INFECTION AND IMMUNITY, Sept. 1979, p. 792-796 0019-9567/79/09-0792/05$02.00/0
Vol. 25, No. 3
Serotypes of Beta-Hemolytic Treponema hyodysenteriae D. H. BAUM* AND L. A. JOENS National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010 Received for publication 7 June 1979
Cultures from 13 isolates of pathogenic, beta-hemolytic Treponema hyodysenteriae from 11 geographically separate outbreaks and 2 experimentally induced cases of swine dysentery were lyophilized and extracted with hot phenol-water. The resulting water phases were examined serologically with antisera produced in rabbits against whole-cell bacterins of the 13 isolates for evidence of antigenic classes within the species. Water-phase antigens gave precipitin reactions with homologous antisera. Results from cross-testing of each water phase with each antiserum showed four serologically distinct groups among the isolants examined. Based on precipitin reactions in agarose gel, four serotypes of pathogenic, betahemolytic T. hyodysenteriae are proposed.
Treponema hyodysenteriae has been shown to be the primary etiological agent of swine dysentery (6, 8, 9, 28, 29). Presently, two major classes of T. hyodysenteriae are recognized on the basis of hemolytic activity in blood agar: beta-hemolytic T. hyodysenteriae and weak beta-hemolytic T. hyodysenteriae (16). The beta-hemolytic isolates can induce swine dysentery when given orally to conventional and specific-pathogen-free swine (7-9, 16, 20-23). The weak beta-hemolytic isolates have been isolated from cases of canine diarrhea (2, 4, 16), normal swine (29), and postweaning diarrhea in swine (16). Weak beta-hemolytic T. hyodysenteriae has failed to produce clinical signs and lesions typical of swine dysentery when introduced orally into specific-pathogen-free swine (16). Lipopolysaccharide is a compound from the outer envelope of gram-negative bacteria that is responsible for endotoxicity and antigenic specificity among gram-negative bacteria (12, 30) and is readily extracted with hot phenol-water (2, 30, 31). Electron microscopy of Treponema has shown that this genus has an outer envelope similar to that of other gram-negative bacteria (1, 10, 11, 14, 17, 18, 24-26). It was presumed, therefore, that phenol-water extraction of betahemolytic T. hyodysenteriae could yield lipopolysaccharide-like antigens that would make serological grouping of the organism possible. The objective of this study was to investigate antigens extracted from 13 isolates of pathogenic, beta-hemolytic T. hyodysenteriae to determine whether they could be used effectively for serological classification of beta-hemolytic T. hyodysenteriae.
MATERIALS AND METHODS Isolates. Beta-hemolytic isolates A-1, B78, B140, B169, B204, B234, Dys 7, and 9605 and weak betahemolytic isolates 4/71, B256, B297, and Puppy were obtained from the Veterinary Medical Research Institute, Iowa State University, Ames, Iowa, through J. M. Kinyon; isolates T3 and T4 were submitted by Joe Welter, Dallas Center, Iowa; isolates T5 and T6 were reisolated from pigs experimentally infected with B204 and B234, respectively, at the National Animal Disease Center, Ames, Iowa; isolate T7 was isolated from B140 and designated according to National Animal Disease Center nomenclature. All isolates were received frozen in broth. Media. Blood agar plates were prepared using Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% citrated bovine blood (1 g of sodium citrate per 100 ml of blood). Trypticase soy broth (TSB) was rehydrated and prepared by the aerobic method of Kinyon and Harris (15). This method was also used for the preparation of 200 ml of TSB in 500-ml round-bottom flasks and 1,000 ml of TSB in 2,000-ml adapted, round-bottom flasks (L. A. Joens, Ph.D. thesis, Iowa State University, Ames, 1977). All broth contained a 10% supplement of fetal calf serum (National Animal Disease Center, Ames, Iowa; GIBCO, Grand Island, N.Y.) that was added at the time of inoculation. Broth cultures were incubated under an atmosphere of deoxygenated H2-CO2 (50:50) at 37°C on a reciprocating shaker (90 rpm). Culture preparations. Frozen isolates were thawed at 37°C, transferred to tubed TSB (11% inoculum), and subcultured first into 200 ml of TSB in 500-ml round-bottom flasks and then into 1,000 ml of TSB in 2,000-ml flasks (Joens, Ph.D. thesis, 1977). Cultures in 2,000-ml round-bottom flasks were incubated for 36 to 48 h on a reciprocating shaker at 37°C. Organisms were harvested by centrifugation at 16,000 x g at 0°C for 20 min, washed twice in 200 ml of 0.01 M phosphate-buffered saline (pH 7.2), resuspended in 792
