JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1990, p. 2573-2575
Vol. 28, No. 11
0095-1137/90/112573-03$02.00/0 Copyright © 1990, American Society for Microbiology
Modified Feist Broth as a Serum-Free Alternative for Enhanced Production of Protective Antigen of Erysipelothrix rhusiopathiae M. H. GROSCHUP AND J. F. TIMONEY* Department of Microbiology, Immunology and Parasitology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Received 13 April 1990/Accepted 15 August 1990
The production of protective antigen in modified serum-free nutrient broth (H. Feist, K.-D. Flossmann, and W. Erler, Arch. Exp. Veterinaermed. 30:49-57, 1976) and in brain heart infusion broth supplemented with 10% horse serum (BHIS) was evaluated for six strains of Erysipelothrix rhusiopathiae serotypes la, 2, 2b, 4, and N. All six strains grew to higher cell densities in modified Feist medium than in BHIS and produced larger amounts of 64,000- to 66,000- and 39,000- to 40,000-molecular-weight antigens involved in immunity to erysipelas. A vaccine produced in Feist medium from E. rhusiopathiae SE-9 (serotype 2) was highly effective in a mouse protection test. We therefore suggest that modified Feist medium is an excellent, if not superior, alternative to BHIS for production of erysipelas vaccine.
Erysipelothrix rhusiopathiae is a widely distributed, grampositive bacillus that is found as a facultative pathogen and commensal organism of birds, mammals, and fish. It is a cause of economic loss in the raising of swine, lambs, chickens, and turkeys. In swine, E. rhusiopathiae may cause acute septicemic disease or chronic endocarditis and polyarthritis. Commercial vaccines for pigs and turkeys consisting of either killed cell bacterins, lysates, or live attenuated strains of E. rhusiopathiae serotype 2 are widely used, and 80 million doses were sold in the United States in 1980 (11). These vaccines stimulate a high level of protection against all known serotypes. Eighty percent of the strains isolated from swine belong to serotypes la, lb, and 2b, sheep and goats predominantly harbor types lb and 5, and poultry carry serotypes 1, 2, 5, 6, and 9 (3, 12). An important protective antigen has been shown in recent studies in our laboratories to have a molecular weight of 64,000 to 66,000 (M. H. Groschup, K. Cussler, R. Weiss, and J. F. Timoney, unpublished data). This antigen has also been cloned and expressed in Escherichia coli (6). E. rhusiopathiae grows moderately well on solid serumcontaining media such as blood agar and produces either of two dissociative forms: the highly virulent smooth (S) form, with 1-mm smooth colonies, or the rough (R) form, with 2-mm colonies. The rough form is associated with chronic lesions. Unlike many other gram-positive bacteria, E. rhusiopathiae grows poorly in fluid media such as brain heart infusion broth unless the broth is supplemented with 10% serum (BHIS). However, E. rhusiopathiae does not utilize the major serum protein fraction. Instead, depending on the analytical method used, "peptide B," consisting of arginine, histidine, lysine, glycine, glutamine, alanine, and proline (13), and a lipoprotein in the a-globulin fraction (2) are utilized in the serum-supplemented broth. Arginine, glutamine, threonine, and serine are the only amino acids removed from yeast extract medium. Arginine, which is apparently not synthesized by the organism, is consumed in *
large amounts and metabolized to citrulline and ornithine (5, 7). Oleic acid (Tween 80) (2, 9), as well as glucose (0.1%) (5), has been shown to enhance the growth of E. rhusiopathiae. In an attempt to supply nutritional amino acids and vitamins in a serum-free fluid growth medium, Feist et al. (5) combined glucose, yeast extract, peptone, arginine, and oleic acid in 0.1 M sodium phosphate buffer (pH 8.0) and obtained excellent growth of E. rhusiopathiae. In this paper we describe the growth and the expression of surface antigens of E. rhusiopathiae strains of different serotypes in Feist medium and in BHIS. The production of protective antigen was tested in a mouse protection model. E. rhusiopathiae EI-6P (swine origin; serotype la), Me-7 (unknown