuged at 10 000 g for 20 min. The supernatant was used as the crude antigen extracted by alkaline treatment.
Crude culturefihrate antigen. After incubation, bacterial cells were removed by centrifugation at 10 000 g for 20 min and the supernatant was filtered through a membrane filter.
Fractionation of antigen Salting out with ammonium sulfate, a. Alkaline-extracted antigen. The crude antigen was salted out with 50% saturated a m m o n i u m sulfate and stored at 4 ° C overnight. It was then centrifuged at 15 000 g for 30 rain. The precipitate was dissolved in 10 ml of 0.05 M Tris-HC1 buffer (pH 8.0) containing 0. ! % sodium dodecyl sulfate (SDS). The solution was dialyzed in 0.05 M TrisHC1 buffer (pH 8.0) at 4°C for 24 hr and concentrated to 4 ml with polyethylene glycol (PEG) 20 000 (NaOH Am-S). b. Culture filtrate antigen. The crude culture filtrate antigen was concen-
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
75
trated by the same procedure as for the crude alkaline-extracted antigen and the final product was concentrated to 5 ml by ultrafiltration through a UK-50 membrane (CF Am-S).
Gelfiltration on Sephadex G-200. a. Alkaline-extracted antigen. Concentrated antigen (4 ml) was fractionated on a Sephadex G-200 gel (elution buffer: 0.05 M Tris-HC1 (pH 8.0) containing 0.1% SDS). Three fractions (NaOH P- 1, NaOH P-2 and NaOH P-3 ) were obtained by gel filtration from the crude alkaline-extracted antigen. These fractions were dialyzed and concentrated with PEG 20 000 and the NaOH P-I fraction was rechromatographed on Sephadex G-200 column (NaOH RP-1 ). b. Culture filtration antigen. Three ml of the concentrated antigen was fractionated on a Sephadex G-200 gel by the same procedure as for the alkalineextracted antigen. The protein content was measured by the BCA microplate method (SSrensen and Brodbeck, 1986) and each fraction (CF P-l, CF P-2, CF P-3 and CF P-4) was concentrated with PEG 20 000 after dialysis for 48 hr. Affinity chromatography. The glycoprotein from the CF P-2 fraction was separated with concanavalin A-Sepharose 4B (Con A-Sepharose). The CF P-2 fraction (5 ml) was layered on a Con A-Sepharose column and washed with 0.05 M Tris-HC1 buffer (pH 8.0) containing 0.5 M NaC1 (glycoprotein-free fraction). Subsequent elution (glycoprotein fraction) was obtained with 0.2 M o~-mannoside solution at 10 times the column volume. The protein content of each fraction was determined by the BCA method. The washed fraction (Con A P-W) and the eluted fraction (Con A P-E) were concentrated with PEG 20 000 after dialysis for 48 h. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE of the NaOH P-2 fraction was performed by Laemmli's method (Laemmli, 1970). Fifty/A of the sample and 200/A of marker protein were dispensed to each lane. Electrophoresis was performed at 15 mA/gel for concentration and 30 mA/gel for separation with constant current for about 5 h. The separated proteins in the slab gel were transferred to a polyvinylidene difluoride membrane with a horizon blotting transfer apparatus using constant current of 2 m A / c m 2 for 90 min. After transfer, the membrane was stained with 1% amide black 10B for 4 min and were decolorized with 7% acetic acid. Immunogenicity test Challenge with a strain of the same serovar Fujisawa. Fujisawa is the same serovar ( 1a), as the immunizing strains, was used to challenge mice immunized with the following antigens prepared from the Koganei strain: amino-
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nium sulfate precipitated fractions, NaOH Am-S and CF Am-S; protein fractions, NaOH RP-1, NaOH P-2, NaOH P-3, CF P-l, CF P-2, CF P-3 and CF P-4; a glycoprotein fraction, Con A P-E and the glycoprotein-free fraction, Con A P-W. Each of the above antigens was mixed with an equal volume of Freund's incomplete adjuvant and the final protein content was adjusted to 2 5 0 / t g / m l and 50/~g/ml for NaOH Am-S, to 1000 # g / m l and 200/zg/ml for CF Am-S, to 100/~g/ml and 20 #g/ml for NaOH RP-1, NaOH P-2, and NaOH P-3, to 500/zg/ml and 100/~g/ml for CF P- 1, CF P-2, CF P-3 and CF P-4, and to 400/zg/ml and 40 # g / m l for Con A P-E and Con A P-W. Each antigen (500 ml ) was injected subcutaneously into 4-week-old SPF ddY female mice. Three weeks later, the immunized mice were challenged with 0.1 ml of a bacterial suspension (adjusted to a concentration of 6 X l03 C F U / m l ) of strain Fujisawa cultured in HI broth. The mice were observed every day for 14 days after challenge and the i m m u n e responses were expressed as the survival rate.
Challenge. Strain Agata (serovar 5) was used for challenging mice immunized with NaOH RP-1, NaOH P-2 and CF P-2 antigens by the method described above. The protein content of the antigens was adjusted to 50/zg/ml for NaOH RP-1 and NaOH P-2 and to 2 0 0 / t g / m l for CF P-2, and the challenge dose of the bacterial suspension was 0.1 ml containing 2 × 103 CFU. Preparation of antiserum NaOH RP-1, NaOH P-2 and CF P-2 antigens were mixed with equal volumes of Freund's incomplete adjuvant and the final protein content was adjusted to 100 # g / m l for NaOH RP- 1 and NaOH P-2 and to 200/~g/ml for CF P-2. Each antigen was injected into 5-10 mice; two 0.5-ml injections with a 2-week interval. The attenuated Koganei strain was incubated in HI broth at 37°C for 24 h. A bacterial suspension was adjusted to 2X 107 C F U / m l and 0.1 ml was injected subcutaneously into each mouse. Blood was taken from the mice i m m u n i z e d with the Koganei strain 3 weeks and from the others 1 week after the second injection, and the serum was separated.