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10 ml of phosphate-buffered saline, and lyophilized. Whole-cell bacterins were prepared from 36- to 48h, 200-ml cultures of T. hyodysenteriae. Cells were harvested and washed as described, resuspended in 10 ml of formalized phosphate-buffered saline (0.3% formaldehyde in 0.01 M phosphate-buffered saline), inactivated at room temperature for 24 h, and stored at 40C. Cultures of all of the beta-hemolytic isolates except A-1, B169, B204, and B234 were purified by agar plug transfer for this study. In the context of this study, purification of cultures means the transfer of a single hemolytic plaque from blood agar to TSB. After 48-h incubation at 420C under deoxygenated H2-CO2 (50: 50), broth cultures were subcultured onto blood agar and streaked for isolation. Agar plug transfer was then repeated, and the resulting broth culture was used to seed 200 ml of TSB for subculture into 1,000 ml of broth. Antiserum production. Antiserum against each isolate of T. hyodysenteriae was produced in white New Zealand rabbits. Bacterins (approximately 109 cells per ml) were mixed with Freund complete adjuvant 1:1 (vol/vol) at the time of injection. Each rabbit received 0.5 ml subcutaneously and 1.0 ml intramuscularly on days 1 and 7. On day 14, each received an intradermal injection of 1.5 ml of bacterin in Freund incomplete adjuvant, 1:1 (vol/vol). On days 21 and 28 each received intravenous boosters of 0.5 and 1.0 ml,
respectively. Preinjection blood samples were taken from each rabbit on days 1, 14, 21, and 28. Terminal blood samples were obtained on day 35. Sera were sterilized by filtration through a membrane (0.22-jum average pore diameter) and stored at -20°C. Extraction of bacteria. The hot phenol-water method (31) was adapted for the extraction of lyophilized beta-hemolytic T. hyodysenteriae. Hot (68°C) distilled water (10 ml) was added to 200 mg of lyophilized cells and blended with a Vortex mixer. Vortex mixing was followed by adding 10 ml of hot (68°C) 88% phenol, remixing, and heating at 68°C for 12 to 15 min. The phenol and water phases were separated by cooling the slurry to 10°C in an ice bath. Cooled extracts were centrifuged at 2,200 x g for 20 min in a swinging-bucket rotor, after which the water phase was carefully drawn from the phenol phase with a 10ml pipette. Ten milliliters of hot distilled water was added to the phenol phase, and the extraction was repeated. The two water phases were combined and dialyzed against 50 volumes of distilled water for 24 h with six changes of water. Dialyzed water phases were concentrated under a vacuum (385 mm of Hg) to onefifth their original volume. Carbohydrate complexes were precipitated (27) with the addition of 6 volumes of 90%o ethanol plus sodium acetate and incubation at -20°C overnight. Precipitates were sedimented by centrifugation (16,000 x g), resuspended in 1.0 ml of distilled water, reprecipitated with 5 volumes of acetone, and sedimented by centrifugation. Acetone precipitation was repeated twice. Final precipitates were suspended in 1.0 ml of distilled water and diluted in distilled water to a concentration of 300 ,ug of hexose per ml.
793
Extract analysis. Protein was estimated by the method of Lowry et al. (19) with bovine serum albumin as the standard; carbohydrate was estimated by the method of Dubois et al. (3) with D-glucose as the hexose standard. Immunodiffusion. Sera and antigens were tested (13) by double immunodiffusion in 1% agarose melted in Karrer's Veronal-buffered saline solution. Melted agarose (3 ml) was applied to a clean 25- by 75-mm frosted-edge slide. Holes were punched in the gel and filled with antisera or extract. Reactions were read after 18 h of incubation at room temperature in a humid chamber. Normal rabbit serum served as a control for each immunodfffusion test.
RESULTS Reactivity of water phases. Thirteen isolates of T. hyodysenteriae were extracted with hot phenol-water. The concentrated water phase from the first extraction of isolant T5 was tested with antisera to whole cells of beta-hemolytic T. hyodysenteriae (Fig. 1). The observed bands of precipitate were later shown to be identical (Fig. 2). Repeated extractions of cells from various lyophilized cultures of T5 yielded an antigen which produced the same types of precipitin reactions in gel. After demonstration that the method of extraction could be repeated, water phases from extracts of the rest ofthe beta-hemolytic isolants were examined. Each water phase produced precipitin reactions in gel against homologous antiserum. Each water phase was cross-tested against each antiserum in agarose gel to determine what, if any, antigenic relationship the isolants had with each other. Final results of these tests are demonstrated in Fig. 3 and summarized in Table 1. Four distinct groups were noted among the isolates examined: B78, B234, Dys 7, T6, and T7 comprising one group; B140, B204, T3, T5, and 9605 comprising another
FIG. 1. Demonstration of preliminary results obtained from phenol-water extraction of beta-hemolytic T. hyodysenteriae isolate T5: 1 = anti-T5; 2 = anti-B234; 3= anti-B169; 4 = anti-B140; 5= normal rabbit serum; 6 = normal rabbit serum; 7= anti-B78; 8 = anti-Dys 7; 9 = anti-A-I; 10 = anti-T6; 11 = normal rabbit serum; 12 = normal rabbit serum. Center wells: Ts water phase.