origin; serotype la), SE-9 (swine origin; serotype 2), T28 (swine origin; serotype 2b), Doggerschabe (fish origin; serotype 4), and Frankfurt XI (swine origin; serotype N) were cultured on blood agar plates (5% sheep blood) from lyophilized stocks and passaged twice in mice (1,000 CFU grown in BHIS and injected intraperitoneally). Smooth colonies (virulent S forms) were grown in Feist medium containing 6 g of glucose per liter, 5 g of proteose peptone no. 2 (Difco, Detroit, Mich.) per liter, 5 g of yeast extract (Difco) per liter, 0.5 g of arginine per liter, and 0.5 ml of oleic acid (Tween 80; Fisher Scientific, Pittsburgh, Pa.) per liter in TABLE 1. Yields of E. rhusiopathiae strains after 20 h of incubation in Feist medium or BHIS Results with: BHIS
Feist medium Strain
No. of CFU/ml
9.6 4.8 7.8 22.4 Doggerschabe 9.3 Frankfurt XI 10.2
EI-6P Me-7 SE-9 T28
Corresponding author. 2573
x 109 x 109 x 109 x 109
x 109 x 109
Dry wt Final of cells pH (mg/ml) 6.2 6.3 4.9 x 109 0.53 5.4 6.3 4.7 x 109 0.40 6.4 2.3 x 109 0.75 6.2 5.8 6.3 1.0 x 109 0.45 6.4 4.6 x 109 0.28 5.6 6.6 5.6 x 109 0.38 6.4
Dry Wt Final of cells pH
(mg/ml) 1.25 0.85 1.45 0.98 0.83 0.78
No. of CFU/ml
2574
NOTES
A 400*
300' c cm, IJ..
co
200
.>
4-
Y
n uu
S~ 10 time (h)
5
0
J. CLIN. MICROBIOL.
15
0
20
5
10 time (h)
15
20
FIG. 1. Growth curves of E. rhusiopathiae T28, El-6P, and Me-7 (A) and Frankfurt XI, Doggerschabe, and SE-9 (B) grown at 37°C in Feist medium and BHIS. Cultures were inoculated with 1% starter culture volumes grown at 37°C for 20 h in the appropriate medium.
0.2 M sodium phosphate buffer (pH 8.0). Preliminary experiments indicated that the use of a buffer at twice the concentration recommended by Feist et al. yielded more viable cells. Other modifications included filtering the medium instead of heating it to 100°C, since it was noticed that even a slight caramelization of the glucose reduced bacterial growth dramatically. Brain heart infusion broth (BBL, Cockeysville, Md.) supplemented with 10% horse serum (BHIS) served as the control for growth in enriched BHIS. Our results showed that the dry weight of E. rhusiopathiae A B C D E F B CD E F A
4
n.w ;
i
~-E97
strains of serotypes la, 2, 2b, 4, and N produced in modified Feist medium was more than twice that generated in BHIS (Table 1). Similarly, the number of viable cells after cultivation in Feist medium (0.5 x 1010 to 2.2 x 1010 bacteria per ml) for 20 h at 37°C was in general twice that in BHIS (1 X 109 to 5 x 109 bacteria per ml). The pH after 20 h of cultures of all strains tested remained well above 6.0 in the strongly buffered Feist medium (Table 1); such a pH is quite compatible with survival of the cells and preservation of the acid-sensitive protective antigens (4). Growth curves of six E. rhusiopathiae strains were prepared with a Klett-Summerson photometer (Klett, New York, N.Y.) equilibrated with the corresponding uninoculated broth at 420 nm. The strains entered the logarithmic phase in Feist medium as early as 2 h after inoculation (1%
*K 67
TABLE 2. Mouse survival after immunization with E. rhusiopathiae SE-9 vaccine and challenge with E. rhusiopathiae Frankfurt Xia
31
Vaccine strain growth medium'
Vaccine dilution
No. of surviving mice (n = 10)`
Feist medium
Undiluted 1:3 1:9 1:27
10 10 10 10
1:81
8
Undiluted 1:3 1:9 1:27 1:81
10 10 9 9 7
Undiluted
0
< 21
FEIST BHI+SERUM FIG. 2. Immunoblot analysis of antigens of E. rhusiopathiae SE-9 (lanes A), EI-6P (lanes B), Me-7 (lanes C), T28 (lanes D), Doggerschabe (lanes E), and Frankfurt Xi (lanes F) expressed in Feist medium and in serum-supplemented brain heart infusion (BHI) broth. Washed pellets of 20-h cultures (1 ml) at 37°C were incubated in Laemmli buffer for 20 min at 56°C before separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide). After transfer to nitrocellulose, the sheet was blocked with 3% gelatin in PBS-Tween, incubated with porcine hyperimmune serum (diluted 1:100 in PBS-Tween), washed three times in PBSTween, and incubated once more with peroxidase-conjugated protein A (diluted 1:1,000 in PBS-Tween). After repeated washings in PBS-Tween and PBS, the immunoblot was developed with 4-chloro1-naphthol.