Passive immunization test Antisera prepared as described above were injected intraperitoneally in 0.2ml quantities into mice. After 4 h, the mice were challenged subcutaneously with 1 X 103 C F U (0.1 ml) of strain Fujisawa cells. The mice were observed every day for 10 days and the responses were expressed as the survival rate.
Growth agglutination test (GA test) Antibody levels were tested by a GA test based on the method of Seto et al. ( 1971 ) using live cells of strain Marienfelde; the titers were expressed as the reciprocal of the m a x i m u m serum dilution.
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Preparation of sonicated antigen Strains Koganei, Fujisawa and Agata were incubated in TT-TS broth (pH 7.6) at 37 °C for 24 h. After incubation, the bacterial cells were washed three times with PBS and 1 g of wet cells was resuspended in 10 ml of PBS. The bacteria were sonicated at 200 W, 9 kHz, for 30 min and centrifuged at 15 000 g for 30 min. The supernatant was used as sonicated cell antigen.
Western blot analysis Protein components of the NaOH P-2 antigen and sonicated cell antigen of strains Koganei, Fujisawa and Agata were separated by SDS-PAGE and these antigens were analyzed by the Western blotting method (Towbin et al., 1979 ). RESULTS
Fractionation of antigens Alkaline-extracted antigen. The elution profile of Sephadex G-200 gel filtration of an alkaline-extracted antigen showed some major peaks, the first of which (NaOH P-I fraction) was rechromatographed using Sephadex G-200. The resulting fraction was recovered as NaOH RP- 1.
Culture filtrate antigen. The elution profile of the culture filtrate antigen obtained by gel filtration using a Sephadex G-200 column showed four peaks, CF P-l, CF P-2, CF P-3 and CF P-4. The CF P-2 fraction was subjected to affinity chromatography with Con A-Sepharose 4B and two fractions were obtained (Con A P-W and Con A P-E). TABLEI lmmunogenicity of alkaline-extracted antigen derived from strain Koganei Antigen fraction
Immunizing dose ( protein/~g/mouse )
No. of surviving mice/No, tested (%)
NaOH Am-S ~
NaOH P-3
250 50 100 20 100 20 100
2/ 2 4/4 4/ 4 4/ 4 4/ 4 4/ 4 1/ 4
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (25.0)
20 Control 3
-
0 / 4 0 / 4
(0.0) (0.0)
NaOH RP-12 NaOH P-2
~Antigen fractionated with ammonium sulfate from alkaline-extracted antigen. 2Rechromatographed preparation of fraction NaOH P- 1. 30.05 M Tris-HC1 buffer (pH 8.0).
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S. KOBAYASHIET AL.
TABLE 2 lmmunogenicity of culture filtrate antigen derived from strain Koganei Antigen fraction
Immunizing dose (protein/xg/mouse )
No. of surviving mice/No, tested (%)
CFAm-S ~
1000 200 500
2/3 (66.7) 3/4 (75.0) 4 / 4 (100.0)
CF P-1 CFP-2 CF P-3 CF P-4 Control ~
100
0/4
500 100 500 100 500 100
3 / 3 (100.0) 4 / 4 (100.0) 0/ 4 (0.0) 1 / 4 (25.0) 0/ 4 (0.0) 0/4 (0.0) 0/ 4 (0.0)
-
(0.0)
~Fractionated antigen with a m m o n i u m sulfate from culture filtrate antigen. 2Non-inoculated culture medium was used for control. TABLE 3 l mmunogenicity of glycoprotein and glycoprotein free fractions derived from culture filtrate of strain Koganei Antigen fraction
Immunizing dose (Protein/zg/mouse)
No. of surviving mice/No, tested (%)
Con A P-E 1
400 40 400 40 -
0/ 4 (0.0) 0/ 4 (0.0) 4 / 4 ( 100.0 ) 4 / 4 (100.0) 0/ 4 (0.0)
Con A P-W 2 Control 3
~Glycoprotein fraction was eluted from a Con A-Sepharose 4B affinity column. 2Glycoprotein free fraction was the washing solution of the Con A-Sepharose 4B affinity column. 30.05 M Tris-HCl buffer (pH 8.0).
SDS-PAGE On SDS-PAGE of the NaOH P-2 fraction, 10 main protein bands, 65, 58, 55, 54, 42, 40, 38, 35, 30 and 26 kDa were obtained. Immunogenicity test Challenge with the same serovar. NaOH Am-S, NaOH RP- 1, NaOH P-2 and
NaOH P-3 antigens derived from strain Koganei were injected into mice, which were challenged with the virulent strains Fujisawa. As shown in Table 1, NaOH Am-S, NaOH RP-1 and NaOH P-2 antigens gave 100% protection. Results of the immunogenicity test with CF Am-S, CF P-l, CF P-2, CF P-3
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
79
TABLE 4
Protective effect of antigens derived from strain Koganei against challenge with a different serovar strain Antigen fraction
Immunizing dose ( Protein ktg/mouse )
No. of surviving mice/No, tested (%)
NaOH RP-I NaOH P-2 CF P-2 Control
50 50 200 -
0 3 4 0
/ / / /
3 (0.0) 4 (75.0) 4 (100.0) 4 (0.0)
~Strain Agata (serovar: 5 ) of E. rhusiopathiae. 20.05 M Tris-HCl buffer (pH 8.0). TABLE5
Passive protective effect of antisera to antigen fractions derived from strain Koganei Antiserum
GA titer
No. of surviving mice/No, tested (%)
Anti-NaOH RP-I serum Anti-NaOH P-2 serum Anti-CF P-2 serum Anti-Koganei strain serum Control ~
640 640 2560 640 < l0
0 4 4 4 0
/ / / / /
4 (0.0) 4 (100.0) 4 (100.0) 4 (100.0) 4 (0.0)
INormal mouse serum.