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group; B169 alone; A-1 alone. Furthermore, none of the water phases reacted with the antisera produced in this study against weak beta-hemolytic isolates.
FIG. 2. Precipitin test in agarose gel demonstrating the identity of the reactions seen in Fig. 1. Reactants: 1 = T5 water phase; 2 = Tr, antiserum; and 3 = B140 antiserum.
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Analysis of extracts. The water-phase precipitates contained 85 to 90% carbohydrate (based on dry weight) as estimated by the method of Dubois et al. (3) with D-glucose as the standard. Protein accounted for 5 to 10% of the precipitate (based on dry weight) estimated by the Lowry method (19). DISCUSSION The results presented in this report indicate that an antigen in the water phase from the phenol-water extraction of each beta-hemolytic T. hyodysenteriae isolate reacts with homologous antisera in gel diffusion precipitin tests. Results from immunodiffusion tests showed that certain isolants have the same variants of this extractable antigen in common. Weak but identical precipitin bands were observed among the B78-B234-Dys 7-T6-T7 group and were probably a result of less than optimal antigen concentration. However, the identity of these reactions provides the basis for inclusion of the isolates in the same group. With that, we propose that the reactions summarized in Table 1 be used as the
FIG. 3. Precipitin reactions among all available reactants. Peripheral wells: 1 = anti-B78; 2 = anti-B234; 3 = anti-Dys 7; 4 = anti-T6; 5 = anti-T7; 6 = anti-B140; 7= anti-B204; 8 = anti-TI:,; 9 = antiT4; 10 = antiT5; 11 = normal rabbit serum; 12 = anti-9605; 13 = anti-B169; 14 = anti-A-I; 15 = normal rabbit serum. Center wells filled with standardized antigen: A = B78; B= B234; C = Dys 7; D = T6; E = T7; F = B140; G = B204; H = TI3; I = T4; J = T5; K = 9605; L = B169; M = A-i.
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TABLE 1. Results of double-diffusion precipitin reactions among water-phase antigens and antisera of T. hyodysenteriae isolatesa Antisera Antigen
B78
B234
Dys 7
T6
T7
B140
B204
T3
T4
T5
9605
B169
A-1
+ + + + B78 + + + + + B234 + + + + + Dys7 + + + + + T6 + + + + + T7 + + + + + + B140 + + + + + + B204 + + + + + + T3 + + + + + + T4 + + + + + + T5 + + + + + + 9605 + B169 + A-1 a Reactions were run at 250C and at antigen concentration of 300 Llg of hexose per ml. Blank areas = no band of precipitin; + = band of precipitin reacting identically with homologous systems.
TABLE 2. Proposed serotype designations of betahemolytic T. hyodysenteriae and included isolates Serotype
Isolates
1 2 3 4
B78, B234, Dys 7, T6, T7 B140, B204, T3, T4, T5, 9605 B169 A-1
basis for establishing serotypes of beta-hemolytic T. hyodysenteriae as outlined in Table 2. The stability of the proposed serotypes remains to be verified. However, isolates T5 and T6 have maintained their serotype after having been isolated from pigs experimentally infected with B204 and B234, respectively. Also, in vitro passage may not alter serological specificity in that isolates B78 and Dys 7 were capable of being serotyped after having been passaged more than 100 times in broth (J. M. Kinyon, personal communication). Finally, the inability of the extracts to react with antisera to weak beta-hemolytic isolates indicates that these antigens could be useful in developing a specific serological test for swine dysentery which would not produce positive results from pigs harboring the weak beta-hemolytic isolates. LITERATURE CITED 1. Bladen, H. A., and E. G. Hampp. 1974. Ultrastructure
of Treponema microdemtium and Borrelia vincentii. J. Bacteriol. 87:1180-1191. 2. Craige, J. E. 1948. Spirochetes associated with dysentery in dogs. J. Am. Vet. Med. Assoc. 113:247-249. 3. Dubois, M., K. A. Giles, J. K. Hamilton, P. D. Rebers, and T. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 4. Goudswaard, J., and J. L. Cornelisse. 1973. The agent
5. 6. 7.
