Presence of Vi antigen in a virulent strain of Leptospira interrogans serovar pomona and relation of Vi antigens of Leptospiras to resistance to leptospiricidal activity mediated by antiserum plus complement

Zbl. Bakt. Hyg., I. Abt. Orig. A 252, 557-565 (1982) Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan (Head: Prof. Dr. R. Yanagawa)

Presence of Vi Antigen in a Virulent Strain of Leptospira interrogans Serovar pomona and Relation of Vi Antigens of Leptospiras to Resistance to Leptospiricidal Activity Mediated by Antiserum plus Complement Vorkommen von Vi-Antigenen in einem virulenten Stamm von Leptospira interrogans Serovar pomona und das Verhalrnis des Vi-Antigens von Leptospiren in bezug auf die durch Antiserum plus Komplement vermittelte leptospirizide Aktivitat KOUKO DENO, RYO YANAGAWA, and HIROSHI KIDA

With 5 Figures' Received March 25, 1982

Abstract Vi antigen was found in a virulent strain of Leptospira interrogans serovar pomona. The presence of Vi antigen was shown by the agglutinin-absorption and precipitin-absorption tests. Each strain of pomona and copenhageni which possessed Vi antigen and was resistant to the leptospiricidal activity mediated by the antiserum plus complement lost Vi antigen and became susceptible to the leptospiricidal activity when cultured in the presence of 8-azaguanine. The strains thus treated with 8-azaguanine recovered the resistance and Vi antigen when cultured in medium without 8-azaguanine.

Zusammenfassung In einem virulenten Stamm von Leptospira interrogans Serovar pomona wurde ein ViAntigen festgestellt, das mit Hilfe des Agglutinin- sowie des Prazipitin-Absorptionstestes nachgewiesen werden konnte. Die Vi-Antigen aufweisenden und gegen die durch Antiserum plus KompIement vermittelte leptospirizide Aktivitat resistenten pomona- und copenhageniStarnme verIoren ihr Vi-Antigen und wurden gegen Ieptospirizide Aktivitat empfindlich, wenn sie in Gegenwart von 8-Azaguanin kultiviert wurden. Die mit 8-Azaguanin behandeIten Stamme erlangten ihre Resistenz und ihr Vi-Antigen dann wieder, wenn sie in Nahrboden ohne 8-Azaguanin gcziichter wurden.

558

K. Dena, R. Yanagawa, and H. Kida

Introduction Vi antigen of leptospiras has been reported to be present in the virulent strains belonging solely to serogroup Icterohaemorrhagiae (5). Vi antigen has been inactivated by heating at 56 DC for 5-10 min, and it has been detected by the agglutininabsorption test (5). In the course of the studies of leptospiricidal activity tests mediated by antiserum plus complement, we noticed that Vi antigen was found in a strain belonging to serogroup Pomona. The presence of Vi antigen was shown also by the precipitin-absorption test. These findings are described in the present communication, which also describes the 8-azaguanine treatment used, which resulted in the decrease of resistance to the leptospiricidal activity mediated by antiserum plus complement and the decrease of Vi antigen.

Material and Methods Strains: Leptospira interrogans serovar pomona strain MLS, which is virulent and hamster lethal, and strain Pomona, which is avirulent, were used. Pomona MLS was passaged through hamsters twice a year. Two lines of Leptospira interrogans serovar copenbageni strain Shibaura with different histories of passage (16) were also used. Line No.1, the virulent line, was passaged through guinea pigs every 2 months and was found to be hamster and guinea pig lethal (LD5 0 was 104 cells/ml in hamsters). Line No.2, the moderately virulent line, was passaged through guinea pigs once a year and found to cause leptospiremia in guinea pigs (LD5 0 was> 108 cells/ml in hamsters). These strains were cultured in the serum medium at 30 DC for 5 to 7 days. Serum medium: 0.2% tryptose phosphate broth (Difco) containing 10% normal rabbit serum and a small amount of phenol red as the pH indicator were used as the serum medium (13). Immune sera: Each rabbit was hyperimmunized with pomona MLS, pomona Pomona and two lines of copenhageni Shibaura following the method described previously (15). Complement: Pooled normal guinea pig serum used as complement was obtained from adult guinea pigs. Examination of Vi antigen of leptospiras: The presence of Vi antigen was examined following the method described by Kmety (5). The ,agglutinin titers of antiserum absorbed with heated homologous leptospiras (56 in 10 min) and living homologous leptospiras were examined. Microscopic agglutination test and agglutinin-absorption test: These tests were done as described previously (2, 6). Immunodiffusion and precipitin-absorption test: Sodium dodecyl sulfate (SDS)-extracted antigen (1 x 108 organisms/ml) was prepared following the method described previously (15). Immunodiffusion and precipitin-absorption tests were conducted as described previously (14, 15). Immunoelectrophoresis: The antigens in the SDS-extracts were examined by rocket immunoelectrophoresis following the method described in an earlier report (11). Leptospiricidal activity test: The test was done as described previously (12).Homologous antiserum was used for the test. Titers were expressed as more than 90% of lysis. Cultivation of virulent leptospiras in presence of 8-azaguanine: 8-azaguanine was added to the serum medium: The final concentration of 8-azaguanine in the growth tubes varied from 62.5 to 1000 ,ug/ml, which were the subinhibitory concentrations (4). The virulent strain (an initial cell count of approximately 1 X 107 celIs/mil was cultured in the medium at 30°C for 5 to 7 days. Growth was measured every 2 days by counting the number of leptospiras per ml under darkfield microscope (X 150). DC

