Comparison of antigenic properties among several strains of weak beta-hemolytic treponeme isolated from swine and a dog

Zbl. Bakr. Hyg., I. Abr. Orig. A 248, 541-547 (1981)

The Na tiona l Institute of Ani ma l H ealth 1 Kyushu Bran ch Lab orato ry, 2702, Chuzan , Kagoshima, 2 Cent ral Laboratory, 1-1, Tsukub a, Ibar aki, 3 Mi yaza ki Livestock H ygiene Service Center, Sadha ra, M iyazaki, Japan

Comparison of Antigenic Properties Among Several Strains of Weak Beta-hemolytic Treponeme Isolated from Swine and a Dog Eine Vergleichsstudie von Antigeneigenschaften bei einigen Stammen von schwach betahamolytischen aus Schweinen und einem Hund isolierten Treponemen YOSH IKAZU ADACH I 1, TSUNEO KUME 2, SHINICHI RO YAMAM OT O 3, and HIR OSHI WAT ASE 1 Wit h 7 Figures' Received M ay 9, 1980

Abstract Agglutinin-absorp tion and pr ecipitin-abso rption studies dem on str ated th at three strains of wea k beta -hemolytic treponeme isolated from cases o f di arrhea o f swi ne and from a clin icall y normal dog wer e ant igenically differe nt from each o ther.

Z usammenfassung Agglut inin-Absorp tions- und Prezipitin -Absorptionsunters uchun gen ha ben gezeigt, daR dr ei Stam me von schwach beta hamolytischen Treponemen, die aus Fall en von Schweinediarrhoe sowie aus eine m klin isch gesu nden Hund isoliert worden war en, unterscheiden sich in ihren Antigeneigenschaften.

Introduction

T he etiological agent of swine dysentery is a spirochete, T reponema byodysenteriae (4, 5, 11). Recently, tw o major classes of T. hyodysenteriae have been reported mainly on th e basis of th e hemolytic activity in blood aga r: beta-hemolytic and weak beta-hemolytic (7). Kinyo n et al. (8) also proposed the terms T . innocens for the weak beta-hemolytic isolates and T . byodysenteriae for th e betahemolytic isolates. [oens et al. (6) repor ted that the agglutinating activity of the antiseru m against

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Y.Adachi, T.Kume, S.Yamamoto, and H .W ata se

the weak beta -hemol ytic isolates used were considerably different in each of the reactions. Lemcke and Barrows (9) showed that a disc growth-inhibition test was useful for differentiating T. byodysentetiae from oth er intestinal spirochetes. Th e tests with antisera against six spirochet es, including two strains of T. byodysenteriae, revealed four serological types among the six strains. On the basis of these results, as mentioned above, the antigenic differences of weak beta-hemolytic strains were further investigated in detail by the agglutination and immunodiffusion tests. Thi s report indicates the stud ies. Materials and Methods Strains and culture. Beta-hem olytic strains S7312 (kindl y supplied by Dr. D.]. Taylor, University of Glasgow , Scotland), and D]70 were isolated from the cases of swine dysenter y. Weak beta-hemolytic strains NS8 and NK2 were isolated from the cases of diarrhea in swine and stra in CFl was isolated from a clinically normal dog. These five strains were used in the studies. The strains were isolated from one colony and then twice passed through the blood agar cont aining 400 ,u g per ml of spectin omycin. Finally they were cloned many times. The biological and morphological properties of strains S7312 and D]70 were consistent with those of T. hyodsenteriae which Harris et al. (5) showed. Also, th e prop erties of stra ins NS8, N K2, and CFl were similar to th ose of T. innocens which Kinyon et al. (8) showe d. Th e organisms were anae rob ically grown on a trypti case soy agar (TSA) (BBL), suppl emented with 5 % sheep or rabb it blood. Hemolytic patterns. Hem olytic patt erns of these trep onem es were investigated on the agar plates containing a final concentration of 5 % blo od from bovine, horse, human, rabbit or sheep. The hemolytic patterns were then judged as previously described (8). Hyperimmune sera. Immune sera were prepared as previously described (1). The cells of those srrains were harv ested in 0.01 M phosphate buffer (pH 7.2), washed twice by the same buffer and adjusted to approximately 1 X 109 cells per ml and injected into a rabbit intravenously at inter vals of 4 days. When the titer of immune serum reached maximu m by agglutination and immunod iffusion tests, the rabbit was bled and the serum was stored at - 20 °C. Agglutination and agglutinin-absorption tests. Th e reaction was observed under a darkfield microscope. Th e results were described with a reciprocal of maxim um dilut ion of the serum to give a 50 % microscopic agglutination of the organism (8). Th e antigens used for the agglutinatio n test were suspended in a 0.01 M ph osphate buffer containing finally 0.3 Ofo form alin a the density of approxi mately 5 X 107 cells. An agglutininabsorption test was carried out as pre viously described (13). The immun e serum was absorbed with packed cells of the wash ed treponeme. T he absorption studies were done for 18 h at 5 ° C after incubation for 2 h at 37 ° C. Preparation of the antigens for immunodiffusion. The cells were harvest ed and suspended in 0.5 Ofo sodium dode cyl sulfate (SDS, Sigma Co.) and then treated in a water bath for 4 h at 45 °C wh ich were read y to use as the ant igen for immunodiffusion (13, 14). Immunodiffusion and precipitin-absorption tests. An immunodiffusion test in gel was carri ed out as follows (13, 14): The aga r gel was pr epared with a 0.01 M phosphate buffer, 1 0f0 agar (Agar N oble, Difco) and 0.01 0f0 thimerosal. T he diamet er of the well was 4 mm and the distance between the antigen and immune serum wells was 3 mm. The precipitin-absorption test in gel was done as follo ws (13, 14): The center well was filled with an ant igen, and after being dried at room temperature, the same antigen was filled again. The immune serum to be absorbed was added to the same well after ant igen dried. The antigens to be reacted with the absorbed immune serum wer e th en added to the peripher al wells. Control was filled with 0.5 % SDS. Preliminary studie s revealed that by test tube precipitin absorption the same results could be obtained as by agar well absorption.