and CF P-4 are shown in Table 2. Protective activity was demonstrated with the CF Am-S antigen fractions containing 1000 pg and 200 pg protein. Complete protection was provided by both the CF P-I antigen (at 500 pg of protein) and the CF P-2 antigen (at 500/tg and 100/tg of protein). Of the preparations subjected to affinity chromatography, the Con A P-W fraction gave 100% protection at both protein contents tested, while Con A P-E fraction did not show any protective activity (Table 3 ). Challenge with a strain o f a different serovar. N a O H RP- 1, NaOH P-2 and CF P-2 antigens were used for challenge with a different serovar (strain Agata; serovar 5 ). NaOH P-2 and CF P-2 antigens were highly protective, but NaOH RP-1 did not show any protective activity (Table 4). Passive i m m u n i z a t i o n test
Antisera obtained from mice immunized with NaOH RP-1, N a O H P-2, CF P-2 and the vaccine strain Koganei were used for passive immunization. All of the mice passively immunized with anti-NaOH P-2 serum, anti-CF P-2 serum and anti-Koganei strain serum were protected against challenge with
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s. KOBAYASHI ET AL.
MP
1
2
3
4
78K 66K
43K
30K
Fig. 1. Western blot analysis of N a O H P-2 fraction. MP: Marker protein stained with amido black 10B. Lane 1: Anti-NaOH RP-I serum. Lane 2: Anti-NaOH P-2 serum. Lane 3: Anti-CF P-2 serum. Lane 4: Anti-Koganei serum. K = k D a .
strain Fujisawa, but anti-NaOH RP-1 serum did not show any protection effect (Table 5 ) The GA titers of these antisera are also shown in Table 5. The titers ranged from 640 to 2560, whereas normal mouse serum caused no agglutination.
Western blot analysis Western blot analysis was performed with the NaOH P-2 antigen and sonicated cell antigen and the above four antisera. The anti-NaOH RP-1 serum strongly recognized the 65 kDa protein and two proteins over 100 kDa, while the anti-NaOH P-2, and anti-CF P-2 sera recognized proteins over 100, 65, 62, 42-45, 40-42 and 28 kDa in the NaOH P-2 antigen. The antiserum against strain Koganei recognized the same protein bands as did the anti-NaOH P-2 and anti-CF P-2 sera with the exception of proteins over 100 kDa (Fig. 1 ). In the sonicated cell antigens of strain Agata (Fig. 2 ), strain Fujisawa (Fig. 3 ) and strain Koganei (Fig. 4), anti-NaOH RP-1 serum recognized some bands
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
MP
1
2
3
81
4
66K 43K
3OK
Fig. 2. Western blot analysis of sonicated antigen from E. rhusiopathiae strain Agata. See legend for Fig. 1.
of approximately 100 kDa, but anti-NaOH P-2 and anti-CF P-2 sera recognized three protein bands, 62, 42 and 41 kDa. Furthermore, the anitiserum against strains Koganei recognized two protein bands, 32 and 28 kDa. DISCUSSION
Traub ( 1947 ) reported that the culture filtrate ofE. rhusiopathiae could be concentrated with a l u m i n u m hydroxide, and demonstrated that it had a protective effect against E. rhusiopathiae infection in swine and mice. He pointed out that the protective activity required the presence of some soluble antigens in the culture filtrate and bacterial cells. White and Verwey (1970b) showed that the culture filtrate and killed bacterial cells both afforded protection in mice, and also demonstrated cross protection between serovar 1 and 2 strains. In our study, we found that in both the alkaline extract of the cell and the culture filtrate of strain Koganei were productive in mice. White and Verwey
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S. KOBAYASHI ET AL.
MP
1
2
3
4
~K
Fig. 3. Western blot analysis of sonicated antigen from E. rhusiopathiae strain Fujisawa. See legend for Fig. 1.
(1970a,b) and Rothe (1982b) suggested that the protective activity was induced by glycoproteins or a glycoprotein-lipid complex because the activity in the culture filtrate was decreased by muramidase. In the present study, glycoprotein separated from the most immunogenic of the culture filtrate fraction (CF P-2 ) with Con A-Sepharose 4B did not show protective activity. Sawada et al. (1987a,c) reported that the protective antigen was present in the culture filtrate of the Koganei strain and that there was cross protection between serovars 1a and 2. Protective activity was also found in antiserum to the culture filtrate and cross protection also occured to challenge with serovars la, lb, 2, 5, 6, 8, 11, 12, 15, 16, 18, 21 and type N strains (Sawada and Takahashi, 1987; Sawada et al., 1987b). But, in these experiments, they did not use the fractionated antigen of the culture filtrate or alkaline-extracted cell antigen. In our study, we have tested the immunological potency of NaOH P-2 and CF P-2 antigens, which were obtained by alkaline extraction from the bacterial cell components and from the culture filtrate, respectively. Moreover, anti-
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
MP
1
2
3
83
4
66K 43K
3OK
Fig. 4. Western blot analysis of sonicated antigen from E. rhusiopathiae strain Koganei. See legend for Fig. 1.
NaOH P-2 and anti-CF P-2 mice sera, which were obtained by immunization of NaOH P-2 and CF P-2 antigens strongly recognized the three bands of 62, 42 and 41 kDa proteins by NaOH P-2 antigen in western blot analysis. These results suggest that the protective antigen in the culture filtrate and the alkaline-extracted protective antigen are the same, because they are both totally protected in the mouse test and detected as the same bands of 62, 42 and 41 kDa proteins by western blot analysis. These antigens are considered to be closely involved in protective activity. Monoclonal antibodies to these antigens are being sought in order to define the protective antigen.
REFERENCES Dedi6, K., 1949. Die saoureloslichen Antigen von Erysipelothrix rhusiopathiae. Monatsh. Veterinaermed., 4: 7-10. Erler, W., 1973. Serologische, chemische und immunchemische Untersuchungen an Rotlaufbakterien. 13. Mitt. Das immunisierende Antigen. Arch. Exper. Vet. Med., 27:321-326.