Leptospira interrogans Serovar pomona

559

Results Virulent MLS and avirulent Pomona were not antigenically different in usual qualitative and quantitative serological examinations, such as cross agglutination tests and immunodifiusion (Fig. 1 and 2) and rocket immunoelectrophoresis (Fig.3). Anti-MLS antiserum absorbed with Pomona agglutinated MLS at 1:600 while the anti-Pomona antiserum which absorbed with MLS did not agglutinate Pomona (Table 1). The findings indicated that virulent pomona MLS possesses an additional antigen which was not present in avirulent pomona Pomona. Anti-MLS antiserum absorbed with heated MLS (56°C for 10 min) agglutineted MLS at 1: 1200, but

Fig. 1

Fig. 2

Fig. 1. Immunodiffusion pattern of anti-MLS antiserum (MLSS) with MLS antigen (MLS), Pomona antigen (POM) and 8-azaguanine (250 ,Ltg/ml)-treated MLS antigen (8-AZA. 250 MLS). Fig. 2. Immunodiffusion pattern of anti-Pomona antiserum (POMS) with Pomona antigen (POM) and MLS antigen (MLS).

Fig. 3. Rocket immunoelectrophoresis against anti-MLS antiserum of antigens MLS (MLS), Pomona (POM) and 8-azaguanine (250 ,Ltg/ml)-treated MLS (8-AZA. 250 MLS). 36 ZbI. Bakt. Hyg., I. Abt. Orig. A 252

560

K.Ueno, R. Yanagawa, and H. Kida

Pomona did not. Anti-Pomona antiserum absorbed with heated Pomona (56°C for 10 min) but did not agglutinate Pomona. These indicate that MLS has a heat-labile antigen and that Pomona has no such antigen (Table 1). Whether or not anti-MLS antiserum absorbed with Pomona agglutinate heated MLS was not examined successfully, because the leptospiras were destroyed by heating.

Table 1. Agglutinin-absorption test using pomona strain MLS and Pomona Antiserum

Agglutinin titers with antigens

Against

Absorbed with

MLS

Pomona

pomona MLS (Virulent)

Unabsorbed MLS Pomona Heated" MLS 8-azaguanine d -treated MLS

12800

6400

12800

_b

_b

Unabsorbed Pomona MLS Heated Pomona

6400

6400

6400

pomona Pomona (Avirulent)

a c d

8-azaguanine d -treated MLS

_&

600 1200 1200

< 150 except b which is < 300 56°C for 10 min 250,ug/ml

Eg.4

Fig. 5

Fig. 4. Precipitin-absorption test in gel. The central well contained anti-MLS antiserum 1: 2 absorbed with Pomona antigen (MLSS-POM). The peripheral wells contained MLS antigen (MLS), MLS antigens heated at 50°C 10 min (MLS 50' 10'), 56°C 5 min (MLS 56· 5') 56°C 10 min (MLS 56' 10') and 56°C 15 min (MLS 56 . 15') and Pomona antigen (POM). Fig. 5. Precipitin-absorption test in gel using anti-Pomona antiserum 1: 2 absorbed with MLS (POMS-MLS) with antigens Pomona (POM) and MLS (MLS).