Antigenic Properties of Weak Beta-hemolytic Treponeme

543

Results Hemolytic patterns. All strains isolated from the cases of swine dysentery were beta-hemolytic, whereas several strains, which were isolated from the cases of swine diarrhea and from a clinically normal dog, showed weak beta-hemolysis (Fig. 1). The hemolytic patterns of these strains did not change during subcultures in vitro. The results of the comparison of hemolytic patterns among the several kinds of blood are shown in Table 1. Strain S73/2 showed the complete hemolysis in all cases except in the rabbit. But strains CF1, NK2 and NS8 showed incomplete hemolysis on the blood agar plates of five kinds of animals. A

C

B

D

Fig. 1. Hemolytic patterns of these strains used. A: 573/2, B: CF1, C: NK2, D: N58. Table 1. Comparison of hemolytic activity among these strains used Strain

Human RBC

Sheep RBC

Horse RBC

Bovine RBC

Rabbit RBC

573/2 CFl NK2 NS8

++1 + + +

++ + + +

++ +

++ +

+ +

+ + + +,

+ +

+ + +, incomplete

1 Each symbol represents the results of a test: complete hemolysis, hemolysis. The hemolytic reaction was judged after 2 to 4 days. RBC represents red blood cells.

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Agglutinin-absorption test. The results of this test are shown in Table 2. Cells of each strain reacted with unabsorbed homologous sera at high titer. Cells of each strain also reacted with anti-573/2 serum. The serum absorbed with strain D]70 reacted only with the strain 573/2. The serum absorbed with each of weak beta-hemolytic strains strongly reacted with both strains 573/2 and D]70. Anti-CFl serum reacted with each of weak beta-hemolytic strains used. AntiCFl serum absorbed with heterologous strains reacted only with the strain CF1. Anti-NK2 and N58 sera also reacted with strains D]70 and CF1. These sera absorbed with heterologous weak beta-hemolytic strains strongly reacted with their respective homologous strains. Table 2. Comparison of antigenicity among each strain by agglutinin-absorption test Immune serum against

Absorbed with

S73/2

Unabsorbed S73/2 D]70 CF1 NK2 NS8

CF1

NK2

N58

Agglutination titer with antigen S73/2 D]70 CF1 NK2 4096 1

512

1024 2048 2048 2048

512 512 512

Unabsorbed S73/2 CF1 NK2 NS8

128

128

NS8 128 64

2048 1024

64

64

1024 1024-

Unabsorbed S73/2 CF1 NK2 N58

64

Unabsorbed 573/2 CFl NK2 N58

128

64 64

1024 1024 1024 1024

256 64 64

4096 4096 4096 4096

1 Titers are expressed as 50 Ofo microscopic agglutination end point. Dashes represent negative reactions at 1:64 dilution. Strain CF1 was isolated from the clinically normal dog.