8.
associated with swine dysentery and antigenic relationship with Borrelia canis. Vet. Rec. 92:562-563. Hamdy, A. H., and M. W. Glenn. 1974. Transmission of swine dysentery with Treponema hyodysenteriae and Vibrio coli. Am. J. Vet. Res. 35:791-797. Harris, D. L. 1974. Current status of research on swine dysentery. J. Am. Vet. Med. Assoc. 164:809-812. Harris, D. L., R. D. Glock, C. R. Christensen, and J. M. Kinyon. 1972. Swine dysentery. I. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin. 67:61-69. Hudson, M. J., T. J. L Alexander, R. J. Lysons, and P. D. Wellstead. 1974. Swine dysentery: failures of an attenuated strain of spirochete given orally to protect pigs against subsequent challenge. Br. Vet. J. 130:37-
40. 9. Hughes, R., H. V. Olander, and C. B. Williams. 1975. Swine dysentery: pathogenicity of Treponema hyodysenteriae. Am. J. Vet. Res. 36:971-977. 10. Jackson, S. W., and P. N. Zey. 1973. Ultrastructure of lipopolysaccharide isolated from Treponema pallidum. J. Bacteriol. 114:838-844. 11. Jepsen, 0. B., K. H. Hougen, and A. Birch-Anderson. 1968. Electron microscopy of Treponema pallidum Nichols. Acta Pathol. Microbiol. Scand. 74:241-258. 12. Kadis, S., G. Weinbaum, and S. J. AjI (ed.). 1971. Microbial toxins, vol. 5, Bacterial endotoxins. Academic Press Inc., New York. 13. Karrer, H., K. F. Meyer, and B. Eddie. 1950. The complement fixation inhibition test and its application to the diagnosis of ornithosis in chickens and ducks. I. Principles and techniques of the test. J. Infect. Dis. 87: 13-36. 14. Kawata, T., and T. Inoue. 1964. Fine structure of the Reiter Treponeme as revealed by electron microscopy using thin sectioning and negative staining techniques. Jpn. J. Microbiol. 8:49-65. 15. Kinyon, J. M., and D. L Harris. 1974. Growth of Treponema hyodysenteriae in liquid medium. Vet. Rec. 95:219-220. 16. Kinyon, J. M., D. L. Harris, and R. D. Glock. 1977. Enteropathogenicity of Treponema hyodysenteriae. Infect. Immun. 15:638-646. 17. Listgarten, M. A., W. J. Loesche, and S. S. Socransky. 1963. Morphology of Treponema microdentium as revealed by electron microscopy of ultrathin sections. J.
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Bacteriol. 85:932. 18. Listgarten, M. A., and S. S. Socransky. 1964. Electron microscopy of axial fibrils, outer envelope, and cell division of certain oral spirochetes. J. Bacteriol. 88: 1087-1103. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 20. Meyer, R. C., J. Simon, and C. S. Byerly. 1974. The etiology of swine dysentery. I. Oral inoculation of germfree swine with Treponema hyodysenteriae and Vibrio coli. Vet. Pathol. 11:515-526. 21. Meyer, R. C., J. Simon, and C. S. Byerly. 1974. The etiology of swine dysentery. II. Effect of a known microbial flora, weaning and diet on disease production in gnotobiotic and conventional swine. Vet. Pathol. 11: 527-534. 22. Meyer, R. C., J. Simon, and C. S. Byerly. 1974. The etiology of swine dysentery. III. The role of selected gram-negative obligate anaerobes. Vet. Pathol. 12:4654.
23. Olujic, M., D. Sofrenovic, B. Trbic, M. Iffic, M. Markovic, and M. Dordevic-Matejic. 1973. Investigations of dysentery in swine. I. Isolation and determination of a spirochete (Treponema hyodysenteriae) in affected
INFECT. IMMUN. swine. Vet. Glas. 4:241-245. 24. Oveinnokov, N. M., and V. V. Delektorskij. 1966. Morphology of Treponema pallidum. Bull. W.H.O. 35: 223-229. 25. Ovcinnikov, N. M., and V. V. Delektorskij. 1968. Morphology of Treponema pallidum and Leptospira (an electron microscope study) (English summary). Zh. Mikrobiol. Epidemiol. Immunobiol. 45:131-135. 26. Ryter, A., and J. Pilot. 1963. etude an microscope electronique de la structure externe et interne du Treponema Reiter (English summary). Ann. Inst. Pasteur (Paris) 104:496-501. 27. Slade, H. D. 1965. Extraction of cell-wall polysacchbride antigen from streptococci. J. Bacteriol. 90:667-672. 28. Taylor, D. J. 1970. An agent possibly associated with swine dysentery. Res. Vet. Sci. 12:177-179. 29. Taylor, D. J., and T. J. L. Alexander. 1971. The production of dysentery in swine by feeding cultures containing a spirochete. Br. Vet. J. 127:58-61. 30. Weinbaum, G., S. Kadis, and S. J. AjI (ed.). 1971. Microbial toxins, vol. 4, Bacterial endotoxins. Academic Press Inc., New York. 31. Westphal, O., 0. Luderitz, and R. Bister. 1952. Olber die Extraktion von Bakterien mit Phenol/Wasser. Z. Naturforsch. 76:148-155.