Leptospira interrogans Serovar pomona

561

Whether MLS possesses Vi antigen was examined by the precipitin-absorption test (Fig. 4). Anti-MLS antiserum absorbed with the Pomona antigen produced a precipitin line against the MLS antigen. The line was diminished progressively when MLS antigen was heated from 50 °c for 5 min to 56°C for 10 min and was no longer detectable when it was heated at 56 °C for 15 min. Anti-Pomona antiserum absorbed with MLS antigen did not produce any precipitin line against Pomona antigen (Fig.5). Anti-MLS antiserum absorbed with heated MLS antigen produced a precipitin line against MLS antigen but not against Pomona antigen. These findings indicate that the additional antigen of virulent MLS is heat-labile and accordingly Vi antigen. We attempted to show the presence of Vi antigen in two lines of copenhageni Shibaura, virulent and moderately virulent. As shown in Table 2, both lines possessed Vi antigen. Virulent line No.1 had more Vi antigen than moderately virulent line No.2 as the agglutinin titer of anti-line No.1 antiserum absorbed with heated line No.1 was 1: 800 while the agglutinin titer of anti-line No.2 antiserum absorbed with heated line No.2 was 1 :200.

Table 2. Agglutinin-absorption test using two lines of copenhageni Shibaura Agglutinin titers with antigens

Antiserum Against

Absorbed with

No.1

No.2

Unabsorbed No.1 No.2 Heated" No.1

12800

12800

400 800

800

Unabsorbed No.2 No.1 Heated No.2

12800

12800

200

200

copenhageni Shibaura Line No.1 (Virulent)

Line No.2 (Moderately virulent)

a b

_a

< 200 56°C for 10 min

The sensitivity of the virulent, moderately virulent and avirulent strains to the leptospiricidal activity mediated by the homologous antiserum plus complement was examined (Table 3). Virulent pomona MLS and copenhageni Shibaura line No.1, both possessing Vi antigen, were resistant to the leptospiricidal activity. Avirulent pomona Pomona, which did not possess Vi antigen, and moderately virulent copenhageni Shibaura line No.2, which showed a small amount of Vi antigen, were sensitive to the leptospiricidal activity. Pomona MLS and copenhageni Shibaura line No.1 were changed from resistant to sensitive to the leptospiricidal activity when they were cultured in the presence of 125,ug/ml or more of 8-azaguanine (Table 4).

562

K. Ueno, R. Yanagawa, and H. Kida

Table 3. Resistance of leptospiras to leptospiricidal activity (LA) mediated by homologous antiserum plus complement Strains or lines

Presence of Vi antigen

Resistant or sensitive

pomona MLS (Virulent) pomona Pomona (Avirulent) copenhageni Shibauta Line No.1 (Virulent) Line No.2 (Moderately virulent)

+ (1200-)

Resistant «

to

LA

100 b)

Sensitive (16000)

+

Resistant «

(800)

+ (200)

100)

Sensitive (16000)

a Agglutinin titer of anti-Vi antiserum, prepared by absorption with heated homologous leptospiras. b Titer of homologous antiserum caused 90% leptospirolysis.

Table 4. Agglutinin-absorption test and resistance to leptospiricidal activity (LA) mediated by antiserum plus complement of virulent strains grown in presence of 8-azaguanine Strains

pomona MLS (Virulent)

copenbageni Shibaura line No.1 (Virulent)

Concentration LA titers with of 8-azaguanine homologous antiserum (fig/ml) caused 90% leptospirolysis

62.5 125 250 500 1000 62.5 125 250 500 1000

< 100 < 100

> >

8000 16000 32000 32000

< 100 < 100 8000

> 32000 > 32000 > 32000

Resistant or sensitive to LA

Agglutinin titers against anti-Vi antiserum prepared by absorbing homologous antiserum- with Avirulent or moderately virulent leptospiras"

Resistant Resistant Sensitive Sensitive Sensitive Sensitive

600 600 150

Resistant Resistant Sensitive Sensitive Sensitive Sensitive

400 400 200

Heated virulent [eptospiras? 1200 1200 150

_b

800 800 200

_c

a Agglutinin titers against the unabsorbed antisera of 8-azaguanine-treated pomona MLS and copenbageni Shibaura line No.1 were 6400-12800. b < 150 c < 200 d Anti-MLS antiserum was absorbed with avirulent Pomona. Anti-line No.1 was absorbed with moderately virulent line No.2. e Anti-MLS antiserum was absorbed with heated MLS. Anti-line No.1 was absorbed with heated line No. 1.