BHIS
Controls
The mice were challenged intraperitoneally with 1,000 50% lethal doses of strain Frankfurt XI 21 days after immunization. ' The vaccine consisted of Formalin-killed bacterins adsorbed to aluminum hydroxide (20%). The controls were aluminum hydroxide (20%) in phosphatebuffered solution. Mouse mortality was calculated 10 days after intraperitoneal challenge. `
`
VOL. 28, 1990
inoculation volume) and reached maximal optical densities at about 10 h (strain EI-6P). In BHIS, the logarithmic phase was not attained until 4 h and optical densities peaked at 13 h (strain EI-6P) (Fig. 1). In general, the peak optical densities of the E. rhusiopathiae strains were approximately three times higher in Feist medium than in BHIS. However, a decrease of up to 35% in the optical density of strain SE-9 during the 10 h following the attainment of peak density in Feist medium suggests lysis of cells. This lysis may be due to exhaustion of a nutrient necessary for the extended viability of the organism. Alternatively, overproduction of certain proteins or toxic metabolites as a result of the very high rate of cell synthesis may also stress the bacteria to the point of fatal lysis. Interestingly, strain T28 appeared to be less susceptible to lysis than the other strains. The effect of Feist medium on the expression of antigenic proteins on the bacterial surface was investigated by heating washed cells from equal culture volumes of each strain in Laemmli buffer (8) for 20 min at 56°C. Proteins in the extracts were separated on a sodium dodecyl sulfate-10% polyacrylamide gel and transferred to nitrocellulose. Immunoblots (1) were prepared by incubation for 90 min in commercial protective E. rhusiopathiae sera from horses and pigs (WDT, Hoyerhagen, Federal Republic of Germany) diluted 1:100 in phosphate buffer, pH 7.2, with 0.1% Tween 20 (PBS-Tween). After being washed in PBS-Tween, the nitrocellulose sheet was incubated with horseradish peroxidase-conjugated protein A (Bio-rad, Richmond, Calif.) or protein G (Zymed, South San Francisco, Calif.) at a dilution of 1:1,000 in PBS-Tween. The immunoblot was developed in 4-chloro-1-naphthol solution. Reactive bands with approximate molecular weights of 94,000, 72,000, 66,000, 64,000, 50,000, 40,000 to 39,000, 35,000, and less than 35,000 were uniformly observed in extracts of cells of all strains from both Feist medium and BHIS. However, in all strains of serotypes 1, 2, 4, and N, the expression of bacterial antigens of E. rhusiopathiae was much greater in modified Feist medium than in BHIS (Fig. 2). Of special interest on the immunoblots was the large amount of 66,000- to 64,000-molecular-weight antigen that is protective in mice (Groschup et al., unpublished data). Mice (strain NMRI; 16 to 18 g) were immunized subcutaneously with 0.5 ml of Formalin-killed aluminum hydroxide (20%)-adsorbed bacterins of the official U.S. vaccine strain, SE-9, grown in Feist medium or in BHIS and adjusted for equivalent cell densities. The mice, challenged after 21 days by the intraperitoneal inoculation of 1,000 50% lethal doses of strain Frankfurt XI, showed very high levels of protection against E. rhusiopathiae infection (Table 2). In conclusion, we have shown that Feist broth is an excellent alternative for production of E. rhusiopathiae vaccines with excellent expression of protective antigen. The medium is easy to prepare from inexpensive and widely
NOTES
2575
available components. However, although the peptone used in our study was of high quality and supported excellent growth, care must be taken in the choice of this component because some peptones have been known to greatly reduce the growth of E. rhusiopathiae (5). This research was supported in part by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to M.H.G.). We gratefully acknowledge K. Cussler, Paul-Ehrlich-Institut, Federal Agency for Sera and Vaccines, Langen, Federal Republic of Germany, for his assistance in the mouse protection test.
