Comparative classification of leptospira serovars of the Pomona group by monoclonal antibodies and restriction-endonuclease analysis

Zbl. Bakt. Hyg. A 266,412-421 (1987)

Comparative Classification of Leptospira Serovars of the Pomona Group by Monoclonal Antibodies and Restriction-Endonuclease Analysis W. J. TERPSTRA, H. KORVER, G. J. SCHOONE, J. v. LEEUWEN, C. E. SCHONEMANN, S. DE JONGE-AGLIBUT, and A. H. J. KOLK N. H. Swellengrebel Laboratory of Tropical Hygiene and WHO/FAO Collaborating Centre for Reference and Research on Leptospirosis, Royal Tropical Institute, Amsterdam, The Netherlands

With 2 Figures· Received December 10, 1986 . Accepted February 16, 1987

Summary The serovars of the Pomona group of Leptospira interrogans are antigenically closely related and can be classified only with difficulty by conventional typing methods. Monoclonal antibodies (MCAs) were prepared to serovars of the Pomona group. The MCAs were directed against antigens of polysaccharide nature. A battery of six MCAs was selected for the classification of Pomona group reference strains. These MCAs could be used for the typing of all Pomona group strains and unknown isolates. Alternatively, DNA was extracted from the same strains and isolates and digested with restriction enzymes. The patterns that were obtained after gel separation of the DNA digests were characteristic and also allowed classification. Restriction enzyme analysis was complicated but gave detailed information. Classification with MCAs could be easily and rapidly performed. Zusammenfassung Die Serovare der Pomonagruppe der Leptospira interrogans besitzen eine enge Antigenverwandtschaft. Daher lassen sie sich mit den iiblichen Methoden nur schwer typisieren. In der vorliegenden Arbeit wurde eine Differenzierung mit monoklonalen Antikorpern versucht. Mit 6 monoklonalen Antikorpern konnten die Referenzstiimme klassifiziert werden. Es gelang damit auch die Typisierung einiger bekannter, neu isolierter Stiimme. Dariiber hinaus wurde eine DNA-Analyse mit Restriktionsenzymen durchgefuhrt, Die damit erhaltenen Muster waren auch fur eine Klassifizierung geeignet. Das Verfahren ist jedoch kompliziert. Die Klassifizierung mit monoklonalen Antikorpern kann dagegen einfach und schnell vorgenommen werden. Introduction The classification of Pomona group strains of Leptospira interrogans has always been difficult. The close antigenic relationship between various strains such as Pomona

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(pomona), LTl026 (kennewick i), Monjakov (monjakov) and 5621 (mozdok) makes it difficult to distinguish the different serovars (Hathaway et al., 1985a, 1985b). The work of Kmety (1967) and Manev (1976) on the analysis of antigenic factors has led to a better understanding of the antigenic structure of the various serovars. However, one of the problems with factor analysis is that of standardizing different batches of factor sera. Recent developments in technology provide two other methods for classification of leptospira serovars; monoclonal antibodies (MCAs) for the recognition of different surface antigens (Terpstra et al., 1985) and the analysis of the leptospiral DNA by restriction enzyme digestion (Marshall et al., 1981; Thiermann et al., 1985, 1986). In this report, we describe the production of MCAs to Pomona group serovars and their use in the classification of laboratory reference strains and field isolates. Further, we compare the classification of Pomona group strains using MCAs with the results of restriction enzyme analysis.

