Serological titres to Leptospira fainei serovar hurstbridge in human sera in Australia.

A set of 723 diagnostic sera from human patients, submitted for the microscopic agglutination test (MAT) for antibodies to a group of 6 leptospiral serovars, was also tested by MAT for antibodies to the recently-discovered Leptospira fainei serovar hurstbridge. MAT titres of > or = 128 to serovar hurstbridge were detected in 13.4% of these sera, and titres of > or = 512 in 7.2%. In contrast, none of 62 sera obtained from a control population of laboratory staff gave titres of > or = 128. The difference between the number of titres of > or = 128 given by the two groups of sera was highly significant (P < 0.01). The titres observed may have been due to cross-reactions with other leptospiral serovars, but this could not be demonstrated. An alternative explanation is that serovar hurstbridge is present in the human population.

Identification and characterization of the dTDP-rhamnose biosynthesis and transfer genes of the lipopolysaccharide-related rfb locus in Leptospira interrogans serovar Copenhageni.

Immunity to leptospirosis is principally humorally mediated and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. The only protective antigen identified to date is the leptospiral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little is known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragment from the chromosome of serovar Copenhageni was cloned in pUC18 in Escherichia coli, after which flanking regions were cloned from a genomic library constructed in bacteriophage lambda GEM12. Sequence analysis identified four open reading frames which showed similarity to the rfbC, rfbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the assembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showed similarity with the rhamnosyltransferase genes of Shigella and Yersinia enterocolitica but not Salmonella. Comparison of deduced amino acid sequences showed up to 85% similarity of the leptospiral proteins with those of other gram-negative bacteria. Polyacrylamide gel electrophoresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira. Moreover, it could restore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexneri which was deficient in all four genes, thereby confirming the functional identification of a part of the leptospiral rfb locus.

Characterization of an antigenic oligosaccharide from Leptospira interrogans serovar pomona and its role in immunity.

An antigenic oligosaccharide fraction derived from the lipopolysaccharide of Leptospira interrogans serovar pomona was isolated by endo-glycosidase H digestion and column chromatography. The oligosaccharide contained rhamnose, ribose, glucose, and glucosamine and inhibited the binding of opsonic, protective monoclonal antibodies directed against the lipopolysaccharide. When conjugated to diphtheria toxoid, the oligosaccharide elicited the production of agglutinating, opsonic antibodies.

Immunochemical studies of opsonic epitopes of the lipopolysaccharide of Leptospira interrogans serovar hardjo.

Leptospiral lipopolysaccharides (LPS) are the main antigens responsible for immunity in leptospirosis. In this investigation we studied the nature of the antigenic determinants of LPS extracted from Leptospira interrogans serovar hardjo (reference strain Hardjoprajitno). The reactions of anti-LPS monoclonal antibodies (mAbs) MUM/F1-4/hardjo (IgM) and MUM/F1-6/hardjo (IgG) with whole cell lysates in Western immunoblotting analysis were unaffected by proteinase K treatment. Periodate treatment of the LPS destroyed the binding of MUM/F1-6/hardjo but preserved that of MUM/F1-4/hardjo. Alkaline phosphatase decreased significantly the binding of MUM/F1-4/hardjo to the LPS but only slightly that of MUM/F1-6/hardjo. On the other hand, phosphodiesterase totally destroyed the binding capacity of both monoclonal antibodies in enzyme immunoassays (EIA). A number of mono- and oligosaccharides was used in EIA inhibition studies. Mannose-6-phosphate and galactose-6-phosphate inhibited the binding of MUM/F1-4/hardjo (50% inhibition at a concentration of 5 mM) to the antigen, but glucose-6-phosphate did not. Galactosamine and mannosamine inhibited the binding of MUM/F1-6/hardjo (50% inhibition at a concentration of 3-4 mM), whereas only a weak inhibition was observed with glucosamine. In contrast, N-acetylated amino sugars did not show any inhibition. An O-acetyl group also appears to be involved in the antigen-antibody binding process.

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni.

A chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with R alpha P64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4 kb EcoRI-Bam HI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5 kDa (Hsp10) and 58 kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an E sigma 70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat-shock conditions as determined by [35S]-methionine pulse labelling experiments. As no functional heat-shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat-shock conditions, similar to mechanisms described for several Gram-positive organisms.

Antigens recognized by the human immune response to severe leptospirosis in Barbados.

Serum samples obtained from patients hospitalized in Barbados with severe leptospirosis were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting with leptospires that had been isolated from these patients. While serum samples taken a few days after onset of symptoms often showed no apparent correlation between MAT and EIA, later sequential serum samples produced similar profiles in both tests during the course of infection. Immunoblotting sonicate from Leptospira interrogans serovars arborea, copenhageni and bim with patients' sera, revealed reactions with a number of bands that corresponded with outer envelope components. These components included lipopolysaccharide (LPS), flagella and other outer membrane proteins, in addition to a low-molecular-weight (MW) carbohydrate cross-reactive with members of the Leptospiraceae. IgM antibodies elicited in the first to second week after infection reacted mainly with LPS and the low-MW cross-reactive carbohydrate. Comparative analysis of isolates of the same serovar by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting showed that while two serovar arborea isolates were identical, serovar bim isolates differed significantly from each other. This difference was also observed in comparative MAT testing.

Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein.

A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L. interrogans) serovar hardjo was cloned and expressed in Escherichia coli. Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18. Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa. The G + C content of the flaB gene was 54.7 mol%. Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins.

Vaccination of mice with lipopolysaccharide (LPS) and LPS-derived immuno-conjugates from Leptospira interrogans.

Mice were vaccinated with lipopolysaccharide (LPS) from Leptospira interrogans serovar pomona or hardjo, or with the polysaccharide (PS) fraction of the LPS, or with an immunoconjugate of PS and diphtheria toxoid (DT). Maximum agglutinin titres were found 6-10 weeks after vaccination with LPS or PS-DT conjugate; the latter elicited antibody titres at least 10 times higher than those produced in response to LPS. Animals failed to react significantly to PS. Titres elicited by antigens of serovar pomona were higher than those elicited by serovar hardjo.

Antigens recognized by the human immune response to vaccination with a bivalent hardjo/pomona leptospiral vaccine.

Serum from volunteer subjects vaccinated with a bivalent whole cell vaccine of Leptospira interrogans serovar hardjo/serovar pomona grown in protein-free medium, was tested by the microscopic agglutination test (MAT), enzyme-immunoassay (EIA) and immunoblotting. Specific IgM antibodies to either serovars hardjo or pomona were detected in some subjects as early as 6 days after vaccination with peak antibody levels occurring 13-68 days after vaccination. Whereas all subjects produced specific IgM to both serovars, not all produced specific IgG to both serovars. Immunoblotting with hardjo sonicate revealed that all subjects produced IgM antibodies reacting with the 15, 23 and 28 kDa components of hardjo lipopolysaccharide (LPS), and most produced IgM antibodies that reacted with the 34.5 kDa flagellar doublet. In contrast, not all sera immunoblotted against pomona sonicate reacted with the 29 and 35 kDa components of pomona LPS. However all subjects produced antibodies reacting with a diffuse 14.4-27 kDa band. These antibodies appeared early in the immune response. Serum from the one vaccinated subject tested protected hamsters from acute lethal infection with serovar pomona.

Purification and characterization of a protein antigen from Leptospira interrogans serovar hardjo, common to a wide range of bacteria.

A protein with a molecular mass of 64 kDa (P64) from Leptospira interrogans serovar hardjo was partially purified by using successively, phase partitioning with Triton X-114, ion-exchange chromatography and sucrose gradient centrifugation. Purification to homogeneity was obtained by electroelution of P64 from SDS-polyacrylamide gels. Monospecific rabbit antiserum (R alpha P64) was prepared using the purified protein preparation. P64 had a native molecular mass of greater than 670 kDa and was recognized by R alpha P64 as well as by human antisera. Western blotting of leptospiral serovars and 18 other bacterial species with R alpha P64 showed that P64 was cross-reactive with an equivalent antigen in a wide range of bacteria, indicating that it belongs to a family of antigens previously designated 'common antigen'. This putative common antigen from Leptospira appears to have a sub-surface location, but its function is not yet known.

Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo.

Lipopolysaccharides (LPSs) from Leptospira interrogans serovar hardjo (reference strain hardjoprajitno and strain hardjobovis) were prepared by the hot phenol-water procedure. High yields of LPSs were found in the phenol phase. Gel electrophoresis of the phenol phase LPSs showed similar patterns for all strains in contrast to the different patterns found in the water phase LPSs. Sugar composition was also similar among all strains with rhamnose as the predominant sugar. Mannosamine was detected by high performance thin layer and gas-liquid chromatography. 2-Keto-3-deoxyoctonic acid (KDO) was comparable with authentic KDO by paper chromatography. Periodate oxidation at near neutral pH with or without prior hydrolysis showed that most of the KDO was substituted. The fatty acid composition of strain hardjobovis LPS was slightly different from that of the reference strain hardjoprajitno. Myristic and 3-hydroxymyristic acid were not detected in any of the LPS preparations. In conjunction with genetic and other data, the two strains are sufficiently different to be regarded as members of two separate species sharing common antigens. There is sufficient evidence to rename the hardjoprajitno strain type L. interrogans hardjo-p, and the hardjobovis strain type L. borgpeterseni hardjo-b.

Experimental immunisation of hamsters with lipopolysaccharide antigens of Leptospira interrogans.

Hamsters were immunised with leptospiral lipopolysaccharide (LPS) or the polysaccharide (PS) fraction of LPS from Leptospira interrogans serovar copenhageni and the antibody responses were measured by agglutination tests. Maximum titres were observed approximately 6 weeks after immunisation and protection against lethal challenge with the homologous strain was afforded by immunisation with as little as 2.5 micrograms of LPS or PS. All animals produced IgM agglutinins but a higher proportion of-animals immunised with PS produced IgG agglutinins than did those immunised with LPS. Immunisation of guinea-pigs with autoclaved PS showed that the preparation retained some but not all of its immunogenic activity.