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Serotypes of Beta-Hemolytic Treponema hyodysenteriae D. H. BAUM* AND L. A. JOENS National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010 Received for publication 7 June 1979
Cultures from 13 isolates of pathogenic, beta-hemolytic Treponema hyodysenteriae from 11 geographically separate outbreaks and 2 experimentally induced cases of swine dysentery were lyophilized and extracted with hot phenol-water. The resulting water phases were examined serologically with antisera produced in rabbits against whole-cell bacterins of the 13 isolates for evidence of antigenic classes within the species. Water-phase antigens gave precipitin reactions with homologous antisera. Results from cross-testing of each water phase with each antiserum showed four serologically distinct groups among the isolants examined. Based on precipitin reactions in agarose gel, four serotypes of pathogenic, betahemolytic T. hyodysenteriae are proposed.
Treponema hyodysenteriae has been shown to be the primary etiological agent of swine dysentery (6, 8, 9, 28, 29). Presently, two major classes of T. hyodysenteriae are recognized on the basis of hemolytic activity in blood agar: beta-hemolytic T. hyodysenteriae and weak beta-hemolytic T. hyodysenteriae (16). The beta-hemolytic isolates can induce swine dysentery when given orally to conventional and specific-pathogen-free swine (7-9, 16, 20-23). The weak beta-hemolytic isolates have been isolated from cases of canine diarrhea (2, 4, 16), normal swine (29), and postweaning diarrhea in swine (16). Weak beta-hemolytic T. hyodysenteriae has failed to produce clinical signs and lesions typical of swine dysentery when introduced orally into specific-pathogen-free swine (16). Lipopolysaccharide is a compound from the outer envelope of gram-negative bacteria that is responsible for endotoxicity and antigenic specificity among gram-negative bacteria (12, 30) and is readily extracted with hot phenol-water (2, 30, 31). Electron microscopy of Treponema has shown that this genus has an outer envelope similar to that of other gram-negative bacteria (1, 10, 11, 14, 17, 18, 24-26). It was presumed, therefore, that phenol-water extraction of betahemolytic T. hyodysenteriae could yield lipopolysaccharide-like antigens that would make serological grouping of the organism possible. The objective of this study was to investigate antigens extracted from 13 isolates of pathogenic, beta-hemolytic T. hyodysenteriae to determine whether they could be used effectively for serological classification of beta-hemolytic T. hyodysenteriae.
MATERIALS AND METHODS Isolates. Beta-hemolytic isolates A-1, B78, B140, B169, B204, B234, Dys 7, and 9605 and weak betahemolytic isolates 4/71, B256, B297, and Puppy were obtained from the Veterinary Medical Research Institute, Iowa State University, Ames, Iowa, through J. M. Kinyon; isolates T3 and T4 were submitted by Joe Welter, Dallas Center, Iowa; isolates T5 and T6 were reisolated from pigs experimentally infected with B204 and B234, respectively, at the National Animal Disease Center, Ames, Iowa; isolate T7 was isolated from B140 and designated according to National Animal Disease Center nomenclature. All isolates were received frozen in broth. Media. Blood agar plates were prepared using Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% citrated bovine blood (1 g of sodium citrate per 100 ml of blood). Trypticase soy broth (TSB) was rehydrated and prepared by the aerobic method of Kinyon and Harris (15). This method was also used for the preparation of 200 ml of TSB in 500-ml round-bottom flasks and 1,000 ml of TSB in 2,000-ml adapted, round-bottom flasks (L. A. Joens, Ph.D. thesis, Iowa State University, Ames, 1977). All broth contained a 10% supplement of fetal calf serum (National Animal Disease Center, Ames, Iowa; GIBCO, Grand Island, N.Y.) that was added at the time of inoculation. Broth cultures were incubated under an atmosphere of deoxygenated H2-CO2 (50:50) at 37°C on a reciprocating shaker (90 rpm). Culture preparations. Frozen isolates were thawed at 37°C, transferred to tubed TSB (11% inoculum), and subcultured first into 200 ml of TSB in 500-ml round-bottom flasks and then into 1,000 ml of TSB in 2,000-ml flasks (Joens, Ph.D. thesis, 1977). Cultures in 2,000-ml round-bottom flasks were incubated for 36 to 48 h on a reciprocating shaker at 37°C. Organisms were harvested by centrifugation at 16,000 x g at 0°C for 20 min, washed twice in 200 ml of 0.01 M phosphate-buffered saline (pH 7.2), resuspended in 792