Fig. 4. Immunodiffusion pattern of the anti-CFl serum absorbed with well). The peripheral wells contained the antigens of the three strains. Fig. 5. Immunodiffusion pattern of the anti-Cfl serum absorbed with well). The peripheral wells contained the antigens of the three strains. Fig. 6. Immunodiffusion pattern of the anti-N58 serum absorbed with well). The peripheral wells contained the antigens of the three strains. Fig. 7. Immunodiffusion pattern of the anti-N58 serum absorbed with well). The peripheral wells contained the antigens of the three strains.

CF1 (the central CaNT: Control. N58 (the central CaNT: Control. N58 (the central caNT: Control. CFl (the central caNT: Control.

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2

545

3

Fig. 2. Immunodiffusion pattern of the anti-CF1 serum (the central well). The peripheral wells contained the anitgens of the three strains. CONT: Control. Fig. 3. Immunodiffusion pattern of the anti-NS8 serum (the central well). The peripheral wells contained the antigens of the three strains. CONT: Control.

4

6

5

7

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Immunodiffusion and precipitin-absorption tests in gel. The antigenic properties of these strains were compared by immunodiffusion using SDS-extracted antigens. With the anti-CFl serum, strain CFl formed two lines but the other strains formed no line (Fig. 2). With the anti-NS8 serum, the homologous strain formed about three lines, while strain CF1 formed weakly one line which seemed to be partially identical with the line on antiserum well side produced by strain NS8 (Fig. 3). The results of the precipitin-absorption test in gel were shown in Figs. 4 to 7. Anti-CFl serum absorbed with the homologous strain showed no reaction (Fig. 4) but the antiserum absorbed with strain NS8 reacted only with strain CF1 (Fig. 5). Anti-NS8 serum absorbed with strain NS8 showed no reaction (Fig. 6) but the antiserum absorbed with strain CF1 reacted with strain NS8 (Fig.7). These findings indicate that these strains which showed weak beta-hemolysis may be different from each other.

Discussion The results presented in this report indicated that the strains used showed weak beta-hemolysis, are antigenically different from each other. The antigenicity of these strains were also distinguished from that of beta-hemolytic strains. The immunodiffusion test demonstrated that strain NS8 cross-reacted only with strain CFl. However, the agglutination test indicated that strains CFl, NS8, and NK2 might possess the antigens in common, respectively. These findings indicate that the antiserum which shows higher titer should be used for the investigation of the common antigens in immunodiffusion in gel. In prior reports, Kinyon et al. (7) reported that the weak beta-hemolytic isolates which were isolated from clinically normal swine, postweaning diarrhea of swine, and clinically normal dogs were nonpathogenic and serologically cross-reacted with beta-hemolytic isolates. They also proposed the terms T. innocens for the weak beta-hemolytic isolates and T. hyodysenteriae for the beta-hemolytic isolates (8). Adachi et al. (1) and Baum and Joens (2) showed that the antigenic properties of beta-hemolytic strains used were different from each other and were distinguished from those of the weak beta-hemolytic strains. Baum and Joens (3) also partially purified a specific antigen of T. hyodysenteriae. This antigen may be the same as those of beta-hemolytic strains which was shown by Adachi et al. (1). There have been only two reports on the antigenic differences of weak betahemolytic isolates. Joens et al. (6) showed that the agglutinating activity of the antisera against the weak beta-hemolytic isolates was different among these isolates, respectively. Lemcke and Barrows (9) reported that a disc growth-inhibition test was useful for differentiating T. hvodysenteriae from other intestinal spirochetes. The tests with antisera against six spirochetes, including two strains of T. hyodysenteriae revealed four serological types among the six strains. The two strains of T. hyodysenteriae also represented one type. Then, the antigenic properties of weak beta-hemolytic treponemes were investigated in detail by agglutininabsorption and precipitin-absorption tests. The results showed that these strains used were antigenically different from each other. These results indicate that there may be a great number of antigenically different types of weak beta-hemolytic strains. On the other hand, the genetic relationship among these weak beta-hemelytic strains is stilI unclear.