Materials and Methods Leptospira strains

All currently recognized leptospira reference strains and several former reference strains were examined together with a variety of Pomona group isolates from different parts of the world which had been sent to our laboratory for examination and identification. The following reference strains were examined; serovar pomona strain Pomona, former serovar monjakov strain Monjakov, former serovar kennewicki strain LT 1026, serovar tsaratsova strain B 81/7, serovar mozdok strain 5621, former serovar dania strain P 26, serovar tropica strain CZ 299, serovar proechimys strain 1161 K and serovar butembo strain Butembo. The following field isolates were examined: a. strain]T 12 was isolated from cattle in Surabaya and strains] 1,] 2,] 24, U 18, U 22, U 23 were isolated from pigs in Solo (Java). The strains were submitted by Dr. Scott-Orr, Balai Penelitian Hewan, Bogor, Indonesia. b. strain Ukkel 60 was isolated from a pig and was submitted by Dr. Desmecht, Nationaal Instituut voor Diergeneeskundig Onderzoek, Brussels, Belgium. c. strains LC 80/20 and G 129 were isolated respectively from a calf and a vole and submitted by Dr. Hathaway, Central Veterinary Laboratory, New Haw, Weybridge, United Kingdom. d. strain c 374 was isolated from a pig and submitted by Centro de Investigaciones "Miguel C. Rubino", Montevideo, Uruguay. Production of monoclonal antibodies

BALBlc mice were immunized against Pomona serogroup reference strains by injecting live leptospires cultured in EM]H medium. All mice received one intraperitoneal injection of 0.5 mllive leptospires in culture medium and a second booster injection 3--4 days before the fusion (Terpstra et al., 1985). The spleen cells were fused with mouse myeloma cells SP 2/0Ag-14 according to standard techniques (Fazekas de St. Groth and Scheidegger, 1980; Galfre and Milstein, 1981; Kohler and Milstein, 1975; Kolk et al., 1984). The resulting hybridomas were screened by the modified microscopic agglutination test (MAT) as described below. Hybridomas secreting antibodies of the desired specificity were cloned three times by limiting dilution and cultured in the peritoneal cavities of pristane-primed BALB/c mice and ascites were harvested from these mice. The titres of the MCAs in the ascites were determined by the conventional MAT. The immunoglobulin class of each MCA was determined with specific goat antisera (Nordic Diagnostics, The Netherlands) by double diffusion in gels.

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The following serovars were used as the individual immunogen in four different fusions:

pomona, fusion 43 (F43); proechimys, F46; tropica, F48 and mozdok, F58. Antibodies to the homologous serovars and to other Pomona group serovars were produced.

Microscopic agglutination test a. Modified MA T for the selection of hybridomas. Hybridomas were tested by mixing 10 1-11 of culture supernatant with an equal volume of live leptospires at a concentration of 2 X 10~ per ml culture medium (EMJH) in the wells of Terasaki microtitre plates (Greiner, FRG). After 2-4 h incubation, the plates were examined under a dark field microscope using a long distance objective at a total magnification of 160 x. A positive reaction was indicated by the presence of agglutinated leptospires. The screening and selection was based on the five recognized Pomona serogroup reference strains: mozdok 5621, pomona Pomona, proechimys 1161 K, tropica CZ 299 and tsaratsova B8117. Hybridomas secreting MCAs with a desired specificity were selected. Criteria for desired specificity were reactivity with a serovar-specific antigen or reactivity with an antigenic determinant that is part of the characteristic antigenic mosaic of a certain serovar.

b. Conventional MAT for titration of MCAs in ascites and typing of unknown leptospires. Serial twofold dilutions in PBS pH 7.2 were made of each ascites. Fifty 1-11 of this diluted ascites was placed in each well of a microtitre plate, to which was added an equal volume of live leptospires at a concentration of 5-8 X 10 8 per ml culture medium (EMJH). After 2-4 h incubation the agglutination titre was determined under the darkfield microscope according to standard methods (Wolff, 1957). Apart from Pomona group strains, the MCAs were tested with reference strains of the following serovars to test for possible cross- reacti vity: australis and bratislava (Australis); autumnalis and rachmat (Autumnalis); ballum (Ballum); bataviae (Bataviae); carlos (Bufonis); butembo (Butembo); canicola (Canicola); celledoni (Celledoni); cynopteri (Cynopteri); djasiman(Djasiman); grippotyphosa (Grippotyphosa); hebdomadis (Hebdomadis); huanuco (Huanuco); icterohaemorrhagiae and copenhageni (Icterohaemorrhagiae); poi (Javanica); kenya (Kenya); louisiana (Louisiana); manhao (Manhao); mini (Mini); panama (Panama); pyrogenes (Pyrogenes); ranarum (Ranarurn); hardjo (Sejroe): shermant (Shermani); tarassovi (Tarassovi).