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10 ml of phosphate-buffered saline, and lyophilized. Whole-cell bacterins were prepared from 36- to 48h, 200-ml cultures of T. hyodysenteriae. Cells were harvested and washed as described, resuspended in 10 ml of formalized phosphate-buffered saline (0.3% formaldehyde in 0.01 M phosphate-buffered saline), inactivated at room temperature for 24 h, and stored at 40C. Cultures of all of the beta-hemolytic isolates except A-1, B169, B204, and B234 were purified by agar plug transfer for this study. In the context of this study, purification of cultures means the transfer of a single hemolytic plaque from blood agar to TSB. After 48-h incubation at 420C under deoxygenated H2-CO2 (50: 50), broth cultures were subcultured onto blood agar and streaked for isolation. Agar plug transfer was then repeated, and the resulting broth culture was used to seed 200 ml of TSB for subculture into 1,000 ml of broth. Antiserum production. Antiserum against each isolate of T. hyodysenteriae was produced in white New Zealand rabbits. Bacterins (approximately 109 cells per ml) were mixed with Freund complete adjuvant 1:1 (vol/vol) at the time of injection. Each rabbit received 0.5 ml subcutaneously and 1.0 ml intramuscularly on days 1 and 7. On day 14, each received an intradermal injection of 1.5 ml of bacterin in Freund incomplete adjuvant, 1:1 (vol/vol). On days 21 and 28 each received intravenous boosters of 0.5 and 1.0 ml,
respectively. Preinjection blood samples were taken from each rabbit on days 1, 14, 21, and 28. Terminal blood samples were obtained on day 35. Sera were sterilized by filtration through a membrane (0.22-jum average pore diameter) and stored at -20°C. Extraction of bacteria. The hot phenol-water method (31) was adapted for the extraction of lyophilized beta-hemolytic T. hyodysenteriae. Hot (68°C) distilled water (10 ml) was added to 200 mg of lyophilized cells and blended with a Vortex mixer. Vortex mixing was followed by adding 10 ml of hot (68°C) 88% phenol, remixing, and heating at 68°C for 12 to 15 min. The phenol and water phases were separated by cooling the slurry to 10°C in an ice bath. Cooled extracts were centrifuged at 2,200 x g for 20 min in a swinging-bucket rotor, after which the water phase was carefully drawn from the phenol phase with a 10ml pipette. Ten milliliters of hot distilled water was added to the phenol phase, and the extraction was repeated. The two water phases were combined and dialyzed against 50 volumes of distilled water for 24 h with six changes of water. Dialyzed water phases were concentrated under a vacuum (385 mm of Hg) to onefifth their original volume. Carbohydrate complexes were precipitated (27) with the addition of 6 volumes of 90%o ethanol plus sodium acetate and incubation at -20°C overnight. Precipitates were sedimented by centrifugation (16,000 x g), resuspended in 1.0 ml of distilled water, reprecipitated with 5 volumes of acetone, and sedimented by centrifugation. Acetone precipitation was repeated twice. Final precipitates were suspended in 1.0 ml of distilled water and diluted in distilled water to a concentration of 300 ,ug of hexose per ml.
793
Extract analysis. Protein was estimated by the method of Lowry et al. (19) with bovine serum albumin as the standard; carbohydrate was estimated by the method of Dubois et al. (3) with D-glucose as the hexose standard. Immunodiffusion. Sera and antigens were tested (13) by double immunodiffusion in 1% agarose melted in Karrer's Veronal-buffered saline solution. Melted agarose (3 ml) was applied to a clean 25- by 75-mm frosted-edge slide. Holes were punched in the gel and filled with antisera or extract. Reactions were read after 18 h of incubation at room temperature in a humid chamber. Normal rabbit serum served as a control for each immunodfffusion test.
RESULTS Reactivity of water phases. Thirteen isolates of T. hyodysenteriae were extracted with hot phenol-water. The concentrated water phase from the first extraction of isolant T5 was tested with antisera to whole cells of beta-hemolytic T. hyodysenteriae (Fig. 1). The observed bands of precipitate were later shown to be identical (Fig. 2). Repeated extractions of cells from various lyophilized cultures of T5 yielded an antigen which produced the same types of precipitin reactions in gel. After demonstration that the method of extraction could be repeated, water phases from extracts of the rest ofthe beta-hemolytic isolants were examined. Each water phase produced precipitin reactions in gel against homologous antiserum. Each water phase was cross-tested against each antiserum in agarose gel to determine what, if any, antigenic relationship the isolants had with each other. Final results of these tests are demonstrated in Fig. 3 and summarized in Table 1. Four distinct groups were noted among the isolates examined: B78, B234, Dys 7, T6, and T7 comprising one group; B140, B204, T3, T5, and 9605 comprising another
FIG. 1. Demonstration of preliminary results obtained from phenol-water extraction of beta-hemolytic T. hyodysenteriae isolate T5: 1 = anti-T5; 2 = anti-B234; 3= anti-B169; 4 = anti-B140; 5= normal rabbit serum; 6 = normal rabbit serum; 7= anti-B78; 8 = anti-Dys 7; 9 = anti-A-I; 10 = anti-T6; 11 = normal rabbit serum; 12 = normal rabbit serum. Center wells: Ts water phase.