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Gledhill, A.W., 1947. Some properties ofa thermolabile antigen ofErysipelothrix rhusiopathiae. J. Gen. Microbiol., 1:211-220. Gledhilk A.W., 1952. The immunizing antigen of Erysipelothrix rhusiopathiae. The role of Lantigen. J. Gen. Microbiol., 7: 179-191. Heuner, F., 1957. Zur Technik serologischer Rotlaufuntersuchungen. Berl. Muench. Tier~irztl. Wochenschr., 70:341-344. Lachmann, P.G. and Deicher, H., 1986. Solubilization and characterization of surface antigenic components ofErysipelothrix rhusiopathiae T28. Infect. Immun., 52:818-822. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Rothe, F., 1982a. Das protektive Antigen des Rotlaufbakteriums (Erysipelothrix rhusiopalhiae) 1. Mitteilung: Spezifischer Nachweis des protektiven Antigens. Arch. Exper. Vet. Med., 36: 243-253. Rothe, F., 1982b. Das protective Antigen des Rotlaufbakteriums (Erysipelothrix rhusiopathiae) 2. Mitteilung: Die weitere Charakteisierung des protektiven Antigen. Arch. Exper. Vet. Med., 36: 255-267. Sawada,T. and Takahashi, T., 1987. Cross-protection of mice and swine inoculated with culture filtrate of Erysipelothrix rhusiopathiae and challenge exposure to strains of various serovars. Am. J. Vet. Res., 48: 239-242. Sawada,T., Takahashi, T. and Tamura, Y., 1987a. Protective effect of sera from swine immunized with different fractions from broth culture of an attenuated strain of Erysipelothrix rhusiopathiae. Jpn. J. Vet. Sci., 49: 37-42. Sawada,T., Takahashi, T. and Tamura, Y., 1987b. Antiserum against culture filtrate is crossprotective for various serovars ofErysipelothrix rhusiopathiae. Vet. Microbiol., 14: 87-93. Sawada,T., Takahashi, T. and Seto, K., 1987c. Immunogenicity of different fractions from broth culture of an attenuated strain ofErysipelothrix rhusiopathiae in mice and swine. Jpn. J. Vet. Sci., 49: 151-154. Seto, K., Ando, K., Nishimura, Y., Fujiki, M. and Matsuno, T., 1965. Studies on the swine erysipelas. Evaluation of growth agglutination and killed cell agglutination. Ann. Rep. Nat. Vet. Assay Lab., 4: 54-62. (in Japanese). Seto, K., Nishimura, Y., Fujiki, M., Azechi, H. and Suzuki, K., 1971. Studies on acriflavin-fast attenuated El:vsipelothrix insidiosa. Comparison on pathogenicity and immunogenicity between mice and pigs. Jpn. J. Vet. Sci., 33:161-171. (in Japanese). Smith, P.K., Krohn, R.L., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., 1985. Measurement of protein using bichinchoninic acid. Anal. Biochem., 150: 76-85. SSrensen, K. and Brodbeck, U., 1986. A sensitive protein assay method using micro-titer plates. Experientia, 42:161-162. Towbin, H., Staebelin, T. and Gorden, J., 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci., USA 76: 4350-4354. Traub, E., 1947. Immunisierung gegen Schweinerotlasuf mit Konzentrierten Adsorbatimpstoffen. Monatsh. Veterinaermed., 10:165-173. Truszczynsky, M., 1961. The antigenic structure of virulent and avirulent strain of Er.vsipelothrix rhusiopathiae. 2. Immunochemic and serologic investigations. Am. J. Vet. Res., 22: 839845. Wellmann, G., 1955. Die subklinische Rotlaufinfektion und Bedeutung ftir die Epidemiologie des Schweinelaufs. Zentralbl. I. Orig., 162: 265-274. White, R.R. and Verwey, W.F., 1970a. Isolation and characterization of a protective antigencontaining particle from culture supernatant fluid ofErysipelothrix rhusiopathiae. Infec. Immun., 1: 380-386.
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White, R.R. and Verwey, W.F., 1970b. Solubilization and characterization of a protective antigen ofErysipelothrix rhusiopathiae. Infect. Immun., 1: 387-394. Wood, R.L., 1984. Swine erysipelas--a review of prevalence and research. J. Am. Vet. Meal. Assoc., 184: 944-949.
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Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix
rhusiopathiae Shigeki Kobayashi, Hisaaki Sato, Kazuhiko Hirose and Hiroshi Saito Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan (Accepted 20 June 1991 ) ABSTRACT Kobayashi, S., Sato, H., Hirose, K. and Saito, H., 1992. Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae. Vet. Microbiol., 30: 73-85. Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar la) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5 ). Anti-NaOH P-2 and antiCF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and lhe antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.
INTRODUCTION
Swine erysipelas is widespread through the world and constitutes a serious problem to the swine-raising industry. A live-cell vaccine of an acriflavin-fast attenuated strain (Koganei) of Erysipelothrix rhusiopathiae has been commonly used for prophylaxis in Japan (Seto et al., 1971 ). Recently, Sawada et al. (1987b) and Sawada and Takahashi (1987) reported that protective antigens were present in the culture filtrate of strain Koganei. Traub (1947), White and Verwey (1970a) and Rothe (1982b) reported that immunization against erysipelas with a killed vaccine required antigens in both the bacterial cells and the culture filtrates. Dedi6 (1949), Erler ( 1973 ) and Rothe (1982b) demonstrated that the cell antigens extracted by alkaline treatment have moderate immunizing potency. These antigenic substances have been detected in protein fractions, but they are more difficult to separate from those 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
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S. KOBAYASHI ET A L
fractions. The protective antigens from the culture filtrate and from cell components extracted by alkaline treatment were compared in this study. MATERIALS AND METHODS
Bacterial strains. Strains Koganei 65-0.15 (serovar 1a) of acriflavin-fast attenuated E. rhusiopathiae was used for the preparation of antigens. Virulent strains Fujisawa (serovar la) and Agata (serovar 5 ) were used for challenge. The Marienfelde strain was used in the growth agglutination (GA) test.
