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At least 19 recordsLinked to original sources
PubMed1987

Enzyme activities of the strains belonging to family Leptospiraceae detected by the API ZYM system.

A total of 32 strains of the family Leptospiraceae (23 strains of Leptospira interrogans, 6 strains of Leptospira biflexa, 2 strains of Leptonema and 1 strain of Leptospira parva) were examined for enzyme activities using 89 substrates (API ZYM system). More than 90% of the strains belonging to the family Leptospiraceae possessed strong activities of beta-D-galactosidase, beta-D-glucosidase and 5 esterases (C5, C6, C8, C9 and C10). More than 90% of the strains belonging to the genus Leptospira, except L. parva, had strong activities of L-lysine arylamidase and alpha-L-glutamate arylamidase. L. biflexa strains, except serovar andamana, were different from the other strains examined in that they possessed glycyl-glycine arylamidase, glycyl-phenylalanine arylamidase and L-tryptophan arylamidase. L. biflexa strains, except andamana, L. parva and Leptonema strains possessed strong activities of glycine arylamidase and leucyl-glycine arylamidase. Two strains of the genus Leptonema were different from the strains belonging to the genus Leptospira in that they possessed strong activities of beta-D-lactosidase. L. parva lacked alpha-D-galactosidase which other strains belonging to the family Leptospiraceae possessed. Dendrogram analysis revealed that strains belonging to the family Leptospiraceae were divided into 4 groups. The first group consisted of all strains belonging to L. interrogans and serovar andamana of L. biflexa; the second group consisted of the remaining 5 serovars of L. biflexa; the third group consisted of the genus Leptonema; and the fourth group consisted of only L. parva.

Aminopeptidases↗
PubMed1996

Structural analysis of the Leptospiraceae and Borrelia burgdorferi by high-voltage electron microscopy.

Spirochetes are an evolutionary and structurally unique group of bacteria. Outermost is a membrane sheath (OS), and within this sheath are the protoplasmic cell cylinder (PC) and periplasmic flagella (PFs). The PFs are attached at each end of the PC and, depending on the species, may or may not overlap in the center of the cell. The precise location of the PFs within the spirochetal cells is unknown. The PFs could lie along the cell axis. Alternatively, the PFs could wrap around the PC in either a right- or a left-handed sense. To understand the factors that cause the PFs to influence cell shape and allow the cells to swim, we determined the precise location of the PFs in the Leptospiraceae (Leptonema illini) and Borrelia burgdorferi. Our approach was to use high-voltage electron microscopy and analyze the three-dimensional images obtained from thick sections of embedded cells. We found that a single PF in L. illini is located in a central channel 29 nm in diameter running along the helix axis of the right-handed PC. The presence of the PFs is associated with the end being hook shaped. The results obtained agree with the current model of Leptospiraceae motility. In B. burgdorferi, which forms a flattened wave, the relationship between the PFs and the PC is more complicated. A multistrand ridge 67 nm in diameter, which was shown to be composed of PFs by cross-sectional and mutant analysis, was found to extend along the entire length of the cell. We found that the PFs wrapped around the PC in a right-handed sense. However, the PFs formed a left-handed helix in space. The wavelength of the cell body and the helix pitch of the PFs were found to be identical (2.83 microm). The results obtained were used to propose a model of B. burgdorferi motility whereby backward-propagating waves, which gyrate counterclockwise as viewed from the back of the cell, are generated by the counterclockwise rotation of the internal PFs. Concomitant with this motion, the cell is believed to rotate clockwise about the body axis as shown for the Leptospiraceae.

Borrelia burgdorferi Group↗
PubMed1993

The use of 16S rDNA sequence analysis to investigate the phylogeny of Leptospiraceae and related spirochaetes.

The 16S rDNA sequences from 15 Leptospiraceae were determined by automated PCR-directed cycle sequencing. Nucleotide comparisons, including those from published sequences for Leptospira canicola Moulton and Serpulina spp., were used to construct phylogenetic trees. Serpulina hyodysenteriae and S. innocens were related to each other but were distinct from the Leptospiraceae comprising Leptospira parva incertae sedis (Turneria parva H), Leptonema illini and Leptospira spp. The pathogenic and the saprophytic leptospires were distinct and separated from each other. Leptospira inadai occupied an intermediate position between the two forms. The pathogens formed three groups. Group I was represented by L. interrogans sensu stricto and L. kirschneri, Group II by L. weilii, L. borgpetersenii and L. santarosai, and Group III comprised L. noguchii and L. meyeri. The saprophytic species, L. wolbachii and L. biflexa sensu stricto shared about 99% sequence similarity. The freshwater isolates were distinct from the marine isolate L. biflexa sensu lato ancona Ancona Porto.

Base Sequence↗
PubMed1992

Presence of putative sphingomyelinase genes among members of the family Leptospiraceae.

