Vol. 29, No. 12

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2794-2797 0095-1137/91/122794-04$02.00/0 Copyright © 1991, American Society for Microbiology

Serotyping of Canadian Isolates of Treponema hyodysenteriae and Description of Two New Serotypes ZUSHENG LI, MYRIAM BÉLANGER, AND MARIO JACQUES*

Groupe de Recherche

sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada

Received 27 June 1991/Accepted 10 September 1991

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec. were

Treponema hyodysenteriae (12) is the etiological agent of swine dysentery (7, 10, 12, 16, 25), a mucohemorrhagic diarrheal disease in which lesions are confined to the large intestine of pigs (6). T. hyodysenteriae, a gram-negative spirochete, is widely distributed in herds throughout most swine-rearing countries of the world (10, 11, 14). This disease has led to heavy economic losses in pig industries (10, 11). The most distinctive characteristics of T. hyodysenteriae are its strong beta-hemolytic activity and its enteropathogenicity in swine (20). Treponema innocens (18), another species of Treponema, is morphologically identical to T. hyodysenteriae (13, 15, 20), but it is different in that it is weakly beta-hemolytic and nonenteropathogenic for swine (18, 19, 25). We have reported a rapid method for differentiation of the two species. The method is based on hemolysis and the ring phenomenon test, in conjunction with an indole-spot test (4). Reclassification of T. hyodysenteriae and T. innocens into a new genus, Serpula, was recently reported by Stanton et al. (24). Because of the illegitimacy of the name Serpula, a new genus designation, Serpulina, has been proposed (23). Serotyping of bacterial isolates is often of great importance in terms of diagnosis and epidemiological evaluation of infectious diseases. It is known that T. hyodysenteriae has heterogeneous antigens in the lipopolysaccharide (LPS) portion of the outer membrane (3, 5, 8, 9, 17, 22, 26). So far, seven serotypes of T. hyodysenteriae have been described on the basis of agar gel double immunodiffusion precipitation (AGDIP) with extracted LPS and hyperimmune rabbit antisera (2, 21). A number of T. hyodysenteriae isolates have been collected in the Saint-Hyacinthe (Quebec, Canada) area and have been biochemically characterized (4). The objective of the present study was to serotype these local isolates of T. hyodysenteriae.

*

MATERIALS AND METHODS Bacteria. A total of 44 field isolates of Treponema, including 30 isolates of T. hyodysenteriae and 14 isolates of T. innocens obtained from rectal swabs, feces, or mucosal scrapings of colons, were collected from pigs in various herds in the Saint-Hyacinthe area. In these herds, swine dysentery was clinically apparent or was considered as a differential diagnosis. Isolates were obtained from S. Messier, Agriculture Canada, Saint-Hyacinthe, Quebec, Canada. T. hyodysenteriae reference strains representing serotypes 1 (B234), 2 (B204), 3 (B169), and 4 (A-1) were provided by L. A. Joens from the Department of Veterinary Science, University of Arizona, Tucson. T. hyodysenteriae serotypes 5 (B8044), 6 (B6933), and 7 (ACK 300/8) and T. innocens B256 were obtained from M. J. Wannemuehler, Veterinary Medical Research Institute, Iowa State University, Ames. Growth conditions. Bacteria were grown on solid medium by using blood agar base no. 2 (Oxoid Ltd., Hampshire, England) containing 5% bovine blood. Plates were incubated anaerobically at 37°C for 4 days in jars (Oxoid) by using a GasPak Plus generator atmosphere (BBL, Becton Dickinson and Co., Cockeysville, Md.) (4). Antisera production. Antisera raised against each reference strain or isolate of T. hyodysenteriae and T. innocens were produced as described previously (21), except that rabbits received a 0.2-ml subcutaneous injection of bacterin in Freund's incomplete adjuvant on day 1; on day 14 they received 1.4-ml subcutaneous injection, and on day 21 they received a 1.0-ml intramuscular injection of bacterin in Freund's complete adjuvant. Adsorption of antisera was done as described previously (21). Preparation of LPS. Extraction of LPS from all reference strains and isolates of T. hyodysenteriae and T. innocens was performed as described by Baum and Joens (2). Immunodiffusion. AGDIP was performed by the method of Mapother and Joens (21), with some modifications. Briefly, 3 ml of melted agarose (1% agarose gel in Tris-NaCI buffer; Bio-Rad Laboratories, Richmond, Calif.) was pipetted into each plastic petri dish (35 by 10 mm; Falcon Plastics, Lincoln Park, N.J.), which was precoated overnight with