Mice. Four-week-old ddY SPF mice were purchased from the Japan SLC Co. Medium. Tryptic soy agar (Difco) containing 0.3% Tris aminomethane and 0.1% Tween 80 (TT-TS broth) was used for maintenance of the strains. The test m e d i u m was HI broth (Difco) containing 0.1% Tween 80, 0.25% glucose, 0.3088% NaRHPO4 and 0.442% K H 2 P O 4 with the pH adjusted to 7.6. Bacterial suspension (0.1 ml; 1 X 108 C F U / m l ) was added to 1000 ml of the test m e d i u m and incubated, without shaking, at 37 °C for 24 hr.
Preparation of crude antigen Crude alkaline-extracted antigen. After incubation, the bacteria were centrifuged at 10 000 g for 20 min and the pellets were washed in Dulbecco's phosphate buffered saline (PBS, pH 7.4). The bacterial cell components were extracted with gentle stirring at a ratio of 10 g per pellet to 100 ml of 0.01 N NaOH solution at 4 °C for 16 h. After extraction, the suspension was centrifuged at 10 000 g for 20 min. The supernatant was used as the crude antigen extracted by alkaline treatment.
Crude culturefihrate antigen. After incubation, bacterial cells were removed by centrifugation at 10 000 g for 20 min and the supernatant was filtered through a membrane filter.
Fractionation of antigen Salting out with ammonium sulfate, a. Alkaline-extracted antigen. The crude antigen was salted out with 50% saturated a m m o n i u m sulfate and stored at 4 ° C overnight. It was then centrifuged at 15 000 g for 30 rain. The precipitate was dissolved in 10 ml of 0.05 M Tris-HC1 buffer (pH 8.0) containing 0. ! % sodium dodecyl sulfate (SDS). The solution was dialyzed in 0.05 M TrisHC1 buffer (pH 8.0) at 4°C for 24 hr and concentrated to 4 ml with polyethylene glycol (PEG) 20 000 (NaOH Am-S). b. Culture filtrate antigen. The crude culture filtrate antigen was concen-
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
75
trated by the same procedure as for the crude alkaline-extracted antigen and the final product was concentrated to 5 ml by ultrafiltration through a UK-50 membrane (CF Am-S).
Gelfiltration on Sephadex G-200. a. Alkaline-extracted antigen. Concentrated antigen (4 ml) was fractionated on a Sephadex G-200 gel (elution buffer: 0.05 M Tris-HC1 (pH 8.0) containing 0.1% SDS). Three fractions (NaOH P- 1, NaOH P-2 and NaOH P-3 ) were obtained by gel filtration from the crude alkaline-extracted antigen. These fractions were dialyzed and concentrated with PEG 20 000 and the NaOH P-I fraction was rechromatographed on Sephadex G-200 column (NaOH RP-1 ). b. Culture filtration antigen. Three ml of the concentrated antigen was fractionated on a Sephadex G-200 gel by the same procedure as for the alkalineextracted antigen. The protein content was measured by the BCA microplate method (SSrensen and Brodbeck, 1986) and each fraction (CF P-l, CF P-2, CF P-3 and CF P-4) was concentrated with PEG 20 000 after dialysis for 48 hr. Affinity chromatography. The glycoprotein from the CF P-2 fraction was separated with concanavalin A-Sepharose 4B (Con A-Sepharose). The CF P-2 fraction (5 ml) was layered on a Con A-Sepharose column and washed with 0.05 M Tris-HC1 buffer (pH 8.0) containing 0.5 M NaC1 (glycoprotein-free fraction). Subsequent elution (glycoprotein fraction) was obtained with 0.2 M o~-mannoside solution at 10 times the column volume. The protein content of each fraction was determined by the BCA method. The washed fraction (Con A P-W) and the eluted fraction (Con A P-E) were concentrated with PEG 20 000 after dialysis for 48 h. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE of the NaOH P-2 fraction was performed by Laemmli's method (Laemmli, 1970). Fifty/A of the sample and 200/A of marker protein were dispensed to each lane. Electrophoresis was performed at 15 mA/gel for concentration and 30 mA/gel for separation with constant current for about 5 h. The separated proteins in the slab gel were transferred to a polyvinylidene difluoride membrane with a horizon blotting transfer apparatus using constant current of 2 m A / c m 2 for 90 min. After transfer, the membrane was stained with 1% amide black 10B for 4 min and were decolorized with 7% acetic acid. Immunogenicity test Challenge with a strain of the same serovar Fujisawa. Fujisawa is the same serovar ( 1a), as the immunizing strains, was used to challenge mice immunized with the following antigens prepared from the Koganei strain: amino-
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nium sulfate precipitated fractions, NaOH Am-S and CF Am-S; protein fractions, NaOH RP-1, NaOH P-2, NaOH P-3, CF P-l, CF P-2, CF P-3 and CF P-4; a glycoprotein fraction, Con A P-E and the glycoprotein-free fraction, Con A P-W. Each of the above antigens was mixed with an equal volume of Freund's incomplete adjuvant and the final protein content was adjusted to 2 5 0 / t g / m l and 50/~g/ml for NaOH Am-S, to 1000 # g / m l and 200/zg/ml for CF Am-S, to 100/~g/ml and 20 #g/ml for NaOH RP-1, NaOH P-2, and NaOH P-3, to 500/zg/ml and 100/~g/ml for CF P- 1, CF P-2, CF P-3 and CF P-4, and to 400/zg/ml and 40 # g / m l for Con A P-E and Con A P-W. Each antigen (500 ml ) was injected subcutaneously into 4-week-old SPF ddY female mice. Three weeks later, the immunized mice were challenged with 0.1 ml of a bacterial suspension (adjusted to a concentration of 6 X l03 C F U / m l ) of strain Fujisawa cultured in HI broth. The mice were observed every day for 14 days after challenge and the i m m u n e responses were expressed as the survival rate.