The presence of multiple DNA elements in pathogenic members of the family Leptospiraceae, similar to the sphA sphingomyelinase gene from Leptospira borgpetersenii, was demonstrated by low-stringency hybridization experiments. These DNA elements were designated putative sphingomyelinase genes. Grouping of strains by similarity of hybridization patterns corresponds to the species subdivision of the family Leptospiraceae on the basis of genetic characteristics. Therefore, hybridization with the sphA gene can be used as a taxonomic tool. These hybridization experiments indicate the presence of two groups of genetically related pathogenic Leptospira species.

Blotting, Southern↗
PubMed1992

Detection of Leptospiraceae by amplification of 16S ribosomal DNA.

The polymerase chain reaction (PCR) was developed to detect Leptospiraceae. Primers were used to amplify 1 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira interrogans sensu stricto, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, L. inadai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species. L. biflexa, L. wolbachii and L. parva were not amplified. There was no PCR product from 23 phylogenetically unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as 10-1 leptospires could be detected using the PCR analysis. Isolates of leptospires from clinical sources gave a positive PCR band, but those from surface waters did not.

Base Sequence↗
PubMed2006

Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing.

The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases < or =10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.

Bacterial Typing Techniques↗
PubMed1990

Fatty acid profiles in the family Leptospiraceae.

Fatty acid profiles of six leptospira strains representative of genera, species, and serogroups within the family Leptospiraceae were determined by gas liquid chromatography (GLC) of fatty acid methyl ester (FAME) derivatives. The influence of methodological and biological variables on FAME profiles of the same strain was tested. FAME profiles were sharply affected by the fatty acid composition of the culture medium but not by the growth phase. Twenty-four FAME peaks were selected on the basis of their presence in repeated gas chromatographic runs of single strains. Inter-strain divergences of FAME profiles were quantified by linear regression analysis (LR). Step-wise divergences in FAME profiles were observed between strains at serogroup, species, and genus levels.

Chromatography, Gas↗
PubMed1999

Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies.

DNA relatedness was determined among 303 strains of Leptospira and Leptonema. Included in the analysis were reference strains from 228 well-characterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (11 strains). The largest groups included Leptospira interrogans (91 strains from 82 serovars), Leptospira santarosai (65 strains from 59 serovars), Leptospira borgpetersenii (49 strains from 43 serovars), Leptospira kirschneri (29 strains from 26 serovars) and Leptospira noguchii (20 strains from 20 serovars). The new genomospecies include Leptospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Leptospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Leptospira genomospecies 3 (one strain, serovar holland), Leptospira genomospecies 4 (one strain, serovar hualin) and Leptospira genomospecies 5 (one strain, serovar saopaulo). With the exception of Ballum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Leptospira alexanderi sp. nov. is proposed for Leptospira genomospecies 2 (type strain L 60T = ATCC 700520T, serovar manhao 3).

Animals↗
PubMed1993

Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms.

Chromosomal DNA from 37 Leptospiracae representing genetic species, groups and reference strains together with five leptospire isolates and Escherichia coli were digested with the restriction endonucleases BamHI, ClaI and EcoRI. The Southern blots were hybridized with a biotinylated E. coli 1.5 kb 16S rDNA probe and gave 36 reproducible and unique patterns. With the exception of the type strain (Leptospira interrogans serovar icterohaemorrhagiae RGA) and neotype strain (serovar icterohaemorrhagiae Ictero no. 1) all the species and taxa examined could be differentiated from each other on the basis of their BamHI, ClaI and EcoRI restriction fragment length polymorphisms (RFLP). L. interrogans and L. borgpetersenii reference strains were heterogeneous, whereas Leptonema illini and L. parva incertae sedis were distinct and separate. Strains representing L. biflexa sensu lato presented divergent RFLP patterns. A porcine isolate was identified to be L. interrogans pomona Pomona.

Base Sequence↗
PubMed1990

Multiple-exposure photographic analysis of a motile spirochete.

The Leptospiraceae are thin spirochetes with a unique mode of motility. These spiral-shaped bacteria have internal periplasmic flagella that propel the cells in low-viscosity and gel-like high-viscosity media. A model of Leptospiraceae motility has been previously proposed that states that the subterminally attached periplasmic flagella rotate between the outer sheath and the helical protoplasmic cylinder. The shape of the cell ends and the direction of gyration of these ends are determined by the direction of rotation of the internal periplasmic flagella. Rotation of the periplasmic flagella in one direction causes that end to be spiral-shaped, and rotation in the other direction causes that end to be hook-shaped. One prediction of the model is that these right-handed spirochetes roll clockwise when swimming away from an observer. For maximum swimming efficiency, the model predicts that the sense of the spiral-shaped end is left-handed and gyrates counterclockwise. The present study presents direct evidence that the cell rolls clockwise (protoplasmic cylinder helix diameter = 0.24 micron; pitch = 0.69 micron), the ends gyrate counterclockwise, and the spiral-shaped end is left-handed (helix diameter = 0.6 micron; pitch = 2.7 microns)--as predicted by the model. The hook-shaped end appears approximately planar. The approach used was to illuminate stroboscopically cells slowed by Ficoll and analyze the resultant multiple-exposure photographs focused above and below the axis of the cell. The methodology used should be helpful in analyzing the motility of the larger and more complex spirochetes.

