Vol. 16, No. 2
INFECTON AND IMMUNITY, May 1977, p. 547-549 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Serological Cross-Reactivity Between Group B Streptococcus and Sporothrix schenckii, Ceratocystis Species, and Graphium Species YOICHI NAKAMURA,* HIROSHI ISHIZAKI, AND ROBERT W. WHEAT Department of Dermatology, Kanazawa University School of Medicine, Kanazawa 920, Japan,* and Department of Microbiology and Immunology, Duke University, Durham, North Carolina 27710
Received for publication 5 January 1977
Serological cross-reactivity of a group B Streptococcus (H36B) with Sporothrix schenckii and 39 different Ceratocystis and Graphium species was investigated by double immunodiffusion. Rabbit anti-H36B serum reacted with antigens from S. schenckii and from 36 of 39 Ceratocystis and Graphium species. It is speculated that low-titer agglutinins to S. schenckii in normal sera are due to antibodies raised against various bacteria which share common antigens with S. schenckii. Neil et al. (6) reported serological cross-reactions between Sporothrix schenckii and types 10, 22, 23, 31, and 32 of Streptococcus (Diplococcus) pneumoniae in precipitin and agglutinin tests. They also commented that a number of other bacteria, particularly certain strains of Leuconostoc mesenteroides and a number of streptococci of human origin, cross-react with S. schenckii. In this paper, the serological cross-reactivity of group B Streptococcus with S. schenckii, Ceratocystis species, and Graphium species is reported. MATERIALS AND METHODS Preparation of antigens. The group B Streptococcus S. agalactiae H36B (ATCC 12401, J. H. Brown strain Baby B), S. schenckii (ATCC 10268, from lymphangitis of arm), and 39 species of the genera Ceratocystis and Graphium (Table 1) were grown on a dialysate medium (prepared by dialyzing 0.5% peptone and 0.5% yeast extract at 60% for 6 h) to which 2% glucose was added before autoclaving. After inoculation, the group B Streptococcus (H36B) was incubated for 4 days at 35°C with constant shaking, whereas S. schenckii and the Ceratocystis and Graphium species were incubated for 1 week at room temperature. Formalin was added to a final concentration of 1%, and the cultures were held overnight at room temperature. Culture filtrate was obtained by centrifugation. It was dialyzed against running tap water for 5 days and then concentrated to 0.05 volume by pervaporation. To the concentrated material, 5 volumes of 99% ethanol were added, and the precipitate obtained by centrifugation was then dried with acetone. The acetone-dried precipitates were dissolved in physiological saline (5 mg/ml for the H36B antigen, and 1 mg/ml each for S. schenckii, Ceratocystis, and Graphium antigens) as double-immunodiffusion test antigen. The production of
precipitin lines between H36B antiserum and antigen wells was recorded as positive (Table 1). Preparation of antisera. S. schenckii and Streptococcus H36B cells separated from culture filtrates were washed with distilled water three times and then dried with acetone. These organisms were suspended in physiological saline (1 mg/ml), and 2-ml amounts were injected into white rabbits intravenously twice a week for 2 weeks. The rabbits were bled 1 week after the final injection, and the antisera were stored at -20°C until used.
RESULTS The group B Streptococcus H36B antiserum produced at least two broad precipitin lines against the H36B antigen and two or three against S. schenckii and 36 of the 39 Ceratocystis and Graphium species (Fig. 1A and 1B). It seemed that the fusion of all common antigenantibody reactions was not always complete on double diffusion. Several of the figures showed spurring, and some showed lines of complete nonidentity. The results are summarized in Table 1. S. schenckii antiserum yielded at least two precipitin lines against the S. schenckii antigen and each of 36 Ceratocystis and Graphium species, but no precipitin lines with the H36B antigen. The H36B antiserum, absorbed with S. schenckii organisms, gave precipitin lines only against H36B antigen (Fig. 10). The S. schenckii antiserum, absorbed with H36B organisms, also gave precipitin lines only against S. schenckii antigen.
DISCUSSION In active sporotrichosis, sporotrichin-reactive antibody rises to a high titer and diminishes as the infection resolves (7). The course of the 547
548
NAKAMURA, ISHIZAKI, AND WHEAT
INFECT. IMMUN.