Challenge. Strain Agata (serovar 5) was used for challenging mice immunized with NaOH RP-1, NaOH P-2 and CF P-2 antigens by the method described above. The protein content of the antigens was adjusted to 50/zg/ml for NaOH RP-1 and NaOH P-2 and to 2 0 0 / t g / m l for CF P-2, and the challenge dose of the bacterial suspension was 0.1 ml containing 2 × 103 CFU. Preparation of antiserum NaOH RP-1, NaOH P-2 and CF P-2 antigens were mixed with equal volumes of Freund's incomplete adjuvant and the final protein content was adjusted to 100 # g / m l for NaOH RP- 1 and NaOH P-2 and to 200/~g/ml for CF P-2. Each antigen was injected into 5-10 mice; two 0.5-ml injections with a 2-week interval. The attenuated Koganei strain was incubated in HI broth at 37°C for 24 h. A bacterial suspension was adjusted to 2X 107 C F U / m l and 0.1 ml was injected subcutaneously into each mouse. Blood was taken from the mice i m m u n i z e d with the Koganei strain 3 weeks and from the others 1 week after the second injection, and the serum was separated.
Passive immunization test Antisera prepared as described above were injected intraperitoneally in 0.2ml quantities into mice. After 4 h, the mice were challenged subcutaneously with 1 X 103 C F U (0.1 ml) of strain Fujisawa cells. The mice were observed every day for 10 days and the responses were expressed as the survival rate.
Growth agglutination test (GA test) Antibody levels were tested by a GA test based on the method of Seto et al. ( 1971 ) using live cells of strain Marienfelde; the titers were expressed as the reciprocal of the m a x i m u m serum dilution.
IMMUNOLOGICALCHARACTERIZATIONOF PROTECTIVEANTIGENS
77
Preparation of sonicated antigen Strains Koganei, Fujisawa and Agata were incubated in TT-TS broth (pH 7.6) at 37 °C for 24 h. After incubation, the bacterial cells were washed three times with PBS and 1 g of wet cells was resuspended in 10 ml of PBS. The bacteria were sonicated at 200 W, 9 kHz, for 30 min and centrifuged at 15 000 g for 30 min. The supernatant was used as sonicated cell antigen.
Western blot analysis Protein components of the NaOH P-2 antigen and sonicated cell antigen of strains Koganei, Fujisawa and Agata were separated by SDS-PAGE and these antigens were analyzed by the Western blotting method (Towbin et al., 1979 ). RESULTS
Fractionation of antigens Alkaline-extracted antigen. The elution profile of Sephadex G-200 gel filtration of an alkaline-extracted antigen showed some major peaks, the first of which (NaOH P-I fraction) was rechromatographed using Sephadex G-200. The resulting fraction was recovered as NaOH RP- 1.
Culture filtrate antigen. The elution profile of the culture filtrate antigen obtained by gel filtration using a Sephadex G-200 column showed four peaks, CF P-l, CF P-2, CF P-3 and CF P-4. The CF P-2 fraction was subjected to affinity chromatography with Con A-Sepharose 4B and two fractions were obtained (Con A P-W and Con A P-E). TABLEI lmmunogenicity of alkaline-extracted antigen derived from strain Koganei Antigen fraction
Immunizing dose ( protein/~g/mouse )
No. of surviving mice/No, tested (%)
NaOH Am-S ~
NaOH P-3
250 50 100 20 100 20 100
2/ 2 4/4 4/ 4 4/ 4 4/ 4 4/ 4 1/ 4
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (25.0)
20 Control 3
-
0 / 4 0 / 4
(0.0) (0.0)
NaOH RP-12 NaOH P-2
~Antigen fractionated with ammonium sulfate from alkaline-extracted antigen. 2Rechromatographed preparation of fraction NaOH P- 1. 30.05 M Tris-HC1 buffer (pH 8.0).
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S. KOBAYASHIET AL.
TABLE 2 lmmunogenicity of culture filtrate antigen derived from strain Koganei Antigen fraction
Immunizing dose (protein/xg/mouse )
No. of surviving mice/No, tested (%)
CFAm-S ~
1000 200 500
2/3 (66.7) 3/4 (75.0) 4 / 4 (100.0)
CF P-1 CFP-2 CF P-3 CF P-4 Control ~
100
0/4
500 100 500 100 500 100
3 / 3 (100.0) 4 / 4 (100.0) 0/ 4 (0.0) 1 / 4 (25.0) 0/ 4 (0.0) 0/4 (0.0) 0/ 4 (0.0)
-
(0.0)
~Fractionated antigen with a m m o n i u m sulfate from culture filtrate antigen. 2Non-inoculated culture medium was used for control. TABLE 3 l mmunogenicity of glycoprotein and glycoprotein free fractions derived from culture filtrate of strain Koganei Antigen fraction
Immunizing dose (Protein/zg/mouse)
No. of surviving mice/No, tested (%)
Con A P-E 1
400 40 400 40 -
0/ 4 (0.0) 0/ 4 (0.0) 4 / 4 ( 100.0 ) 4 / 4 (100.0) 0/ 4 (0.0)
Con A P-W 2 Control 3
~Glycoprotein fraction was eluted from a Con A-Sepharose 4B affinity column. 2Glycoprotein free fraction was the washing solution of the Con A-Sepharose 4B affinity column. 30.05 M Tris-HCl buffer (pH 8.0).