Cell Movement↗
PubMed1988

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, Bacterial↗
PubMed1989

[Classification of spirochetes infecting man].

The systematics od spirochetes must from now own fulfil the genomic criteria recently laid down. Formerly, these parasites were classified on the basis of various phenotypic features, among which the pathogenicity and epidemiological criteria played the predominant role. At present, the order of Spirochaetales is divided into two families: Spirochaetaceae and Leptospiraceae. Two of the four genera of Spirochaetaceae, Treponema and Borrelia, include species that are pathogenic to man. Among Leptospiraceae, only one genus, Leptospira, has pathogenic species. The phenotypic characteristics of the various taxons are detailed, the role of each actor (causative agent, vector, reservoir) in the main pathological complexes and the progress of genotypic studies are briefly described.

Humans↗
PubMedDate not supplied

[Prevalence of the gene encoding the outer membrane lipoprotein LipL32 in leptospires of different taxons].

Primers flanking the fragment sized 677 bp have been constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to reveal the prevalence of gene lipL32 among 73 Leptospiraceae family strains representing different genera and genomic species. The gene lipL32 appeared to be conservative across the pathogenic species. In contrast, it was not detected in the genome of nonpathogenic free-living leptospires. Thus the developed PCR test-system with primers LEP21/LEP22 may be efficiently used to differentiate these two distinct ecological groups of leptospires.

Animals↗
PubMed1999

Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product.

Sequence analysis of 16S rRNA genes extracted from nucleic acids databases enabled the identification of a Leptospira biflexa (L. biflexa) signature sequence, against which a reverse primer designated L613, was designed. This primer, when used in conjunction with a universal bacterial specific forward primer designated Fd1, enabled the development of a LightCycler-based PCR protocol in which fluorescence emission due to binding of SYBR Green I dye to amplified products could be detected and monitored. A melting temperature (Tm), determined from the melting curve of the amplified product immediately following the termination of thermal cycling, confirmed that the product was that of L. biflexa. Agarose gel electrophoresis therefore was not necessary for identification of PCR products. The PCR protocol was very rapid, and consisted of 30 cycles with a duration of 20 s for each cycle with the monitoring of the melting curve requiring an additional 3 min. The whole protocol was completed in less than 20 min. The PCR protocol was also specific and enabled the identification of 18 strains of L. biflexa, whilst excluding 14 strains of L. interrogans and Leptonema illini. Two examples of its utility in improving work flow of a Leptospira reference laboratory are presented in this article. The use of a simple boiling method for extraction of DNA from all the members of the Leptospiraceae family DNA further simplifies the procedure and makes its use conducive to diagnostic laboratories.

Base Sequence↗
PubMed1991

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.

Agglutination Tests↗
PubMed2000

Phylogenetic foundation of spirochetes.

The spirochetes are free-living or host-associated, helical bacteria, some of which are pathogenic to man and animal. Comparisons of 16S rRNA sequences demonstrate that the spirochetes represent a monophyletic phylum within the bacteria. The spirochetes are presently classified in the Class Spirochaetes in the order Spirochetales and are divided into three major phylogenetic groupings, or families. The first family Spirochaetaceae contains species of the genera Borrelia, Brevinema, Cristispira, Spirochaeta, Spironema, and Treponema. The second family Brachyspiraceae contains the genus Brachyspira (Serpulina). The third family Leptospiraceae contains species of the genera Leptonema and Leptospira. Novel spirochetal species, or phylotypes, that can not be presently cultivated in vitro, have been identified from the human oral cavity, the termite gut, and other host-associated or free-living sources. There are now over 200 spirochetal species or phylotypes, of which more than half is presently not cultivable. It is likely that there is still a significant unrecognized spirochetal diversity that should be evaluated.

Borrelia↗
PubMed1993

Genomic techniques for identification of Leptospira strains.

Within the Leptospiraceae family, the genus Leptospira is divided into the pathogenic L. interrogans sensu lato and the saprophytic L. biflexa sensu lato. Based on DNA-DNA hybridization, L. interrogans sensu lato has been shown to contain 7 different genomic species. Each genomic species contains numerous serovars. Pulsed-field genetic studies performed during this work demonstrated a great heterogeneity of serovars, within genomic species, based on restriction length polymorphism analysis. In contrast, an identified serovar, despite the time and region of isolation, has been shown to be highly stable in its genomic structure. The most likely reasons for this finding include the long generation time of these bacteria and the lack of acquisition of heterologous DNA. New identification techniques, based on gene amplification, have been used for Leptospira strains. These techniques represent the first available to facilitate the study of the epidemiology of Leptospira.

DNA, Bacterial↗