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group; B169 alone; A-1 alone. Furthermore, none of the water phases reacted with the antisera produced in this study against weak beta-hemolytic isolates.
FIG. 2. Precipitin test in agarose gel demonstrating the identity of the reactions seen in Fig. 1. Reactants: 1 = T5 water phase; 2 = Tr, antiserum; and 3 = B140 antiserum.
INFECT. IMMUN.
Analysis of extracts. The water-phase precipitates contained 85 to 90% carbohydrate (based on dry weight) as estimated by the method of Dubois et al. (3) with D-glucose as the standard. Protein accounted for 5 to 10% of the precipitate (based on dry weight) estimated by the Lowry method (19). DISCUSSION The results presented in this report indicate that an antigen in the water phase from the phenol-water extraction of each beta-hemolytic T. hyodysenteriae isolate reacts with homologous antisera in gel diffusion precipitin tests. Results from immunodiffusion tests showed that certain isolants have the same variants of this extractable antigen in common. Weak but identical precipitin bands were observed among the B78-B234-Dys 7-T6-T7 group and were probably a result of less than optimal antigen concentration. However, the identity of these reactions provides the basis for inclusion of the isolates in the same group. With that, we propose that the reactions summarized in Table 1 be used as the
FIG. 3. Precipitin reactions among all available reactants. Peripheral wells: 1 = anti-B78; 2 = anti-B234; 3 = anti-Dys 7; 4 = anti-T6; 5 = anti-T7; 6 = anti-B140; 7= anti-B204; 8 = anti-TI:,; 9 = antiT4; 10 = antiT5; 11 = normal rabbit serum; 12 = anti-9605; 13 = anti-B169; 14 = anti-A-I; 15 = normal rabbit serum. Center wells filled with standardized antigen: A = B78; B= B234; C = Dys 7; D = T6; E = T7; F = B140; G = B204; H = TI3; I = T4; J = T5; K = 9605; L = B169; M = A-i.
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TABLE 1. Results of double-diffusion precipitin reactions among water-phase antigens and antisera of T. hyodysenteriae isolatesa Antisera Antigen
B78
B234
Dys 7
T6
T7
B140
B204
T3
T4
T5
9605
B169
A-1
+ + + + B78 + + + + + B234 + + + + + Dys7 + + + + + T6 + + + + + T7 + + + + + + B140 + + + + + + B204 + + + + + + T3 + + + + + + T4 + + + + + + T5 + + + + + + 9605 + B169 + A-1 a Reactions were run at 250C and at antigen concentration of 300 Llg of hexose per ml. Blank areas = no band of precipitin; + = band of precipitin reacting identically with homologous systems.
TABLE 2. Proposed serotype designations of betahemolytic T. hyodysenteriae and included isolates Serotype
Isolates
1 2 3 4
B78, B234, Dys 7, T6, T7 B140, B204, T3, T4, T5, 9605 B169 A-1
basis for establishing serotypes of beta-hemolytic T. hyodysenteriae as outlined in Table 2. The stability of the proposed serotypes remains to be verified. However, isolates T5 and T6 have maintained their serotype after having been isolated from pigs experimentally infected with B204 and B234, respectively. Also, in vitro passage may not alter serological specificity in that isolates B78 and Dys 7 were capable of being serotyped after having been passaged more than 100 times in broth (J. M. Kinyon, personal communication). Finally, the inability of the extracts to react with antisera to weak beta-hemolytic isolates indicates that these antigens could be useful in developing a specific serological test for swine dysentery which would not produce positive results from pigs harboring the weak beta-hemolytic isolates. LITERATURE CITED 1. Bladen, H. A., and E. G. Hampp. 1974. Ultrastructure
of Treponema microdemtium and Borrelia vincentii. J. Bacteriol. 87:1180-1191. 2. Craige, J. E. 1948. Spirochetes associated with dysentery in dogs. J. Am. Vet. Med. Assoc. 113:247-249. 3. Dubois, M., K. A. Giles, J. K. Hamilton, P. D. Rebers, and T. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 4. Goudswaard, J., and J. L. Cornelisse. 1973. The agent
5. 6. 7.