Corresponding author. 2794

VOL. 29, 1991

SEROTYPING OF T. HYODYSENTERIAE ISOLATES

TABLE 1. Serotyping of 30 Canadian isolates of T. hyodysenteriae by using antisera raised against reference strains of T. hyodysenteriae representing serotypes 1 to 7 Serotype

Adsorption of antisera with cross-reacting cells resulted in serotype-specific antisera. Each extract from local isolates of Treponema was then tested with each antiserum or adsorbed antiserum against the T. hyodysenteriae reference strains representing serotypes 1 to 7. As summarized in Table 1, only 17% (5 of 30) of the isolates showed precipitations. The other 25 isolates did not precipitate with antisera raised against the seven reference strains of T. hyodysenteriae and were considered untypeable. Immunodiffusion using antisera raised against local isolates. Antisera against 11 local isolates of T. hyodysenteriae were then produced and tested against extracts from reference strains and isolates. The results are summarized in Table 2. Two distinct serological groups were observed among 21 untypeable local isolates. Fourteen isolates of T. hyodysenteriae (recovered from eight herds in 1988), which strongly reacted with antisera raised against seven isolates, made up one group; seven other isolates (recovered from five other herds in 1989), which reacted with antisera raised against four isolates, made up another group. The remaining four untypeable isolates were antigenically different from these two groups, and from serotypes 1 to 7 as well; these four untypeable isolates were recovered in 1988 (two isolates), 1989 (one isolate), and 1990 (one isolate). It is interesting that on two occasions, more than one serotype was found in a single herd. There were no detectable reactions between the 11 antisera raised against local isolates and LPS extracted from the seven reference strains of T. hyodysenteriae, except that antisera against one isolate, 89-440, reacted weakly with LPS extracted from all reference strains and all other isolates of T. hyodysenteriae and T. innocens. Adsorption of this antiserum with T. innocens 89-1608D removed the weak cross-reactions, leaving only specific precipitations, as indicated in Table 2. Precipitate bands in each group were shown to be identical (Fig. 1). These two groups represent two new serotypes, serotypes 8 and 9. We propose isolates FM 88-90 and FMV 89-3323 as the reference strains for serotypes 8 and

No. of isolatesa

1 2 3 4 5

2 0 1 0 i

i

6 7 Untypeable Total

2795

0 25 30

a Serotypes 1 to 7 made up 17% of isolates, and strains with an untypeable serotype made up 83% of isolates.

0.2% agarose (Sigma Chemical Co., St. Louis, Mo.). Fourmillimeter-diameter holes were punched in the gel 7 mm apart (center to center) and filled with 20 ,ul of antiserum or LPS extract. The results were read after an overnight incubation at room temperature in a moist chamber. Normal rabbit sera and preimmune sera were used as controls. RESULTS Immunodiffusion using antisera raised against reference strains representing serotypes 1 to 7. We examined the reactivities of antisera against seven reference strains of T. hyodysenteriae cross-tested with each LPS extract before we serotyped our isolates of T. hyodysenteriae. The results showed that strong homologous precipitation occurred in each case. Cross-reactions between serotypes 5 and 2, serotypes 1 and 6, and serotype 7 and both serotypes 1 and 2 were observed. In addition, antisera against serotypes 3, 4, and 7 showed weak cross-reactions with LPS extracted from all seven reference strains of T. hyodysenteriae and T. innocens B256.

TABLE 2. AGDIP by using LPS extracts and antisera raised against untypeable Canadian isolates of T. hyodysenteriae LPS from isolate:

FM88-76

FM88-104 FM88-116

FM88-90 FM88-105 P9 FM88-91 FM88-81P6 FM88-95P6 FM88-86P7 FM88-88P10 FM88-89 FM88-94 FM88-106P6 FM88-77P6 FMV 89-3323 89-1066 D81 89-1066 C21 FMV 89-1614 D 89-1066 C2

FM 88-90

FM 88-116

FM 88-91

FM 88-86P7

+ + + + + + +

+ + + + + + +

+ + + + + +

+ + + + + +

+ + + + + + +

+ + + + + + +

+ + + + + + + +

+ + + + + + +

Antisera against isolate: FM FM FM 88-77P6 88-89 88-95P6 + + + + + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + +

+ + +

+

+

+

+

+

+

+

+

+

FMV 89-3323

89-10 66C2

89440a

89-1020P4

+

+

+

+

+ + +

+ + +

+ + +

+ + +

+

+

+

89-440

+

+

+

FMV 89-1020 P4

+

+

+

a

Antisera against 89-440 was adsorbed with T. innocens 89-1608D.

FMV

+

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J. CLIN. MICROBIOL.

LI ET AL.