1.
2.
3.
4. 5.
6. 7.
8. 9. 10.
11. 12.
13.
LITERATURE CITED Burnette, W. N. 1982. "Western-blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gel to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:192-203. Durmer, K., H. Guttner, R. Kamieth, P. Schmidt, H. Winkler, and K. H. Kludas. 1972. Supplements as components of nutrient media for bacteria and cells. Arch. Exp. Veterinaermed. 26:2531. Eames, G. J., M. J. Turner, and R. E. Catt. 1988. Serotypes of Erysipelothrix rhusiopathiae in Australian pigs, small ruminants, poultry and captive wild birds and animals. Aust. Vet. J. 65:249-252. Erler, W. 1973. Serological, chemical and immunochemical investigations with erysipelas bacteria. XIII. The immunizing antigen. Arch. Exp. Veterinaermed. 27:321-326. Feist, H., K.-D. Flossmann, and W. Erler. 1976. Investigations on the nutritional requirements of erysipelas bacteria. Arch. Exp. Veterinaermed. 30:49-57. Galan, J. E., and J. F. Timoney. 1990. Cloning and expression in Escherichia coli of a protective antigen of Erysipelothrix rhusiopathiae. Infect. Immun. 58:3116-3121. Kludas, K. H., and M. Meese. 1968. The significance of various supplements in nutrient media for the appearance of immunizing antigen of Erysipelothrix rhusiopathiae. 1. Characterization of effective serum components. Folia Microbiol. 13:515-517. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. O'Leary, W. M. 1962. The fatty acids of bacteria. Bacteriol. Rev. 26:421-447. White, R. R., and W. F. Verwey. 1970. Isolation and characterization of a protective antigen-containing particle from culture supernatant fluids of Erysipelothrix rhusiopathiae. Infect. Immun. 1:380-386. Wood, R. L. 1984. Swine erysipelas-a review of prevalence and research. J. Am. Vet. Med. Assoc. 184:944-948. Wood, R. L., and R. Harrington. 1978. Serotypes of Erysipelothrix rhusiopathiae isolated from swine and from soil and manure of swine pens in the United States. Am. J. Vet. Res. 39:1833-1840. Zimmermann, G., and K. H. Kludas. 1956. A growth stimulating peptide for Erysipelothrix rhusiopathiae. Arch. Exp. Veterinaermed. 10:237-245.
Vol. 28, No. 11
0095-1137/90/112573-03$02.00/0 Copyright © 1990, American Society for Microbiology
Modified Feist Broth as a Serum-Free Alternative for Enhanced Production of Protective Antigen of Erysipelothrix rhusiopathiae M. H. GROSCHUP AND J. F. TIMONEY* Department of Microbiology, Immunology and Parasitology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Received 13 April 1990/Accepted 15 August 1990
The production of protective antigen in modified serum-free nutrient broth (H. Feist, K.-D. Flossmann, and W. Erler, Arch. Exp. Veterinaermed. 30:49-57, 1976) and in brain heart infusion broth supplemented with 10% horse serum (BHIS) was evaluated for six strains of Erysipelothrix rhusiopathiae serotypes la, 2, 2b, 4, and N. All six strains grew to higher cell densities in modified Feist medium than in BHIS and produced larger amounts of 64,000- to 66,000- and 39,000- to 40,000-molecular-weight antigens involved in immunity to erysipelas. A vaccine produced in Feist medium from E. rhusiopathiae SE-9 (serotype 2) was highly effective in a mouse protection test. We therefore suggest that modified Feist medium is an excellent, if not superior, alternative to BHIS for production of erysipelas vaccine.