TABLE 1. Cross-reactivity of S. schenckii, Ceratocystis, and Graphium antigens with group B Streptococcus (H36B) antiserum Serological Organism Sourcea Strain no. cross-reaction Ceratocystis ambrosia Columbia 1099-45 + C. brunneociliata CBS 149-54 C. californica Colorado 633 + C. cana CBS 133-51 + C. catonianum CBS 263-35 + C. capillifera CBS 134-51 + C. europhioides Colorado 723-C + C. fagi CBS 236-32 + C. galeiformis CBS 156-54 + C. gossypina Colorado 600-A + C. gossypina var. robusta Colorado 609-D + C. ips Colorado 445-A + C. minor Colorado 811 + C. minuta Columbia + 1099-50 C. minuta-bicolor Colorado 247-A + C. multiannulata Colorado 827 + C. narcissi CBS + 774-73 C. obscura CBS 125-39 + C. olivaceapini Colorado + 548-A C. penicillata CBS 443-69 + C. pilifera Colorado 501-A + C. pluriannulata Columbia + 1099-39 C. radicicola CBS 146-59 C. retusi Colorado 888 + C. sagmatospora Colorado 947-A + C. serpens CBS + 141-36 C. seticollis Colorado + 435-A C. stenoceras Colorado 922 + C. tetropii CBS 140-51 + C. tremuroaurea Colorado + 565-A C. ulmi Duke + Graphium album CBS + 276-54 G. aureum + Colorado 471-B G. cartwrightii CBS 123-41 G. erubescens CBS + 278-54 G. fragrans CBS + 279-54 G. rigidum + CBS 209-34 G. robustum + Colorado 137-B G. rubrum + CBS 210-34 Sporothrix schenckii + ATCC 10268 a Columbia = Columbia University; CBS = Centraalbureau voor Schimmel-cultures, Baarn, The Netherlands; Colorado = Colorado State University; Duke = Duke University; ATCC = American Type Culture Collection.
antibody titer is of clinical significance in determining whether the disease is in an active or inactive stage. According to Nishikawa et al. (7), an agglutinin titer above 1:32 is helpful for the diagnosis of sporotrichosis, but it is interesting to note that a low agglutinin titer is demonstrable in all normal sera. Karlin and Nielsen (3) did not consider a titer of 1:40 suggestive of active sporotrichosis, provided that other evidence of infection is not present. B.M. Ashbrook (M.S. thesis, Duke Univ., Durham, N.C., 1964) found
that sera from apparently normal individuals had agglutinins to S. schenckii in low dilutions, and subclinical or previous infection was suggested. This suggestion, however, seems to be an unlikely explanation since sensitization or subclinical infection with S. schenckii (or other related fungi such as a Ceratocystis species) would be expected to result in a positive skin (sporotrichin) test, which is rarely seen in normal individuals. The polysaccharides from S. schenckii, C. stenoceras, C. ulmi, C. minor, and C. pilifera
VOL. 16, 1977
CROSS-REACTIVITY OF GROUP B STREPTOCOCCUS
549
ture was reported to be the immunodominant structure in yeast-form S. schenckii grown at 37°C and in several Ceratocystis species grown at 25°C (5). On the other hand, nonreducing end groups of L-rhamnose are major immunological determinants of the capsular polysaccharide of the type XXIII pneumococcus as well as of group B and G Streptococcus antigens and Aerobacter aerogenes (Klebsiella) 8172 (2). It is possible, therefore, that S. schenckii and closely related Ceratocystis and Graphium species may cross-react serologically with the group G Streptococcus and with A. aerogenes 8172. These bacteria are found as part of the normal and pathogenic human flora (1, 2, 4, 8), and the presence of antibodies to these bacteria in normal human sera would therefore be expected, as indicated by Baker and Kasper (1). In conclusion, we speculate that the agglutinins of low titer to S. schenckii found in normal sera (normal, healthy individuals with no history of sporotrichosis and a negative sporotrichin skin test) are probably due to presence of antibodies produced in response to various bacteria which share common antigens or determinant groups with the S. schenckii, Ceratocystis, and Graphium groups of fungi. LITERATURE CITED 1. Baker, C. J., and D. L. Kasper. 1976. Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294:753-756. 2. Heidelberger, M., J. M. Davie, and R. M. Krause. 1967. Cross-reactions of the group-specific polysaccharides of streptococcal group B and G in anti-pneumococcal sera with reference to type XXIII and its determinants. J. Immunol. 99:794-796. 3. Karlin, J. V., and H. S. Nielsen, Jr. 1970. Serologic aspects of sporotrichosis. J. Infect. Dis. 121:316-322. 4. Lancefield, R. C., and R. Hare. 1935. The serological
FIG. 1. Immunodiffusion of group B Streptococcus H36B, S. schenckii, and Ceratocystis antigens. Antiserum to group B Streptococcus H36B is in the center wells. Antigens are in the outer wells as follows. (A) 1 and 4, H36B; 2, C. ips; 3, C. serpens; 5, C. microspora; 6, C. seticollis. (B) 1 and 4, H36B; 2, C. penicillata; 3, S. schenckii; 5, C. minuta; 6, C. tetropii. (C) 1, H36B; 2, S. schenckii; 3, C. ulmi; 4, C. ips; 5, C. minor; 6, C. stenoceras. The H36B antiserum in C is absorbed with S. schenckii organisms.