Leptospira interrogans Serovar pomona

563

MLS grown in the absence of 8-azaguanine and MLS grown in the presence of 62.5,Ltg/ml of 8-azaguanine were agglutinated, and MLS grown in the presence of 125,Ltg/ml of 8-azaguanine was only partially agglutinated. Also, MLS grown in the presence of 250,Ltg/ml or more of 8-azaguanine was not agglutinated by anti-Vi antiserum (anti-Ml.S antiserum absorbed either with avirulent Pomona or with heated MLS) (Table 4). Resistance to leptospiricidal activity in parallel with the agglutinability with anti-Vi antiserum decreased in inverse proportion to the concentration of 8-azaguanine in the medium. Anti-MLS antiserum absorbed with MLS grown in the presence of 8-azaguanine (250,Ltg/ml) agglutinated MLS at 1: 1200 but did not agglutinate Pomona (Table 1). This result along with those from the immunodiffusion (Fig. 1) and rocket immunoelectrophoresis (Fig. 3) tests using the antigens of leptospiras grown in the presence of 8-azaguanine indicated that the antigen which decreased in the presence of 8-azaguanine was Vi antigen. Copenbageni Shibaura line No.1 grown in the absence of 8-azaguanine and line No.1 grown in the presence of 62.5 ,Ltg/ml of 8-azaguanine were agglutinated; line No. 1 grown in the presence of 125,Ltg/ml of 8-azaguanine was partially agglutinated; and line No.1 grown in the presence of 250,Ltg/ml or more of 8-azaguanine were not agglutinated by anti-Vi antiserum (anti-line No.1 antiserum absorbed either with moderately virulent line No.2 or with heated line No.1) (Table 4). Resistance to leptospiricidal activity and agglutinability with anti-Vi antiserum decreased in inverse proportion to the concentration of 8-azaguanine, as in the case of virulent pomona MLS. The 8-azaguanine(250 ,Ltg/ml)-treated virulent pomona MLS and copenhageni Shibaura line No.1 recovered resistance to leptospiricidal activity and Vi antigen when cultured in the medium without 8-azaguanine.

Discussion Vi antigen of leptospiras, which has been reported to be present in virulent strains belonging solely to serogroup Icterohaemorrhagiae (5), was found in the present study in virulent strain MLS of pomona belonging to serogroup Pomona. Distribution of Vi antigen of leptospiras was thus extended to at least two serogroups. The virulent strains possessing Vi antigen were resistant to the leptospiricidal activity mediated by the antiserum plus complement, while the avirulent strains possessing no Vi antigen were sensitive to the leptospiricidal activity. The virulent strains changed from resistant to sensitive to the leptospiricidal activity in parallel with the decrease of Vi antigen by cultivation in a medium containing 8-azaguanine. The 8azaguanine-treated virulent strains recovered resistance to the leptospiricidal activity and Vi antigen by cultivation in a medium containing no 8-azaguanine. Resistance to the leptospiricidal activity seems to be intimately related to the presence of Vi antigen. Only virulent leptospiras have been reported to resist the leptospiricidal activity mediated by the antiserum plus complement (4). The mechanism of the resistance is not yet clear. The present experiments showed that Vi antigen may be a factor in the resistance to the leptospiricidal activity. Distribution of Vi antigen is, however, limited to members of Icterohaemorrhagiae and Pomona serogroups. Virulent

564

K. Ueno, R. Yanagawa, and H. Kida

leptospiras belonging to the other serogroups might have an antigen resembling Vi antigen; this question is a subject for future study. Reports have been published on the mechanism of the resistance to the complement-dependent killing in Gram-negative bacteria (3,7,8,9,10). In Escherichia coli and Neisseria gonorrhoae it was shown that the production of an outer membrane protein stimulated an increased resistance to normal serum killing (3, 7, 8, 9). Another resistance mechanism was shown in Neisseria gonorrhoeae to be an external structure to coat the outer membrane, for example, the capsule (10). Location of Vi antigen was not examined in the present study. It is clear, however, that Vi antigen is detectable by the agglutinin-absorption test and that Vi antigen has an intimate relationship with the resistance to the leptospiricidal activity. The outer sheath of leptospiras was reported to be the primary target of complement (1). Vi antigen, therefore, must be located on the leptospiral surface. Detection of Vi antigen has been successful only by absorption tests, either agglutinin-absorption or precipitin-absorption. This may indicate that Vi antigen is not easily separable from the remaining surface antigens. Avirulent variants of pomona MLS and copenhageni Shibaura, which possess antigens identical with those of each homologous parent, except Vi antigen, have not yet been available. Use of such variants will facilitate studies of Vi antigen.