Erysipelothrix rhusiopathiae is a widely distributed, grampositive bacillus that is found as a facultative pathogen and commensal organism of birds, mammals, and fish. It is a cause of economic loss in the raising of swine, lambs, chickens, and turkeys. In swine, E. rhusiopathiae may cause acute septicemic disease or chronic endocarditis and polyarthritis. Commercial vaccines for pigs and turkeys consisting of either killed cell bacterins, lysates, or live attenuated strains of E. rhusiopathiae serotype 2 are widely used, and 80 million doses were sold in the United States in 1980 (11). These vaccines stimulate a high level of protection against all known serotypes. Eighty percent of the strains isolated from swine belong to serotypes la, lb, and 2b, sheep and goats predominantly harbor types lb and 5, and poultry carry serotypes 1, 2, 5, 6, and 9 (3, 12). An important protective antigen has been shown in recent studies in our laboratories to have a molecular weight of 64,000 to 66,000 (M. H. Groschup, K. Cussler, R. Weiss, and J. F. Timoney, unpublished data). This antigen has also been cloned and expressed in Escherichia coli (6). E. rhusiopathiae grows moderately well on solid serumcontaining media such as blood agar and produces either of two dissociative forms: the highly virulent smooth (S) form, with 1-mm smooth colonies, or the rough (R) form, with 2-mm colonies. The rough form is associated with chronic lesions. Unlike many other gram-positive bacteria, E. rhusiopathiae grows poorly in fluid media such as brain heart infusion broth unless the broth is supplemented with 10% serum (BHIS). However, E. rhusiopathiae does not utilize the major serum protein fraction. Instead, depending on the analytical method used, "peptide B," consisting of arginine, histidine, lysine, glycine, glutamine, alanine, and proline (13), and a lipoprotein in the a-globulin fraction (2) are utilized in the serum-supplemented broth. Arginine, glutamine, threonine, and serine are the only amino acids removed from yeast extract medium. Arginine, which is apparently not synthesized by the organism, is consumed in *
large amounts and metabolized to citrulline and ornithine (5, 7). Oleic acid (Tween 80) (2, 9), as well as glucose (0.1%) (5), has been shown to enhance the growth of E. rhusiopathiae. In an attempt to supply nutritional amino acids and vitamins in a serum-free fluid growth medium, Feist et al. (5) combined glucose, yeast extract, peptone, arginine, and oleic acid in 0.1 M sodium phosphate buffer (pH 8.0) and obtained excellent growth of E. rhusiopathiae. In this paper we describe the growth and the expression of surface antigens of E. rhusiopathiae strains of different serotypes in Feist medium and in BHIS. The production of protective antigen was tested in a mouse protection model. E. rhusiopathiae EI-6P (swine origin; serotype la), Me-7 (unknown origin; serotype la), SE-9 (swine origin; serotype 2), T28 (swine origin; serotype 2b), Doggerschabe (fish origin; serotype 4), and Frankfurt XI (swine origin; serotype N) were cultured on blood agar plates (5% sheep blood) from lyophilized stocks and passaged twice in mice (1,000 CFU grown in BHIS and injected intraperitoneally). Smooth colonies (virulent S forms) were grown in Feist medium containing 6 g of glucose per liter, 5 g of proteose peptone no. 2 (Difco, Detroit, Mich.) per liter, 5 g of yeast extract (Difco) per liter, 0.5 g of arginine per liter, and 0.5 ml of oleic acid (Tween 80; Fisher Scientific, Pittsburgh, Pa.) per liter in TABLE 1. Yields of E. rhusiopathiae strains after 20 h of incubation in Feist medium or BHIS Results with: BHIS
Feist medium Strain
No. of CFU/ml
9.6 4.8 7.8 22.4 Doggerschabe 9.3 Frankfurt XI 10.2
EI-6P Me-7 SE-9 T28
Corresponding author. 2573
x 109 x 109 x 109 x 109
x 109 x 109
Dry wt Final of cells pH (mg/ml) 6.2 6.3 4.9 x 109 0.53 5.4 6.3 4.7 x 109 0.40 6.4 2.3 x 109 0.75 6.2 5.8 6.3 1.0 x 109 0.45 6.4 4.6 x 109 0.28 5.6 6.6 5.6 x 109 0.38 6.4
Dry Wt Final of cells pH
(mg/ml) 1.25 0.85 1.45 0.98 0.83 0.78
No. of CFU/ml
2574
NOTES
A 400*
300' c cm, IJ..