are composed of a-irhamnopyranosyl (1-..3) and a-L-rhamnopyranosyl (1--2)-L-rhamnopyranosyl side-chain units. The a-irhamnosyl or a-L-rhamnopyranosyl (1-.3)-)-manno-struc-
differentiation of pathogenic and non-pathogenic strains of hemolytic streptococci from parturient women. J. Exp. Med. 61:335-349. 5. Lloyd, K. O., and L. R. Travaos. 1975. Immunochemical studies on L-rhamno-D-mannans of Sporothrix schenckii and related fungi by use of rabbit and human antisera. Carbohydr. Res. 40:89-97. 6. Neil, J. M., C. G. Castillo, and A. H. Pinkes. 1955. Serological relationships between fungi and bacteria.
1. Cross-reactions of Sporotrichum schenckii with
pneumococci. J. Immunol. 74:120-125. 7. Nishikawa, T., T. Harada, T. Shiobara, H. Hatano, and S. Harada. 1975. Serologic aspects of cutaneous sporotrichosis. Jpn. J. Dermatol. 85:467-471. 8. Wilkinson, H. W., R. R. Facklam, and E. C. Wortham. 1973. Distribution by serological type of group B streptococci isolated from a variety of clinical material over a five-year period (with special reference to neonatal sepsis and meningitis). Infect. Immun. 8: 228-235.
INFECTON AND IMMUNITY, May 1977, p. 547-549 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Serological Cross-Reactivity Between Group B Streptococcus and Sporothrix schenckii, Ceratocystis Species, and Graphium Species YOICHI NAKAMURA,* HIROSHI ISHIZAKI, AND ROBERT W. WHEAT Department of Dermatology, Kanazawa University School of Medicine, Kanazawa 920, Japan,* and Department of Microbiology and Immunology, Duke University, Durham, North Carolina 27710
Received for publication 5 January 1977
Serological cross-reactivity of a group B Streptococcus (H36B) with Sporothrix schenckii and 39 different Ceratocystis and Graphium species was investigated by double immunodiffusion. Rabbit anti-H36B serum reacted with antigens from S. schenckii and from 36 of 39 Ceratocystis and Graphium species. It is speculated that low-titer agglutinins to S. schenckii in normal sera are due to antibodies raised against various bacteria which share common antigens with S. schenckii. Neil et al. (6) reported serological cross-reactions between Sporothrix schenckii and types 10, 22, 23, 31, and 32 of Streptococcus (Diplococcus) pneumoniae in precipitin and agglutinin tests. They also commented that a number of other bacteria, particularly certain strains of Leuconostoc mesenteroides and a number of streptococci of human origin, cross-react with S. schenckii. In this paper, the serological cross-reactivity of group B Streptococcus with S. schenckii, Ceratocystis species, and Graphium species is reported. MATERIALS AND METHODS Preparation of antigens. The group B Streptococcus S. agalactiae H36B (ATCC 12401, J. H. Brown strain Baby B), S. schenckii (ATCC 10268, from lymphangitis of arm), and 39 species of the genera Ceratocystis and Graphium (Table 1) were grown on a dialysate medium (prepared by dialyzing 0.5% peptone and 0.5% yeast extract at 60% for 6 h) to which 2% glucose was added before autoclaving. After inoculation, the group B Streptococcus (H36B) was incubated for 4 days at 35°C with constant shaking, whereas S. schenckii and the Ceratocystis and Graphium species were incubated for 1 week at room temperature. Formalin was added to a final concentration of 1%, and the cultures were held overnight at room temperature. Culture filtrate was obtained by centrifugation. It was dialyzed against running tap water for 5 days and then concentrated to 0.05 volume by pervaporation. To the concentrated material, 5 volumes of 99% ethanol were added, and the precipitate obtained by centrifugation was then dried with acetone. The acetone-dried precipitates were dissolved in physiological saline (5 mg/ml for the H36B antigen, and 1 mg/ml each for S. schenckii, Ceratocystis, and Graphium antigens) as double-immunodiffusion test antigen. The production of
precipitin lines between H36B antiserum and antigen wells was recorded as positive (Table 1). Preparation of antisera. S. schenckii and Streptococcus H36B cells separated from culture filtrates were washed with distilled water three times and then dried with acetone. These organisms were suspended in physiological saline (1 mg/ml), and 2-ml amounts were injected into white rabbits intravenously twice a week for 2 weeks. The rabbits were bled 1 week after the final injection, and the antisera were stored at -20°C until used.