8.
associated with swine dysentery and antigenic relationship with Borrelia canis. Vet. Rec. 92:562-563. Hamdy, A. H., and M. W. Glenn. 1974. Transmission of swine dysentery with Treponema hyodysenteriae and Vibrio coli. Am. J. Vet. Res. 35:791-797. Harris, D. L. 1974. Current status of research on swine dysentery. J. Am. Vet. Med. Assoc. 164:809-812. Harris, D. L., R. D. Glock, C. R. Christensen, and J. M. Kinyon. 1972. Swine dysentery. I. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin. 67:61-69. Hudson, M. J., T. J. L Alexander, R. J. Lysons, and P. D. Wellstead. 1974. Swine dysentery: failures of an attenuated strain of spirochete given orally to protect pigs against subsequent challenge. Br. Vet. J. 130:37-
40. 9. Hughes, R., H. V. Olander, and C. B. Williams. 1975. Swine dysentery: pathogenicity of Treponema hyodysenteriae. Am. J. Vet. Res. 36:971-977. 10. Jackson, S. W., and P. N. Zey. 1973. Ultrastructure of lipopolysaccharide isolated from Treponema pallidum. J. Bacteriol. 114:838-844. 11. Jepsen, 0. B., K. H. Hougen, and A. Birch-Anderson. 1968. Electron microscopy of Treponema pallidum Nichols. Acta Pathol. Microbiol. Scand. 74:241-258. 12. Kadis, S., G. Weinbaum, and S. J. AjI (ed.). 1971. Microbial toxins, vol. 5, Bacterial endotoxins. Academic Press Inc., New York. 13. Karrer, H., K. F. Meyer, and B. Eddie. 1950. The complement fixation inhibition test and its application to the diagnosis of ornithosis in chickens and ducks. I. Principles and techniques of the test. J. Infect. Dis. 87: 13-36. 14. Kawata, T., and T. Inoue. 1964. Fine structure of the Reiter Treponeme as revealed by electron microscopy using thin sectioning and negative staining techniques. Jpn. J. Microbiol. 8:49-65. 15. Kinyon, J. M., and D. L Harris. 1974. Growth of Treponema hyodysenteriae in liquid medium. Vet. Rec. 95:219-220. 16. Kinyon, J. M., D. L. Harris, and R. D. Glock. 1977. Enteropathogenicity of Treponema hyodysenteriae. Infect. Immun. 15:638-646. 17. Listgarten, M. A., W. J. Loesche, and S. S. Socransky. 1963. Morphology of Treponema microdentium as revealed by electron microscopy of ultrathin sections. J.
796
BAUM AND JOENS
Bacteriol. 85:932. 18. Listgarten, M. A., and S. S. Socransky. 1964. Electron microscopy of axial fibrils, outer envelope, and cell division of certain oral spirochetes. J. Bacteriol. 88: 1087-1103. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 20. Meyer, R. C., J. Simon, and C. S. Byerly. 1974. The etiology of swine dysentery. I. Oral inoculation of germfree swine with Treponema hyodysenteriae and Vibrio coli. Vet. Pathol. 11:515-526. 21. Meyer, R. C., J. Simon, and C. S. Byerly. 1974. The etiology of swine dysentery. II. Effect of a known microbial flora, weaning and diet on disease production in gnotobiotic and conventional swine. Vet. Pathol. 11: 527-534. 22. Meyer, R. C., J. Simon, and C. S. Byerly. 1974. The etiology of swine dysentery. III. The role of selected gram-negative obligate anaerobes. Vet. Pathol. 12:4654.
23. Olujic, M., D. Sofrenovic, B. Trbic, M. Iffic, M. Markovic, and M. Dordevic-Matejic. 1973. Investigations of dysentery in swine. I. Isolation and determination of a spirochete (Treponema hyodysenteriae) in affected
INFECT. IMMUN. swine. Vet. Glas. 4:241-245. 24. Oveinnokov, N. M., and V. V. Delektorskij. 1966. Morphology of Treponema pallidum. Bull. W.H.O. 35: 223-229. 25. Ovcinnikov, N. M., and V. V. Delektorskij. 1968. Morphology of Treponema pallidum and Leptospira (an electron microscope study) (English summary). Zh. Mikrobiol. Epidemiol. Immunobiol. 45:131-135. 26. Ryter, A., and J. Pilot. 1963. etude an microscope electronique de la structure externe et interne du Treponema Reiter (English summary). Ann. Inst. Pasteur (Paris) 104:496-501. 27. Slade, H. D. 1965. Extraction of cell-wall polysacchbride antigen from streptococci. J. Bacteriol. 90:667-672. 28. Taylor, D. J. 1970. An agent possibly associated with swine dysentery. Res. Vet. Sci. 12:177-179. 29. Taylor, D. J., and T. J. L. Alexander. 1971. The production of dysentery in swine by feeding cultures containing a spirochete. Br. Vet. J. 127:58-61. 30. Weinbaum, G., S. Kadis, and S. J. AjI (ed.). 1971. Microbial toxins, vol. 4, Bacterial endotoxins. Academic Press Inc., New York. 31. Westphal, O., 0. Luderitz, and R. Bister. 1952. Olber die Extraktion von Bakterien mit Phenol/Wasser. Z. Naturforsch. 76:148-155.