co
200
.>
4-
Y
n uu
S~ 10 time (h)
5
0
J. CLIN. MICROBIOL.
15
0
20
5
10 time (h)
15
20
FIG. 1. Growth curves of E. rhusiopathiae T28, El-6P, and Me-7 (A) and Frankfurt XI, Doggerschabe, and SE-9 (B) grown at 37°C in Feist medium and BHIS. Cultures were inoculated with 1% starter culture volumes grown at 37°C for 20 h in the appropriate medium.
0.2 M sodium phosphate buffer (pH 8.0). Preliminary experiments indicated that the use of a buffer at twice the concentration recommended by Feist et al. yielded more viable cells. Other modifications included filtering the medium instead of heating it to 100°C, since it was noticed that even a slight caramelization of the glucose reduced bacterial growth dramatically. Brain heart infusion broth (BBL, Cockeysville, Md.) supplemented with 10% horse serum (BHIS) served as the control for growth in enriched BHIS. Our results showed that the dry weight of E. rhusiopathiae A B C D E F B CD E F A
4
n.w ;
i
~-E97
strains of serotypes la, 2, 2b, 4, and N produced in modified Feist medium was more than twice that generated in BHIS (Table 1). Similarly, the number of viable cells after cultivation in Feist medium (0.5 x 1010 to 2.2 x 1010 bacteria per ml) for 20 h at 37°C was in general twice that in BHIS (1 X 109 to 5 x 109 bacteria per ml). The pH after 20 h of cultures of all strains tested remained well above 6.0 in the strongly buffered Feist medium (Table 1); such a pH is quite compatible with survival of the cells and preservation of the acid-sensitive protective antigens (4). Growth curves of six E. rhusiopathiae strains were prepared with a Klett-Summerson photometer (Klett, New York, N.Y.) equilibrated with the corresponding uninoculated broth at 420 nm. The strains entered the logarithmic phase in Feist medium as early as 2 h after inoculation (1%
*K 67
TABLE 2. Mouse survival after immunization with E. rhusiopathiae SE-9 vaccine and challenge with E. rhusiopathiae Frankfurt Xia
31
Vaccine strain growth medium'
Vaccine dilution
No. of surviving mice (n = 10)`
Feist medium
Undiluted 1:3 1:9 1:27
10 10 10 10
1:81
8
Undiluted 1:3 1:9 1:27 1:81
10 10 9 9 7
Undiluted
0
< 21
FEIST BHI+SERUM FIG. 2. Immunoblot analysis of antigens of E. rhusiopathiae SE-9 (lanes A), EI-6P (lanes B), Me-7 (lanes C), T28 (lanes D), Doggerschabe (lanes E), and Frankfurt Xi (lanes F) expressed in Feist medium and in serum-supplemented brain heart infusion (BHI) broth. Washed pellets of 20-h cultures (1 ml) at 37°C were incubated in Laemmli buffer for 20 min at 56°C before separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide). After transfer to nitrocellulose, the sheet was blocked with 3% gelatin in PBS-Tween, incubated with porcine hyperimmune serum (diluted 1:100 in PBS-Tween), washed three times in PBSTween, and incubated once more with peroxidase-conjugated protein A (diluted 1:1,000 in PBS-Tween). After repeated washings in PBS-Tween and PBS, the immunoblot was developed with 4-chloro1-naphthol.