SDS-PAGE On SDS-PAGE of the NaOH P-2 fraction, 10 main protein bands, 65, 58, 55, 54, 42, 40, 38, 35, 30 and 26 kDa were obtained. Immunogenicity test Challenge with the same serovar. NaOH Am-S, NaOH RP- 1, NaOH P-2 and
NaOH P-3 antigens derived from strain Koganei were injected into mice, which were challenged with the virulent strains Fujisawa. As shown in Table 1, NaOH Am-S, NaOH RP-1 and NaOH P-2 antigens gave 100% protection. Results of the immunogenicity test with CF Am-S, CF P-l, CF P-2, CF P-3
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
79
TABLE 4
Protective effect of antigens derived from strain Koganei against challenge with a different serovar strain Antigen fraction
Immunizing dose ( Protein ktg/mouse )
No. of surviving mice/No, tested (%)
NaOH RP-I NaOH P-2 CF P-2 Control
50 50 200 -
0 3 4 0
/ / / /
3 (0.0) 4 (75.0) 4 (100.0) 4 (0.0)
~Strain Agata (serovar: 5 ) of E. rhusiopathiae. 20.05 M Tris-HCl buffer (pH 8.0). TABLE5
Passive protective effect of antisera to antigen fractions derived from strain Koganei Antiserum
GA titer
No. of surviving mice/No, tested (%)
Anti-NaOH RP-I serum Anti-NaOH P-2 serum Anti-CF P-2 serum Anti-Koganei strain serum Control ~
640 640 2560 640 < l0
0 4 4 4 0
/ / / / /
4 (0.0) 4 (100.0) 4 (100.0) 4 (100.0) 4 (0.0)
INormal mouse serum.
and CF P-4 are shown in Table 2. Protective activity was demonstrated with the CF Am-S antigen fractions containing 1000 pg and 200 pg protein. Complete protection was provided by both the CF P-I antigen (at 500 pg of protein) and the CF P-2 antigen (at 500/tg and 100/tg of protein). Of the preparations subjected to affinity chromatography, the Con A P-W fraction gave 100% protection at both protein contents tested, while Con A P-E fraction did not show any protective activity (Table 3 ). Challenge with a strain o f a different serovar. N a O H RP- 1, NaOH P-2 and CF P-2 antigens were used for challenge with a different serovar (strain Agata; serovar 5 ). NaOH P-2 and CF P-2 antigens were highly protective, but NaOH RP-1 did not show any protective activity (Table 4). Passive i m m u n i z a t i o n test
Antisera obtained from mice immunized with NaOH RP-1, N a O H P-2, CF P-2 and the vaccine strain Koganei were used for passive immunization. All of the mice passively immunized with anti-NaOH P-2 serum, anti-CF P-2 serum and anti-Koganei strain serum were protected against challenge with
80
s. KOBAYASHI ET AL.
MP
1
2
3
4
78K 66K
43K
30K
Fig. 1. Western blot analysis of N a O H P-2 fraction. MP: Marker protein stained with amido black 10B. Lane 1: Anti-NaOH RP-I serum. Lane 2: Anti-NaOH P-2 serum. Lane 3: Anti-CF P-2 serum. Lane 4: Anti-Koganei serum. K = k D a .
strain Fujisawa, but anti-NaOH RP-1 serum did not show any protection effect (Table 5 ) The GA titers of these antisera are also shown in Table 5. The titers ranged from 640 to 2560, whereas normal mouse serum caused no agglutination.
Western blot analysis Western blot analysis was performed with the NaOH P-2 antigen and sonicated cell antigen and the above four antisera. The anti-NaOH RP-1 serum strongly recognized the 65 kDa protein and two proteins over 100 kDa, while the anti-NaOH P-2, and anti-CF P-2 sera recognized proteins over 100, 65, 62, 42-45, 40-42 and 28 kDa in the NaOH P-2 antigen. The antiserum against strain Koganei recognized the same protein bands as did the anti-NaOH P-2 and anti-CF P-2 sera with the exception of proteins over 100 kDa (Fig. 1 ). In the sonicated cell antigens of strain Agata (Fig. 2 ), strain Fujisawa (Fig. 3 ) and strain Koganei (Fig. 4), anti-NaOH RP-1 serum recognized some bands
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
MP
1
2
3
81
4
66K 43K
3OK
Fig. 2. Western blot analysis of sonicated antigen from E. rhusiopathiae strain Agata. See legend for Fig. 1.
of approximately 100 kDa, but anti-NaOH P-2 and anti-CF P-2 sera recognized three protein bands, 62, 42 and 41 kDa. Furthermore, the anitiserum against strains Koganei recognized two protein bands, 32 and 28 kDa. DISCUSSION
Traub ( 1947 ) reported that the culture filtrate ofE. rhusiopathiae could be concentrated with a l u m i n u m hydroxide, and demonstrated that it had a protective effect against E. rhusiopathiae infection in swine and mice. He pointed out that the protective activity required the presence of some soluble antigens in the culture filtrate and bacterial cells. White and Verwey (1970b) showed that the culture filtrate and killed bacterial cells both afforded protection in mice, and also demonstrated cross protection between serovar 1 and 2 strains. In our study, we found that in both the alkaline extract of the cell and the culture filtrate of strain Koganei were productive in mice. White and Verwey
82
S. KOBAYASHI ET AL.
MP
1
2
3
4
~K
Fig. 3. Western blot analysis of sonicated antigen from E. rhusiopathiae strain Fujisawa. See legend for Fig. 1.
(1970a,b) and Rothe (1982b) suggested that the protective activity was induced by glycoproteins or a glycoprotein-lipid complex because the activity in the culture filtrate was decreased by muramidase. In the present study, glycoprotein separated from the most immunogenic of the culture filtrate fraction (CF P-2 ) with Con A-Sepharose 4B did not show protective activity. Sawada et al. (1987a,c) reported that the protective antigen was present in the culture filtrate of the Koganei strain and that there was cross protection between serovars 1a and 2. Protective activity was also found in antiserum to the culture filtrate and cross protection also occured to challenge with serovars la, lb, 2, 5, 6, 8, 11, 12, 15, 16, 18, 21 and type N strains (Sawada and Takahashi, 1987; Sawada et al., 1987b). But, in these experiments, they did not use the fractionated antigen of the culture filtrate or alkaline-extracted cell antigen. In our study, we have tested the immunological potency of NaOH P-2 and CF P-2 antigens, which were obtained by alkaline extraction from the bacterial cell components and from the culture filtrate, respectively. Moreover, anti-
IMMUNOLOGICAL CHARACTERIZATION OF PROTECTIVE ANTIGENS
MP
1
2
3
83
4
66K 43K
3OK
Fig. 4. Western blot analysis of sonicated antigen from E. rhusiopathiae strain Koganei. See legend for Fig. 1.