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547

Miao et al. (10) showed the pathogenic and nonp athogenic isolates were clearly within the same genus (28 (j/o homology) but appear to be distinct species. The reciprocity of the reassociation assays indicated that all of these stra ins had the same size genome. Therefore, we will further investigate the genetic relationships among these weak beta-hemolytic strain s. Acknowledgment. We give thanks to Dr. Suenaga of the Takeda Chemical Industries, Ltd., Osaka, for the supply of can ine strain, and Dr. Takahata of the Daiichi Seiyaku Co., Ltd ., Tokyo, for the supply of weak beta-hemolytic strain isolated from swine.

References 1. Adachi, Y., M. Kashiwazaki, and T. Kume: Comparison of antig enic properties amon g various strains of Treponema hyodysenteriae. Zbl. Bakt. Hyg. , I. Abt. Orig . A 245 (1979) 527-533 2. Baum , D. H. and L. A. Joens: Serot ypes of beta-hemolytic Tr eponema hyodysenteriae. Infect. Immun. 25 (1979) 792-796 3. Baum , D. H. and L. A . [oe s: Partial purification of a specific antigen of Tr eponem a byod ysent eriae. Infect. Immun. 26 (1979) 1211-1213 4. Glock , R. D. and D. L. Harris: Swine dysentery. II. Characte rization of lesion s in pigs inoculated with Tr eponema hyo dysenteriae in pure and mixed culture. Vet. Med. Small Anim, Clin. 67 (1972) 65-68 5. Harris, D. L., R. D. Glock, C. R. Christensen, and ]. M. Kinyon: Swine dysente ry. I. Ino culation of pigs with T reponema hyodyse nte riae (new species) and reproductio n of the disease. Vet. Med . Small Anim. Clin. 67 (1972) 61- 64 6. Joens, L. A., D. L. Harris, J. M. Kinyon, and M. L. Kaeberle: M icrotitration agglutination for dete ction of T reponema hyo dyse nte riae ant ibod y. J. C1in. Microbiol. 8 (1978) 293- 298 7. Kinyon, J. M., D. L. Harris, and R. D. Glock: Enteropathogenicity of var ious isolates of Trep onema hyodysenteriae. Infect. Immun. 15 (1977) 638-646 8. Kinyon, J. M. and D. L. Harris: Trepo nema innocens, a new species of intestinal bacterial, and emend ed description of the type strain of T reponema hyodysent eriae Harris et al. Int. J. system. Bact. 29 (1979) 102-10 9 9. Lemcke, R. M . and M . R. Barrows: A disc growth-inhibition test for different atin g T repo nema byo dysen teriae from other intes tinal spiro chaetes. Vet. Rec. 104 (1979) 548-551 10. Miao, R. M., A. H. Fieldsteel, and D. L. Harris: Geneti cs of Tr eptonema: Characterization of Trepon ema hyodysenteriae and its relationship to Treponema pallidum . Infect. Immun. 22 (1978) 736-739 11. T aylor, D. J. and T. J. L. Alexander: The production of dysentery in swine by feeding cultures conta ining a spirochae te. Brit. vet. ]. 127 (1971) 58-61 12. W orld Health Organ ization: Current probl ems in lept ospiro sis resear ch. Report of a WHO expe rt group. WId. Hlth O rg. Te chn. Rep. Ser. 380 (1967) 13. Ya nagauia, R. and Y. Adachi: Ident ification of some Japanese leptospiral strains as serotypes copenhagen! and icterohaemorrhagiae by pr ecipitin- absorption test in gel. Zbl. Bakt. Hyg., I. Abr. Orig. A 237 (1977) 96-103 14. Yanagawa, R., M . Shin agauia, and I. T ak ashim a: Serological studies of lepto spiras by immunod iffusion. Zbl. Bakt. Hyg., I. Abt. O rig. A 228 (1974) 369-377 Y. Adachi, T he Natio nal Institute of Animal Health, Kyushu Bran ch Laboratory, 2702, Chu zan, Kagoshima , 891- 01, Jap an