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona.

Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona, were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona. In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.

Expression of two conserved leptospiral antigens in Escherichia coli.

The genes encoding two protein antigens of Leptospira interrogans serovar pomona were cloned and expressed in Escherichia coli. Rabbit antisera raised against the cloned proteins, designated p12 and p20, were used to identify the antigens in Western blots of disrupted leptospiral cells. The proteins p12 and p20 were conserved within the genus Leptospira and were not detected in Leptonema illini. Although both proteins were present in leptospiral outer envelope preparations they did not elicit the production of agglutinating or opsonising antibodies.

Experimental syphilitic orchitis. Relationship between Treponema pallidum infection and testis synthesis of proteoglycans.

The relationship between Treponema pallidum infection and the synthesis of proteoglycans by organ cultures of rabbit testes was investigated. Two proteoglycan fractions, large (CL-6B Kav 0.05) and small (Kav 0.25), that were not synthesized at detectable levels by cultures from rabbits infected with T. pallidum for 0 or 5 days, were produced by cultures from 10-, 15- and 20-day infected rabbits. The small proteoglycan appeared to be synthesized first because greater amounts of this fraction were detected in extracts of cultures from 10-day infected animals. The large proteoglycan fraction may have been induced directly by T. pallidum because increased synthesis correlated with maximal treponemal numbers, 15 days after infection. In contrast, the induction of the smaller proteoglycan did not appear to be directly related to numbers of elutable organisms. The proteoglycans synthesized between 10 and 20 days after infection were analyzed for glycosaminoglycan (GAG) size and composition. The size of GAGs beta-eliminated from the proteoglycans generally increased over the 10-20-day infection period. Whereas the composition of the small proteoglycan fraction was largely unchanged during the 10-20-day period (45% chondroitin sulphate (CS), 55% dermatan sulphate (DS)), the amount of DS in the high molecular weight proteoglycan fraction decreased from 50-20% during this period (CS 50-80%). Autoradiography studies revealed that increased proteoglycan synthesis in T. pallidum-infected testes was localized to cells lining the seminiferous tubules and to fibroblasts infiltrating peritubular spaces.

Reaction of monoclonal antibodies with species specific determinants in Leptospira interrogans outer envelope.

A set of 24 monoclonal antibodies (MABs) was produced against an outer envelope preparation from Leptospira interrogans serovar copenhageni. The MABs reacted in enzyme immunoassay with species-specific determinants of an antigen in the leptospiral outer envelope (OE) of pathogenic but not of saprophytic species of Leptospira. The MABs did not agglutinate whole leptospires, nor could they opsonise homologous leptospires for phagocytosis by mouse macrophages or protect new-born guinea-pigs against lethal infection. The MABs reacted by Western blotting with a 35 x 10(3)-mol-wt band in OE separated on SDS-polyacrylamide gels, and also reacted with other bands to a lesser extent. The determinants to which the MABs were directed were localised in the leptospiral OE by immunogold labelling techniques.

Identification of leptospiral flagellar antigens by gel electrophoresis and immunoblotting.

Flagella extracted from five serovars, representative of the pathogenic and saprophytic species of the Leptospiraceae, were morphologically similar. Analysis of Leptospira interrogans flagellar preparations by polyacrylamide gel electrophoresis revealed three common major bands in the (30-40) x 10(3)-mol. wt region, and serovar-specific bands in the lower region of the gels. Although some differences were observed, flagella extracted from L. biflexa serovar patoc and Leptonema illini revealed similar electrophoretic profiles to those seen in L. interrogans flagella. Immunoblot analysis showed that while flagellar components in the (20-30) x 10(3)-mol. wt region were recognised only by homologous rabbit antisera, a major protein doublet of (33-34) X 10(3)-mol. wt, depending on the species, was also demonstrated by heterologous antisera. The serovar-specific bands in the (20-30) x 10(3)-mol. wt region were composed of lipopolysaccharide (LPS). These results show that leptospiral flagella are immunogenic and contain antigens which are conserved among the different genera of the family Leptospiraceae.

Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo.

Serum samples from patients infected with Leptospira interrogans serovar hardjo were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting. There was no apparent correlation between MAT titre and EIA optical density (OD) for individual serum samples, but sequential serum samples produced similar profiles in both tests during the course of an infection. Immunoblotting of hardjo sonicate with patients' sera revealed reactions with a number of bands, in the mol. wt (10(3] range 14.4-95. However, all serum samples reacted with the major 28 x 10(3)-mol. wt sub-unit of hardjo lipopolysaccharide (LPS) and most reacted with a (34.5-35) x 10(3)-mol. wt flagella doublet. Examination of sequential serum samples obtained over a period of about 3 months after infection revealed little change in the antigens detected after the second to third week of infection. Absorption of patients' sera with whole viable leptospires revealed that antibodies to several exposed antigens, including LPS, were produced. Sera which reacted with hardjo flagella also reacted with bands of similar mol. wts in preparations from other serovars.