RESULTS The group B Streptococcus H36B antiserum produced at least two broad precipitin lines against the H36B antigen and two or three against S. schenckii and 36 of the 39 Ceratocystis and Graphium species (Fig. 1A and 1B). It seemed that the fusion of all common antigenantibody reactions was not always complete on double diffusion. Several of the figures showed spurring, and some showed lines of complete nonidentity. The results are summarized in Table 1. S. schenckii antiserum yielded at least two precipitin lines against the S. schenckii antigen and each of 36 Ceratocystis and Graphium species, but no precipitin lines with the H36B antigen. The H36B antiserum, absorbed with S. schenckii organisms, gave precipitin lines only against H36B antigen (Fig. 10). The S. schenckii antiserum, absorbed with H36B organisms, also gave precipitin lines only against S. schenckii antigen.
DISCUSSION In active sporotrichosis, sporotrichin-reactive antibody rises to a high titer and diminishes as the infection resolves (7). The course of the 547
548
NAKAMURA, ISHIZAKI, AND WHEAT
INFECT. IMMUN.
TABLE 1. Cross-reactivity of S. schenckii, Ceratocystis, and Graphium antigens with group B Streptococcus (H36B) antiserum Serological Organism Sourcea Strain no. cross-reaction Ceratocystis ambrosia Columbia 1099-45 + C. brunneociliata CBS 149-54 C. californica Colorado 633 + C. cana CBS 133-51 + C. catonianum CBS 263-35 + C. capillifera CBS 134-51 + C. europhioides Colorado 723-C + C. fagi CBS 236-32 + C. galeiformis CBS 156-54 + C. gossypina Colorado 600-A + C. gossypina var. robusta Colorado 609-D + C. ips Colorado 445-A + C. minor Colorado 811 + C. minuta Columbia + 1099-50 C. minuta-bicolor Colorado 247-A + C. multiannulata Colorado 827 + C. narcissi CBS + 774-73 C. obscura CBS 125-39 + C. olivaceapini Colorado + 548-A C. penicillata CBS 443-69 + C. pilifera Colorado 501-A + C. pluriannulata Columbia + 1099-39 C. radicicola CBS 146-59 C. retusi Colorado 888 + C. sagmatospora Colorado 947-A + C. serpens CBS + 141-36 C. seticollis Colorado + 435-A C. stenoceras Colorado 922 + C. tetropii CBS 140-51 + C. tremuroaurea Colorado + 565-A C. ulmi Duke + Graphium album CBS + 276-54 G. aureum + Colorado 471-B G. cartwrightii CBS 123-41 G. erubescens CBS + 278-54 G. fragrans CBS + 279-54 G. rigidum + CBS 209-34 G. robustum + Colorado 137-B G. rubrum + CBS 210-34 Sporothrix schenckii + ATCC 10268 a Columbia = Columbia University; CBS = Centraalbureau voor Schimmel-cultures, Baarn, The Netherlands; Colorado = Colorado State University; Duke = Duke University; ATCC = American Type Culture Collection.