BHIS
Controls
The mice were challenged intraperitoneally with 1,000 50% lethal doses of strain Frankfurt XI 21 days after immunization. ' The vaccine consisted of Formalin-killed bacterins adsorbed to aluminum hydroxide (20%). The controls were aluminum hydroxide (20%) in phosphatebuffered solution. Mouse mortality was calculated 10 days after intraperitoneal challenge. `
`
VOL. 28, 1990
inoculation volume) and reached maximal optical densities at about 10 h (strain EI-6P). In BHIS, the logarithmic phase was not attained until 4 h and optical densities peaked at 13 h (strain EI-6P) (Fig. 1). In general, the peak optical densities of the E. rhusiopathiae strains were approximately three times higher in Feist medium than in BHIS. However, a decrease of up to 35% in the optical density of strain SE-9 during the 10 h following the attainment of peak density in Feist medium suggests lysis of cells. This lysis may be due to exhaustion of a nutrient necessary for the extended viability of the organism. Alternatively, overproduction of certain proteins or toxic metabolites as a result of the very high rate of cell synthesis may also stress the bacteria to the point of fatal lysis. Interestingly, strain T28 appeared to be less susceptible to lysis than the other strains. The effect of Feist medium on the expression of antigenic proteins on the bacterial surface was investigated by heating washed cells from equal culture volumes of each strain in Laemmli buffer (8) for 20 min at 56°C. Proteins in the extracts were separated on a sodium dodecyl sulfate-10% polyacrylamide gel and transferred to nitrocellulose. Immunoblots (1) were prepared by incubation for 90 min in commercial protective E. rhusiopathiae sera from horses and pigs (WDT, Hoyerhagen, Federal Republic of Germany) diluted 1:100 in phosphate buffer, pH 7.2, with 0.1% Tween 20 (PBS-Tween). After being washed in PBS-Tween, the nitrocellulose sheet was incubated with horseradish peroxidase-conjugated protein A (Bio-rad, Richmond, Calif.) or protein G (Zymed, South San Francisco, Calif.) at a dilution of 1:1,000 in PBS-Tween. The immunoblot was developed in 4-chloro-1-naphthol solution. Reactive bands with approximate molecular weights of 94,000, 72,000, 66,000, 64,000, 50,000, 40,000 to 39,000, 35,000, and less than 35,000 were uniformly observed in extracts of cells of all strains from both Feist medium and BHIS. However, in all strains of serotypes 1, 2, 4, and N, the expression of bacterial antigens of E. rhusiopathiae was much greater in modified Feist medium than in BHIS (Fig. 2). Of special interest on the immunoblots was the large amount of 66,000- to 64,000-molecular-weight antigen that is protective in mice (Groschup et al., unpublished data). Mice (strain NMRI; 16 to 18 g) were immunized subcutaneously with 0.5 ml of Formalin-killed aluminum hydroxide (20%)-adsorbed bacterins of the official U.S. vaccine strain, SE-9, grown in Feist medium or in BHIS and adjusted for equivalent cell densities. The mice, challenged after 21 days by the intraperitoneal inoculation of 1,000 50% lethal doses of strain Frankfurt XI, showed very high levels of protection against E. rhusiopathiae infection (Table 2). In conclusion, we have shown that Feist broth is an excellent alternative for production of E. rhusiopathiae vaccines with excellent expression of protective antigen. The medium is easy to prepare from inexpensive and widely
NOTES
2575
available components. However, although the peptone used in our study was of high quality and supported excellent growth, care must be taken in the choice of this component because some peptones have been known to greatly reduce the growth of E. rhusiopathiae (5). This research was supported in part by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to M.H.G.). We gratefully acknowledge K. Cussler, Paul-Ehrlich-Institut, Federal Agency for Sera and Vaccines, Langen, Federal Republic of Germany, for his assistance in the mouse protection test.
1.
2.
3.
4. 5.
6. 7.
8. 9. 10.
11. 12.
13.
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