Characterization of MCAs by sodiumdodecylsulphate polyacrylamidegel electrophoresis (SDS-PACE) and SDS-PACE immunoperoxidase assay (SCIP) Leptospires grown in EMJH medium were the source of the antigens of pomona, mozdok, proechimys and tropica. Cultures were centrifuged and the pellets sonicated and treated with 5 % mercaptoethanol and 2°;\, sodium dodecyl sulphate at 100°C for 5 minutes. The antigens thus prepared were separated by polyacrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (1970). The gels were stained for protein by coomassie brilliant blue and for glycoconjugates by the silver staining method (Tsai and Frasch, 1982). The SGIP analysis was done according to Klatser (1984). Briefly, leptospira antigens were first separated by SDS-PAGE. The gels were then sliced at -30°C with a freeze microtome and the slices were incubated with MCAs and, after appropriate washings. with anti-mouse peroxidase conjugate (Sigma, USA). Antigen-antibody complexes were visualized by incubation with tetra methyl benzidine.

Biochemical characterization of leptospiral antigens reacting with MCAs Sonicated leptospires were suspended in Tris-HCl buffer pH 8.0 containing 2 mM calcium chloride at a concentration of 2.0 mg leptospires/ml. Pronase (Boehringer, FRG) was then added (3% dry weight) with a further addition at 24 h. Toluene was added to suppress

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bacterial growth. After 48 h incubation at room temperature the mixture was heated for 10 min at 100°C to inactivate pronase. Leptospiral antigens were deglycosylated by treatment of sonicated leptospires with trifluoromethane suiphonic acid (TFMS) (Edge et al., 1981). Restriction endonuclease analysis of leptospiral DNA

a. Purification of DNA. DNA was extracted and purified as described by Marshall et al. (1981), except that the lysate was treated with RNase (100 ug/ml) at 3rC for 30 min. DNA concentrations were determined spectrophotometrically. b. Digestion of DNA. One I-Ig DNA was digested to completion in 2 h with 2 U of EcoR 1 (Boehringer, FRG) in 10 1-11 of 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl b pH 8.0. Digests were electrophoresed at 25 V for 18 h in 40 mM Tris-acetate (pH 7.7) buffer containing 20 mM sodium acetate, 2 mM sodium-EDTA, 1% agarose and ethidium bromide 1 I-Ig/ml. The gel was 19 x 13 ern and 5 mm thick. The gels were photographed with an UV transilluminator in combination with a Kodak Wratten nr. 9 blocking filter and a Polaroid camera using Tri-X Kodak-film.

Results a. Monoclonal antibodies

A battery of six MCAs was selected for the classification of Pomona group strains, which were tested at least twice with the MCA panel in the conventional MAT. The resulting agglutination profiles of all of these strains are presented in Table 1. MCAs, F48C6 and F43C9 reacted with all Pomona group strains but only at a low titre with some strains. These two antibodies delineated a group of pomona-like strains. Pomona, monjakov and kennewicki reacted very similarly, but monjakov had a somewhat lower titre with F48C6. Most isolates from Indonesia and the one from Uruguay had higher tit res with F43C9 than the reference strain. The other Pomona group strains were characterized by their reaction with MCAs F46C9 and F58Cl. Tsaratsova, tropica and proechimys could each be separated from a mozdok-like group of strains by the different pattern of titres. Within the mozdoklike group, mozdok reacted almost similarly to dania but G 129 and LC 80/20 differed by reacting at a lower titre with F58C I. The Belgian isolate Ukkel 60 differed mainly from mozdok by its reaction at a high titre with MCA F46C2. Tsaratsova resembled the mozdok-like strains but differed by reacting at a very high titre with MCA F43C9. Reference strain tropica was characterized by a reaction with MCA F48C3, the only MCA that was serovar-specific, and it was the only strain that reacted only weakly with the group specific MCA F48C6. The main feature of the agglutination profile of proechimys was the very high titre with MCA F46C2. Butembo was the only serovar outside the Pomona group that reacted with MCAs: F48C6 and F46C9. The results of the characterization of MCAs by SDS-PAGE and SGIP are given in Fig. 1. The reaction patterns of the MCAs with the different serovars in the SGIP assay were the same as for the agglutination. MeA F48C3 reacted with tropica only (Fig. lc; Data of other MCAs not shown). All six MCAs reacted with a diffuse zone in the region of 25 to 67 kD proteins. In the SDS-PAGE the polysaccharide bands of the Pomona group were found in the region 14 to 35 kD after silver staining (Fig. Ib). The reaction of MCA F48C3 was abolished after deglycosylation with TFMS but not after pronase digestion (Fig. ] d). SImilarly the other MCAs failed to react with their respec-