antibody titer is of clinical significance in determining whether the disease is in an active or inactive stage. According to Nishikawa et al. (7), an agglutinin titer above 1:32 is helpful for the diagnosis of sporotrichosis, but it is interesting to note that a low agglutinin titer is demonstrable in all normal sera. Karlin and Nielsen (3) did not consider a titer of 1:40 suggestive of active sporotrichosis, provided that other evidence of infection is not present. B.M. Ashbrook (M.S. thesis, Duke Univ., Durham, N.C., 1964) found
that sera from apparently normal individuals had agglutinins to S. schenckii in low dilutions, and subclinical or previous infection was suggested. This suggestion, however, seems to be an unlikely explanation since sensitization or subclinical infection with S. schenckii (or other related fungi such as a Ceratocystis species) would be expected to result in a positive skin (sporotrichin) test, which is rarely seen in normal individuals. The polysaccharides from S. schenckii, C. stenoceras, C. ulmi, C. minor, and C. pilifera
VOL. 16, 1977
CROSS-REACTIVITY OF GROUP B STREPTOCOCCUS
549
ture was reported to be the immunodominant structure in yeast-form S. schenckii grown at 37°C and in several Ceratocystis species grown at 25°C (5). On the other hand, nonreducing end groups of L-rhamnose are major immunological determinants of the capsular polysaccharide of the type XXIII pneumococcus as well as of group B and G Streptococcus antigens and Aerobacter aerogenes (Klebsiella) 8172 (2). It is possible, therefore, that S. schenckii and closely related Ceratocystis and Graphium species may cross-react serologically with the group G Streptococcus and with A. aerogenes 8172. These bacteria are found as part of the normal and pathogenic human flora (1, 2, 4, 8), and the presence of antibodies to these bacteria in normal human sera would therefore be expected, as indicated by Baker and Kasper (1). In conclusion, we speculate that the agglutinins of low titer to S. schenckii found in normal sera (normal, healthy individuals with no history of sporotrichosis and a negative sporotrichin skin test) are probably due to presence of antibodies produced in response to various bacteria which share common antigens or determinant groups with the S. schenckii, Ceratocystis, and Graphium groups of fungi. LITERATURE CITED 1. Baker, C. J., and D. L. Kasper. 1976. Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294:753-756. 2. Heidelberger, M., J. M. Davie, and R. M. Krause. 1967. Cross-reactions of the group-specific polysaccharides of streptococcal group B and G in anti-pneumococcal sera with reference to type XXIII and its determinants. J. Immunol. 99:794-796. 3. Karlin, J. V., and H. S. Nielsen, Jr. 1970. Serologic aspects of sporotrichosis. J. Infect. Dis. 121:316-322. 4. Lancefield, R. C., and R. Hare. 1935. The serological
FIG. 1. Immunodiffusion of group B Streptococcus H36B, S. schenckii, and Ceratocystis antigens. Antiserum to group B Streptococcus H36B is in the center wells. Antigens are in the outer wells as follows. (A) 1 and 4, H36B; 2, C. ips; 3, C. serpens; 5, C. microspora; 6, C. seticollis. (B) 1 and 4, H36B; 2, C. penicillata; 3, S. schenckii; 5, C. minuta; 6, C. tetropii. (C) 1, H36B; 2, S. schenckii; 3, C. ulmi; 4, C. ips; 5, C. minor; 6, C. stenoceras. The H36B antiserum in C is absorbed with S. schenckii organisms.
are composed of a-irhamnopyranosyl (1-..3) and a-L-rhamnopyranosyl (1--2)-L-rhamnopyranosyl side-chain units. The a-irhamnosyl or a-L-rhamnopyranosyl (1-.3)-)-manno-struc-
differentiation of pathogenic and non-pathogenic strains of hemolytic streptococci from parturient women. J. Exp. Med. 61:335-349. 5. Lloyd, K. O., and L. R. Travaos. 1975. Immunochemical studies on L-rhamno-D-mannans of Sporothrix schenckii and related fungi by use of rabbit and human antisera. Carbohydr. Res. 40:89-97. 6. Neil, J. M., C. G. Castillo, and A. H. Pinkes. 1955. Serological relationships between fungi and bacteria.
1. Cross-reactions of Sporotrichum schenckii with
pneumococci. J. Immunol. 74:120-125. 7. Nishikawa, T., T. Harada, T. Shiobara, H. Hatano, and S. Harada. 1975. Serologic aspects of cutaneous sporotrichosis. Jpn. J. Dermatol. 85:467-471. 8. Wilkinson, H. W., R. R. Facklam, and E. C. Wortham. 1973. Distribution by serological type of group B streptococci isolated from a variety of clinical material over a five-year period (with special reference to neonatal sepsis and meningitis). Infect. Immun. 8: 228-235.