NaOH P-2 and anti-CF P-2 mice sera, which were obtained by immunization of NaOH P-2 and CF P-2 antigens strongly recognized the three bands of 62, 42 and 41 kDa proteins by NaOH P-2 antigen in western blot analysis. These results suggest that the protective antigen in the culture filtrate and the alkaline-extracted protective antigen are the same, because they are both totally protected in the mouse test and detected as the same bands of 62, 42 and 41 kDa proteins by western blot analysis. These antigens are considered to be closely involved in protective activity. Monoclonal antibodies to these antigens are being sought in order to define the protective antigen.
REFERENCES Dedi6, K., 1949. Die saoureloslichen Antigen von Erysipelothrix rhusiopathiae. Monatsh. Veterinaermed., 4: 7-10. Erler, W., 1973. Serologische, chemische und immunchemische Untersuchungen an Rotlaufbakterien. 13. Mitt. Das immunisierende Antigen. Arch. Exper. Vet. Med., 27:321-326.
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Gledhill, A.W., 1947. Some properties ofa thermolabile antigen ofErysipelothrix rhusiopathiae. J. Gen. Microbiol., 1:211-220. Gledhilk A.W., 1952. The immunizing antigen of Erysipelothrix rhusiopathiae. The role of Lantigen. J. Gen. Microbiol., 7: 179-191. Heuner, F., 1957. Zur Technik serologischer Rotlaufuntersuchungen. Berl. Muench. Tier~irztl. Wochenschr., 70:341-344. Lachmann, P.G. and Deicher, H., 1986. Solubilization and characterization of surface antigenic components ofErysipelothrix rhusiopathiae T28. Infect. Immun., 52:818-822. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Rothe, F., 1982a. Das protektive Antigen des Rotlaufbakteriums (Erysipelothrix rhusiopalhiae) 1. Mitteilung: Spezifischer Nachweis des protektiven Antigens. Arch. Exper. Vet. Med., 36: 243-253. Rothe, F., 1982b. Das protective Antigen des Rotlaufbakteriums (Erysipelothrix rhusiopathiae) 2. Mitteilung: Die weitere Charakteisierung des protektiven Antigen. Arch. Exper. Vet. Med., 36: 255-267. Sawada,T. and Takahashi, T., 1987. Cross-protection of mice and swine inoculated with culture filtrate of Erysipelothrix rhusiopathiae and challenge exposure to strains of various serovars. Am. J. Vet. Res., 48: 239-242. Sawada,T., Takahashi, T. and Tamura, Y., 1987a. Protective effect of sera from swine immunized with different fractions from broth culture of an attenuated strain of Erysipelothrix rhusiopathiae. Jpn. J. Vet. Sci., 49: 37-42. Sawada,T., Takahashi, T. and Tamura, Y., 1987b. Antiserum against culture filtrate is crossprotective for various serovars ofErysipelothrix rhusiopathiae. Vet. Microbiol., 14: 87-93. Sawada,T., Takahashi, T. and Seto, K., 1987c. Immunogenicity of different fractions from broth culture of an attenuated strain ofErysipelothrix rhusiopathiae in mice and swine. Jpn. J. Vet. Sci., 49: 151-154. Seto, K., Ando, K., Nishimura, Y., Fujiki, M. and Matsuno, T., 1965. Studies on the swine erysipelas. Evaluation of growth agglutination and killed cell agglutination. Ann. Rep. Nat. Vet. Assay Lab., 4: 54-62. (in Japanese). Seto, K., Nishimura, Y., Fujiki, M., Azechi, H. and Suzuki, K., 1971. Studies on acriflavin-fast attenuated El:vsipelothrix insidiosa. Comparison on pathogenicity and immunogenicity between mice and pigs. Jpn. J. Vet. Sci., 33:161-171. (in Japanese). Smith, P.K., Krohn, R.L., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., 1985. Measurement of protein using bichinchoninic acid. Anal. Biochem., 150: 76-85. SSrensen, K. and Brodbeck, U., 1986. A sensitive protein assay method using micro-titer plates. Experientia, 42:161-162. Towbin, H., Staebelin, T. and Gorden, J., 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci., USA 76: 4350-4354. Traub, E., 1947. Immunisierung gegen Schweinerotlasuf mit Konzentrierten Adsorbatimpstoffen. Monatsh. Veterinaermed., 10:165-173. Truszczynsky, M., 1961. The antigenic structure of virulent and avirulent strain of Er.vsipelothrix rhusiopathiae. 2. Immunochemic and serologic investigations. Am. J. Vet. Res., 22: 839845. Wellmann, G., 1955. Die subklinische Rotlaufinfektion und Bedeutung ftir die Epidemiologie des Schweinelaufs. Zentralbl. I. Orig., 162: 265-274. White, R.R. and Verwey, W.F., 1970a. Isolation and characterization of a protective antigencontaining particle from culture supernatant fluid ofErysipelothrix rhusiopathiae. Infec. Immun., 1: 380-386.
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White, R.R. and Verwey, W.F., 1970b. Solubilization and characterization of a protective antigen ofErysipelothrix rhusiopathiae. Infect. Immun., 1: 387-394. Wood, R.L., 1984. Swine erysipelas--a review of prevalence and research. J. Am. Vet. Meal. Assoc., 184: 944-949.