1280-25 60

CZ 299

1161 K

Butembo

tropi ca proechimys

but emb o

- = less th an 1 :20

Ig class

40

B 81/7

tsa ratso va

M

1280-2560

2560

2560 5120 2560 2560- 5 120 5 120- 10240

5621 P 26 G 129 LC 80/20 Ukkel 60

mozdok dan ia Isolate U. K. Isolat e U. K. Isolate Belgium

1280-2560 320-640 1280- 2560 1280- 5120 1280 2560

F48C6

Pomona Monjakov LT 1026 J and U stra ins (7 x ) JT 12 C 374

- -- - ------,------.

Strain

pom on a monjakov kennewicki Isolates Indonesia Isolate Indon esia Isolate Urug uay

Serova r

G3

256 0-5120

G3

5 120- 10240

10240

20480

25 60-5 120 10240-20480 10240 10240 20480

40

1280

10240

20-40 320 320 160- 320 160- 32 0

80-160 80-160 160- 320 160- 5120 640 640

Mon oclon al Antibodies F43C9 F46C9

G1

40 96 0

640-1280

81920

20480- 40960 4096 0 2560 640 8192 0

F58C1

Table I. Agglutina tio n titres of MCA s with Pomo na serogro up reference strai ns and field isolates (tirres in recipr ocals)

G3

10240

-

160

1280

80 80

F46C2

G3

1280

f'48C3

~

~

'" ~

i!i ....

"0

;;1 ....

~ '---'

0--

Classification of Pomona Serogroup

1

2

3

4

5

6

7

1

94

94

67

16 7

43

43

30

30

20

20

2

3

4

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6

2

=1

94

67

43 -

417

7

3

94-

67- .

43-

,

3020-

30 20 Fig. 1. Characterization of MCAs by SDS-PAGE and SGIP. a) SDS-PAGE profile of leptospires stained by coomassie brilliant blue for proteins: lane 1, molecular weight markers phosphorylase B (94kD), albumin (63kD), ovalbumin (40kD), carbonic anhydrase (30kD), trypsin inhibitor (20kD); lane 2, butembo; lane 3, proechimys; lane 4, G 129; lane 5, mozdok; lane 6, tropica and lane 7, pomona. b) SDS-PAGE profile of leptospires stained by silver for glycoconjugates, lane 1-7 the same as in Fig. 1a. c) SGIP profile of MCA F48C3 on leptospires, lane 1-7, the same as in Fig. 1a. Note that F48C3 reacts only with tropica. d) SGIP profile of MCA F48C3 on tropica leptospires. lane 1, tropica; lane 2, tropica digested with pronase; lane 3, tropica after treatment with TFMS.

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tive strains after deglycosylation but not after pronase digestion. These results indicate that the MCAs are directed against polysaccharide antigens.

b. Restriction enzyme analysis The patterns of DNA fragments of Pomona group and related strains, obtained after restriction enzyme digestion and gel separation are presented in Fig. 2. The figure shows a group of pomona-like strains (lanes 2-7) (that had a similar pattern), a group of mozdok-Iike strains (lanes 8-13) and three less related strains (lanes 14-16). Differences in patterns between pomona, monjakov and kennewicki, were located between 10 and 15 kB. In this area pomona had one strong band, whereas monjakov showed 5 faintly visible bands and kennewicki 2 bands. Except for Indonesia strain ]T 12 all Indonesia isolates showed identical patterns. They could be differentiated from pomona in the 12 kB region where the Indonesia strains had one extra band. In the 7.5 kB region all Indonesia strains had one major band in common which was only weakly visible in the pomona pattern. Indonesia strain]T 12 differed in more bands from the rest of the Indonesia strains although the overal pattern was comparable. The Uruguay strain C 374 could easily be differentiated from pomona by one extra minor band situated close to the major 12 kB band. The Uruguay strain was different from the other isolates. Serovars tsaratsova, dania and mozdok were identical and could easily be differenti-

KB

2

3

4

5 6

7

8 9 10 11 12 13 14 15 16 17

23 .1

9.4 6 .6 4.4

Fig. 2. Eco R1 digestion of DNA from Pomona group strains: Lanes 1 and 17 DNA molecular weight marker ADNA, Hind III; lane 2, pomona; lane 3, moniakou, lane 4, kennewicki; lane 5,] 1; lane 6,]T 12; lane 7, C 374; lane 8, mozdok; lane 9, dania; lane 10, G 129; lane 11, LC 80/20; lane 12, Ukkel 60; lane 13, tsaratsova; lane 14, tropica; lane 15, proechimys, lane 16, butembo.

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ated from the pomona-like strains. Strain G 129 was similar to mozdok. LC 80120 differed from mozdok by the absence of a band in the 18 kB region. The Belgian strain Ukkel 60 showed one band in the 10 kB region which could not be demonstrated in the mozdok pattern. Serovars tropica, proechimys and butembo all had unique patterns which could be easily differentiated from all other strains mentioned.

Discussion In this study we have shown the serological profiles of several laboratory reference strains and field isolates of Pomona group strains. Clusters of antigenically interrelated pomona- or mozdok-like strains were characterized by minor differences in agglutination titres with several MCAs. Those differences were reproducible upon re-testing. Since they cannot be explained by test-to-test variability, these differences may be due to true, possibly quantitative, antigenic variation between strains. It would be preferable if more MCAs had been typespecific. To our opinion it did not seem worth the trouble to keep searching for typespecific MCAs as application of the panel of six allowed a rapid characterization. In our earlier study on the classification of Sejroe serogroup strains, we found that only those MCAs which distinguished in a semiqualitative fashion were useful (Terpstra et aI., 1985). This study shows that the application of semiqualitative criteria is too rigid and would probably blur subtle antigenic diversity between closely related strains. The MCAs were directed to antigens which were found to be heterogenously distributed in SDS-PAGE; no specific narrow bands could be detected in the SGIP. The antigens are probably of a polysaccharide nature since the bands were sensitive for deglycosylation but resistant to pronase treatment. The MCAs revealed the existence of groups of pomona-like or mozdok-like strains. Analysis of restriction enzyme digests of leptospiral DNA showed patterns which confirmed these serological observations. Monjakov and kennewicki could not be distinguished by MCAs from pomona. Unlike Marshall et al. (1981) but in accordance with the observations by Thiermann et al. (1985, 1986) we found that these two serovars had DNA digestion patterns which were slightly different from pomona. Thiermann et al. (1985, 1986) analysed isolates by restriction enzyme analysis and found that strains from the USA, Canada and New Zealand all were kennewicki and strains from Northern Ireland were pomona. In conjunction with our observations this suggests that there occur many different varieties all over the world that are closely related to reference strain pomona. We also found, contrary to Marshall et al. (1984), minor differences between the digestion patterns of mozdok, dania and the British isolates LC 80120 and G 129. The DNA digestion pattern of tsaratsova resembled that of mozdok, but tsaratsova could easily be distinguished by MCA analysis. Adler and Faine (1983) reported a Pomona group specific MCA which reacted to almost the same titre with all reference strains that were tested, while our most broadly reactive MCA showed a striking dissimilarity in titre and moreover reacted also with butembo. The reactivity of butembo with two Pomona MCAs shows an interesting relationship which is all the more surprising because our polyclonal, conventionally-prepared rabbit anti-pomona antiserum did not cross-react with butembo (and vice versa); cross-reactivity with pomona was not reported in the original description of butembo (Wolff and Bohlander, 1961) either. It is conceivable 28 Zbl. Bakt. Hyg. A 266/3-4

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that the mouse immune system has the capacity to react with other antigenic determinants than rabbits. In two cases, that is, with F43C9 (pomona) and F48C6 (tropica), the heterologous strains reacted more strongly than did the homologous strain, a phenomenon which was also described by Kobayashi et al. (1984) for MCAs directed against icterohaemorrhagiae. These paradoxical reactions, which may be caused by quantitative differences in cross-reacting antigens, are well known in agglutination reactions (Kmety, 1957) and have been described by Terpstra et al. (1983) also for precipitation reactions in gels. In the present serovar conception the recognized reference strain stands as a representative for what is often a cluster of closely related strains with a slightly different antigenic make-up. This cluster is delineated by falling within the boundaries of a 10% residual titre after repeated absorptions according to the serovar definition by the cross-agglutination absorption using conventional rabbit antisera (WHO, 1967). This test is insufficiently sensitive to clearly reveal the differences between strains. The objective of this study was to find MCAs which could be used for the characterization and identification of strains up to the serovar status as indeed the MCAs were selected by strains representing serovars. If we had had the wish to make MCAs which differentiate between strains we should have selected with antigens of closely related strains belonging to the same serovar instead of reference strains representing different serovars. Usually typing of an isolate to serovar level will be sufficient. In this study it was possible by restriction enzyme analysis to differentiate between strains and in this respect the method is more refined than typing with the Pomona group MCAs. The preparation of MCAs is laborious and time consuming requiring skill and a well equipped laboratory but once they are available they are easy to apply and very precise tools giving quick results as the agglutination test can be completed in 2-4 h. Restriction enzyme analysis is complicated and laborious also requiring skill and a well equipped laboratory while the results are sometimes erratic depending upon the quality of the restriction enzymes. It is an important method in that it gives information about genetic differences between leptospira strains. It is concluded that both, MCAs and restriction enzyme analysis, are useful tools for classification being complementary rather than competitive as the first give quick results and are easy to apply and the second gives more detailed information. Acknowledgement. We wish to thank Dr. E. P. Wright for advice on the manuscript.

References 1. Adler, B. and S. Faine: A Pomona serogroup-specific, agglutinating antigen in Leptospira, identified by monoclonal antibodies. Pathology 15 (1983) 247-250 2. Edge, A. S. B., C. R. Faltynek, 1. Hof, 1. E. Reichert, and P. Weber: Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Analyt. Biochem. 118 (1981) 131-137 3. Fazekas de St. Groth, S. and D. Scheidegger: Production of monoclonal antibodies; strategy and tactics. J. Immunol. Meth. 35 (1980) 1-21 4. Galfre, G. and C. Milstein: Preparation of monoclonal antibodies; strategies and procedures. Meth. Enzymol. 73 (1981) 3-46 5. Hathaway, S. c., R. B. Marshall, T. W. A. Little, S. A. Headlam, and P.J. Winter: Differentiation of reference strains of leptospires of the Pomona serogroup by crossagglutination absorption and restriction endonuclease analysis. Res. Vet. Sci. 39 (1985) 145-150

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6. Hathaway, S. c., R. B. Marshall, T. W. A. Little, S. A. Headlam, and P. j. Winter: Identification by cross-agglutination absorption and restriction endonuclease analysis of leptospires of the Pomona serogroup isolated in the United Kingdom. Res. Vet. Sci. 39 (1985) 151-156 7. Klatser, P. R., M. M. van Rens, and T. A. Eggelte: Immuno-chemical characterization of M.leprae antigens by SGIP using patients' sera. Clin. expoImmunol. 56 (1984) 537-544 8. Kmety, E.: Betrachtungen zum Problem der paradoxen Reaktion und deren Bedeutung in der Serodiagnostik einiger Leptospirosen. Zbl. Bakt. Hyg., I. Abt. Orig. A 170 (1957) 597-608 9. Kmety, E.: Faktorenanalyse von Leptospiren der Icterohaemorrhagiae- und einiger verwandter Serogroupen. Thesis. Slovak Academy of Sciences, Bratislava (1967) 10. Kobayashi, Y., T. Tamai, and T. Oyama: Characterization of monoclonal antibodies against etiological agents of Weil's disease. Microbiol. Immunol. 28 (1984) 359-370 11. Kohler, G. and C. Milstein: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256 (1975) 495-497 12. Kolk, A. H. j., M. L. Ho, P. R. Klatser, T. A. Eggelte, S. Kuyper, S. de ]onge-Aglibut, and j. van Leeuwen: Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. Clin. expo Immunol. 58 (1984) 511-521 13. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage H. Nature 227 (1970) 680-685 14. Manev, c.: Serological characteristics of the Leptospira serogroup Pomona, factor analysis of the reference strains. Zbl. Bakt. Hyg., I. Abt. Orig. A 236 (1976) 316-322 15. Marshall, R. B., B. E. Wilton, and A. j. Robinson: Identification of Leptospira serovars by restriction-endonuclease analysis. ]. Med. Microbiol. 14 (1981) 163-166 16. Marshall, R. B., P. j. Winter, and R. Yanagau/a: Restriction-endonuclease DNA analysis of Leptospira interrogans serovars icterohaemorrhagiae and hebdomadis. ]. Clin. Microbiol. 20 (1984) 808-810 17. Terpstra, W. j. and G. j. Schoone: Analysis of Leptospira antigens by crossed immunoelectrophoresis. Scand.]. Immunol. 18 (1983) 113-121 18. Terpstra, W. j., H. Korver, .f. van Leeuwen, P. R. Klatser, and A. H. ]. Kolk: The classification of the Sejroe group of Leptospira interrogans with monoclonal antibodies. Zbl. Bakt.Hyg., I. Abt. Orig. A 259 (1985) 498-506 19. Thiermann, A. B., A. L. Handsaker, S. L. Moseley, and B. Kingscote: New method for classification of leptospiral isolates belonging to serogroup Pomona by restriction endonuclease analysis: serovar kennewicki. ]. Clin. Microbiol. 21 (1985) 585-587 20. Thiermann, A. B. and W. A. Ellis: Identification of leptospires of veterinary importance by restriction endonuclease analysis. In: The Present State of Leptospirosis. Diagnosis and Control, pp. 99-104, W. A. Ellis and T. W. A. Little (eds.). Martinus Nijhoff Publishers, Dordrecht-Boston-Lancaster (1986) 21. Tsai, C. M. and E. Frasch: A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Analyt, Biochem. 119 (1982) 115-119 22. World Health Organization: Current Problems in Leptospirosis Research. Technical Report Series No. 380. Geneva (1967) 23. Wolff, J. W.: The Laboratory Diagnosis of Leptospirosis. C. C. Thomas, Publisher, Springfield, Illinois/U.S.A. (1957) 24. Wolff, J. W. and H. J. Bohlander: The serological classification of Leptospira Butembo. Trop. Geogr. Med. 13 (1961) 173-174 25. Wolff, J. W. and]. C. Broom: The genus Leptospira Noguchi, 1917. Problems of classification and a suggested system based on antigenic analysis. Doc. Med. Geogr. Trop. 6 (1954) 78-95 Dr. W. j. Terpstra, N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands