Hans Korver

The Classification of Sejroe Group Serovars of Leptospira interrogans with Monoclonal Antibodies

Using the hybridoma technique we produced monoclonal antibodies to serovars of Leptospira interrogans. We focussed on serovar hardjo which is an important pathogen for humans and animals, and on other serovars of the Sejroe group. With combinations of monoclonals, characteristic patterns of agglutination were observed according to the specific pattern of antigenic determinants of that serovar. A set of 13 monoclonal antibodies was composed which allowed the classification of almost all serovars of the Sejroe group.

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

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.

Muskrats as carriers of pathogenic leptospires in The Netherlands

Leptospires were isolated from 24 of 327 (7%) muskrats (Ondatra zibethicus) caught in The Netherlands. All isolates were identified asLeptospira interrogans. One isolate was typed as serovarcopenhageni in the Icterohaemorrhagiae serogroup, one as serovarlora in the Australis serogroup. Twenty-one isolates showed a close relationship with serovarsgrippotyphosa, valbuzzi, muelleri andratnapura from the Grippotyphosa serogroup. One isolate was lost. Sera from 196 muskrats were examined by the microscopic agglutination test. Forty-five (23%) sera reacted positively (titers≧1: 160), 42 (21%) of these 45 sera to Grippotyphosa and 3 (2%) to Sejroe serogroup antigens. This is the first report of serological and cultural evidence of leptospira infection in muskrats in The Netherlands.

Leptospires isolated from toads and frogs on the Island of Barbados.

Four pathogenic strains of leptospires were isolated from the kidneys of toads (Bufo marinus) and seven from frogs (Eleutherodactylus johnstonei). Isolates from two toads and one frog belonged to serovar bim, the causative agent of most cases of severe leptospirosis on Barbados. The other eight strains belonged to a new serovar within the Australis serogroup. The name bajan is proposed for this new serovar of Leptospira interrogans.

Two New Leptospiral Serovars in the Hebdomadis Serogroup Isolated from Zimbabwe Cattle

Four strains belonging to the genus Leptospira serogroup Hebdomadis were isolated from Zimbabwe cattle at slaughter. These isolates were subjected to cross-agglutinin absorption tests and to restriction fragment length polymorphism and pulsed-field gel electrophoresis analyses of their genomic DNAs. One of these strains represents a new serovar, for which the name mhou is proposed; strain SBF 40 is the reference strain of this serovar. The other three strains belong to a second new serovar, for which the name marondera is proposed; the reference strain of this serovar is strain SBF 5. The three strains of serovar marondera could be differentiated by their restriction fragment polymorphism and pulsed-field gel electrophoretic patterns.

Identification of a serogroup bataviae Leptospira strain isolated from an ox in Zimbabwe.

A strain belonging to the genus Leptospira serogroup Bataviae, isolated from an ox at slaughter in Zimbabwe, was identified by using cross-agglutinin absorption, the monoclonal antibody, restriction fragment length polymorphism, and polymerase chain reaction analyses. Results of both serological tests used showed that the isolate (strain SBF 37) was antigenically similar to reference strain Paidjan and therefore belongs to serovar paidjan. Strain SBF 37 was, however, genetically different from strain Paidjan since their chromosomal DNA had different restriction fragment length polymorphism patterns. The Zimbabwe paidjan strain was identified as belonging to the species Leptospira kirschneri while strain Paidjan reacted as a member of one of the non-kirschneri species in the polymerase chain reaction. The Zimbabwe isolate therefore belongs to Leptospira kirschneri serovar paidjan.

A new leptospiral serovar, ngavi, in the Tarassovi serogroup isolated from Zimbabwe oxen

Two strains of the genus Leptospira, belonging to serogroup Tarassovi, were isolated from kidneys of apparently healthy oxen slaughtered at an abattoir in Zimbabwe. Both strains belonged to the same serovar but could not be assigned to previously known serovars using the cross-agglutinin absorption test. The name ngavi is proposed for the new serovar containing these two strains; strain SBF 16 is the reference strain. The Zimbabwe isolates showed some antigenic similarity to serovar gatuni when analyses were carried out using eight monoclonal antibodies, and had restriction patterns similar to those of serovars tarassovi, tunis, moldaviae and guidae when their chromosomal DNAs were analysed using RFLP analysis. The restriction patterns of the two strains could be distinguished from each other and from those of the four serovars when their Southern blots were hybridized with a probe synthesized from a repetitive sequence element cloned from serovar hardjo strain Hardjo-bovis.

Identification of leptospires of the Pomona and Grippotyphosa serogroups isolated from cattle in Zimbabwe

Two strains of the genus Leptospira, isolated from kidneys of oxen slaughtered in Zimbabwe, one belonging to serogroup Pomona (strain SBF 8) and the other to serogroup Grippotyphosa (strain SBF 32), were identified by using cross-agglutinin absorption, monoclonal antibody, restriction fragment length polymorphism and polymerase chain reaction analyses. The identification of the two strains was equivocal. Strain SBF 8 showed a close similarity to both serovars mozdok and proechimys by cross-agglutinin absorption tests and to serovar pomona by monoclonal antibody analysis, but had a distinct DNA restriction pattern. Strain SBF 32 showed a close antigenic similarity to serovars ratnapura, grippotyphosa and valbuzzi by the cross-agglutinin absorption test, and to serovar ratnapura by monoclonal antibody analysis but also had a distinct DNA restriction pattern. Both strains SBF 8 and SBF 32 reacted as members of species Leptospira kirschneri by the polymerase chain reaction. It is concluded that strains SBF 8 and SBF 32 represent new genetic strains in the Pomona and Grippotyphosa groups, respectively.

Classification of serovars of the Sejroe group ofLeptospira interrogans with monoclonal antibodies

The classification of L e p t o s p i r a serovars is based on cross-agglutination absorption tests (Wolff and Broom, 1954). The classification system is satisfactory but test procedures are too complicated and too time-consuming to be of value when rapid results are needed. The introduction, by Kmety (1967), of a classification system based on antigenic factors and subsequent production of factor sera enabling rapid typing meant a considerable improvement. Factor sera however are available in limited quantities of variable quality. Since hybridomas secrete antibodies in virtually unlimited supply, with atI of them having the same specificity (K6hler and MiIstein, 1975), we examined monoclonal antibodies for their capacity to characterize antigens of different leptospira serovars. We concentrated on serovar hard jo as this serovar, being recently isolated from cattle in the Netherlands, was the presumptive cause of disease in humans as well as in cattle. BALB/c mice were immunized with different immunizing procedures involving live whote cells as well as partly purified outer membranes of hardjo . Spleen cells of immunized mice were fused with SP2/O-Ag-14 myeloma cells. The resulting hybridomas were tested for antibody production using viable leptospirae in a Microscopic Agglutination Test (MAT). Antibody-secreting hydridomas were cloned three times and eventually cultured in the peritoneal cavity of BALB/c mice. After a few days, ascites containing large amounts of monoclonal antibodies was harvested.