FIG. 1. AGDIP with antiserum raised against isolate FM 88-90 or FMV 89-3323 and various LPS extracts of T. hyodysenteriae. (A) Center well, antiserum against FM 88-90; peripheral wells, LPS extracted from local isolates FM 88-90 (well 1), FM 88-116 (well 2), FM 88-91 (well 3), FM 88-86 P7 (well 4), FM 88-95 P6 (well 5), FM 88-89 (well 6), and FM 88-77 P6 (well 7); well 8 is a control (distilled water). (B) Center well, antiserum against FMV 89-3323; peripheral

wells, LPS extracted from local isolates FMV 89-3323 (well 1), 89-1066 D81 (well 2), 89-1066 C21 (well 3), FMV 89-1614 D (well 4), 89-1066 C2 (well 5), 89-440 (well 6), and FMV 89-1020 P4 (well 7); well 8 is a control (distilled water). (C) Center well, antiserum against FM 88-90 or FMV 89-3323; peripheral wells 1 to 7, LPS extracted from reference strains of T. hyodysenteriae serotypes 1 to 7, respectively; well 8 is a control (distilled water).

9, respectively. Seventy percent (21 of 30) of the isolates recovered in the Saint-Hyacinthe area belonged to these two new serotypes. Finally, no precipitations in AGDIP could be observed between antisera raised against T. innocens reference strains or isolates and LPS extracted from any T. hyodysenteriae reference strains

or isolates.

DISCUSSION

primary work with antisera against the seven referhyodysenteriae demonstrated strong precipitations for all homologous reactions. Mapother and Joens (21) reported that cross-reactions occurred in AGDIP between serotypes 5 and 2, serotypes 6 and 1, and serotype 7 and both serotypes 1 and 2. Our results support these observations. In addition, weak cross-reactions were found when unadsorbed antisera against serotypes 3, 4, and 7 reacted with LPS extracted from all reference strains of T. hyodysenteriae. Adsorption of antisera raised against reference strains of T. hyodysenteriae with cross-reacting bacteria resulted in serotype-specific antisera which were then used to serotype our local isolates. Only 17% (5 of 30) of the isolates of T. hyodysenteriae were typeable. Thus, the situation in Quebec seems to be quite different from that in the United States, where serotypes 1 and 2 were found to be predominant (2, 17). The results of AGDIP by cross-testing each LPS extract with each antiserum against local isolates revealed that 21 isolates of T. hyodysenteriae belong to two distinct serological groups and that the isolates in each group share identical antigens. This evidence, along with the fact that no reactions could be observed between these 21 isolates and the antisera against the reference strains, and vice versa, suggests that these local isolates represent two new serotypes. Therefore, we propose two new serotypes of T. hyodysenteriae, serotypes 8 and 9. We also propose that isolate FM 88-90 be the reference strain for serotype 8 and isolate FMV 89-3323 be the reference strain for serotype 9. It is clear that T. hyodysenteriae displays heterogeneous antigenicity in geographically different locations, which was confirmed by a number of investigations by using AGDIP (1, 3, 22) or sodium dodecyl sulfate-polyacrylamide gel electroOur

ence strains of T.

phoresis followed by immunoblotting (5, 8, 9, 22, 26). The serotype-specific antigens are located in the LPS portion of the outer membrane of T. hyodysenteriae (21). Considering that more than 13% of our isolates are still untypeable, the present description of nine serotypes of T. hyodysenteriae is probably far from complete, and further work could reveal more new serotypes. Nevertheless, the new serotypes described here, serotypes 8 and 9, which represented 70% of our T. hyodysenteriae isolates, seem to be the major serotypes responsible for swine dysentery in Quebec. ACKNOWLEDGMENTS This work was supported by grant 2371 from the Conseil de Recherche en Pêche et Agro-alimentaire du Québec. We thank Marcelo Gottschalk for helpful discussions and Charles M. Dozois for reading the manuscript. REFERENCES 1. Adachi, V., M. Kashiwazaki, and T. Kume. 1979. Comparison of antigenic properties among various strains of Treponema hyodysenteriae. Zentralbl. Bakteriol. Parasitenkd. Infektiouskr. Hyg. Abt. 1 Orig. Reihe A 245:527-533. 2. Baum, D. H., and L. A. Joens. 1979. Serotypes of betahemolytic Treponeina hyodysenteriae. Infect. Immun. 25:792796. 3. Baum, D. H., and L. A. Joens. 1979. Partial purification of a specific antigen of Treponema hyodysenteriae. Infect. Immun. 26:1211-1213. 4. Bélanger, M., and M. Jacques. 1991. Evaluation of the An-Ident system and an indole spot test for the rapid differentiation of porcine treponemes. J. Clin. Microbiol. 29:1727-1729. 5. Chatfield, S. T., D. S. Fernie, C. Penn, and G. Dougan. 1988. Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponemra innocens. Infect. Immun. 56:1070-1075. 6. Chengappa, M. M., W. H. Fales, R. D. Glock, J. M. Kinyon, R. C. Pottenger, J. G. Songer, and L. J. Hoffman. 1989. Laboratory procedures for diagnosis of swine dysentery. Report of the Committee on Swine Dysentery. American Association of Veterinary Laboratory Diagnosticians, Inc., Columbia, Mo. 7. Glock, R. D., and D. L. Harris. 1972. Swine dysentery. Il. Characterization of lesions in pigs inoculated with Treponema hyodysenteriae in pure and mixed culture. Vet. Med. Small Anim. Clin. 67:65-68. 8. Halter, M. R., and L. A. Joens. 1988. Lipopolysaccharides from Treponema hyodysenteriae and Treponema innocens. Infect. Immun. 56:3152-3156. 9. Hampson, D. J., J. R. L. Mhoma, and B. Combs. 1989. Analysis of lipopolysaccharide antigens of Treponema hyodysenteriae. Epidemiol. Infect. 103:275-284. 10. Harris, D. L. 1974. Current status of research on swine dysentery. J. Am. Vet. Med. Assoc. 164:809-812. 11. Harris, D. L., and R. D. Glock. 1986. Swine dysentery and spirochaetal disease, p. 494-507. Ini A. D. Leman, B. Straw, R. D. Glock, W. L. Mengeling, R. H. C. Penny, and E. Scholl (ed.), Diseases of swine, 6th ed. Iowa State University Press, Ames. 12. 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. 67:61-64. 13. Harris, D. L., and J. M. Kinyon. 1974. Significance of anaerobic spirochetes in the intestines of animals. Am. J. Clin. Nutr. 27:1297-1304. 14. Harris, D. L., J. M. Kinyon, M. T. Mullin, and R. D. Glock. 1972. Isolation and propagation of spirochetes from the colon of swine dysentery affected pigs. Can. J. Comp. Med. 36:74-76. 15. Hudson, M. J., T. J. L. Alexander, and R. J. Lysons. 1976. Diagnosis of swine dysentery: spirochaetes which may be confused with Treponema hyodysenteriae. Vet. Rec. 99:498500.

VOL. 29, 1991 16. Hughes, R., H. J. Olander, D. L. Kanitz, and S. Qureshi. 1977. A study of swine dysentery by immunofluorescence and histology. Vet. Pathol. 14:490-507. 17. Joens, L. A., N. A. Nord, J. M. Kinyon, and I. T. Egan. 1982. Enzyme-linked immunosorbent assay for detection of antibody to Treponemna hyodvsenteriae antigens. J. Clin. Microbiol. 15:249-252. 18. Kinyon, J. M., and D. L. Harris. 1979. Treponema innocens, a new species of intestinal bacteria, and emended description of the type strain of Treponema hvodysenteriae Harris et al. Int. J. Syst. Bacteriol. 29:102-109. 19. Kinyon, J. M., D. L. Harris, and R. D. Glock. 1977. Enteropathogenicity of various isolates of Treponeina hvodvsenteriae. Infect. Immun. 15:638-646. 20. Kinyon, J. M., J. G. Songer, M. Janc, and D. L. Harris. 1976. Isolation and identification of Treponema hyodysenteriae: aid to the diagnosis and treatment of swine dysentery, p. 65-74. Proc. Am. Assoc. Vet. Lab. Diagn.

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21. Mapother, M. E., and L. A. Joens. 1985. New serotypes of Treponema hvodvsenteriae. J. Clin. Microbiol. 22:161-164. 22. Smith, S. C., F. Roddick, S. Ling, N. L. Gerraty, and P. J. Coloe. 1990. Biochemical and immunochemical characterization of strains of Treponema hyodysenteriae. Vet. Microbiol. 24:29-41. 23. Stanton, T. B. (NADC, Ames, Iowa). 1991. Personal communication. 24. Stanton, T. B., N. S. Jensen, T. A. Casey, L. A. Tordoif, F. E. Dewhirst, and B. J. Paster. 1991. Reclassification of Treponema hyodysenteriae and Treponemna innocens in a new genus. Serpula gen. nov., as Serpula hyodvsenteriae comb. nov. and Serpula innocens comb. nov. Int. J. Syst. Bacteriol. 41:50-58. 25. Taylor, D. J., and T. J. L. Alexander. 1971. The production of dysentery in swine by feeding cultures containing a spirochaete. Br. Vet. J. 127:58-61. 26. Wannemuehler, M. J., R. D. Hubbard, and J. M. Greer. 1988. Characterization of the major outer membrane antigens of Treponema hyodysenteriae. Infect. Immun. 56:3032-3039.

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes.

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiff...
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