References 1. Anderson, D.L. and R. C.Johnson: Electron microscopy of immune disruption of leptospires: Action of complement and lysozyme. J. Bact. 95 (1968) 2293-2309 2. Dikken, H. and E. Kmety: Serological typing methods of leptospires. In: Methods in Microbiology, vol. 11, pp. 260-294 (T.Bergan and J.R.Norris, ed). Academic Press, London (1978) 3. Hildebrandt, J.F., L. W. Mayer, S.P. Wang, and T. M. Buchanam : Neisseria gonorrhoeae acquire a new principal outer-membrane protein when transformed to resistance to serum bactericidal activity. Infect. Immun. 20 (1978) 267-273 4. Johnson, R. C. and V.G. Harris: Antileptospiral activity of serum II. Leptospiricidal virulence factor. J. Bact. 93 (1967) 513-519 5. Kmety, E.: Uber ein Vi-Antigen bei Leptospiren. Zbl. Bakt. Hyg., I.Abt. Orig. A 221 (1972) 343-351 6. Kmety, E., M. M. Galton, and C.R. Sulzer: Further standardization of the agglutininabsorption test in the serology of leptospires. Bull. WId Hlth Org. 42 (1970) 733-738 7. Lambden, P.R., J.E.Heckels, L. T.James, and P.J. Watt: Variation in surface protein composition associated with virulence protein properties in opacity types of Neisseria gonorrhoeae. J. gen. Microbiol. 114 (1979) 305-312 8. Moll, A., P.A.Manning, and K.N. Timnis: Plasmid-determined resistance to serum bactericidal activity: A major outer membrane protein, the tra T gene product, is responsible for plasmid-specified serum resistance in Escherichia coli. Infect. Irnmun. 28 (1980) 359-367 9. Ogata, R. T. and R. P.Levine: Characterization of complement resistance in Escherichia coli conferred by the antibiotic resistance plasmid R100. J. Immunol. 125 (1980) 14941498 10. Richardson, R. P. and J. C. Sadoff: production of a capsule by Neisseria gonorrhoeae. Infect. Immun. 15 (1976) 663-664 11. Shimono, E. and R. Yanagatoa: Unstable antigenic variation of leptospiras. Zbl. Bakt. Hyg., I.Abt. Orig. A 249 (1981) 133-141

Leptospira interrogans Serovar pomona

565

12. Veno, K. and R. Yanagawa: Antigenic variants of leptospiras selected by antiserumcomplement mediated-killing. Zbl. Bakt. Hyg., I.Abt. Orig. A 248 (1981) 548-557 13. Yanagaioa, R. and Y. Adachi: Identification of some Japanese leptospiral strains as serotypes copenhageni and icterohaemorrhagiae by precipitin-absorption test in gel. Zbl. Bakt. Hyg., I.Abt. Orig. A 237 (1977) 96-103 14. Yanagawa, R. and E. Shimono: Isolation of antigenic variants from leptospiras grown in vitro and from heart blood of guinea pigs inoculated with a clonized strain of leptospira. Zbl. Bakt. Hyg., I.Abt. Orig. A 245 (1979) 345-355 15. Yanagawa, R., M.Shinagawa, and I. Takashima: Serological studies of leptospiras by immunodiffusion. Zbl. Bakt. Hyg., I.Abt. Orig. A 228 (1974) 369-377 16. Yepez, W. P., R. Yanagau/a, and K. Ueno : Correlation of virulence, susceptibility to leptospiricidal activity test mediated by antiserum plus complement and colonial morphology of five lines of a strain of Leptospira interrogans serovar copenbageni. Zbl. Bakt. Hyg., I.Abt. Orig. A (in press) Prof. Dr. Ryo Yanagaioa, Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan