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PubMed1978

Enzymatic degradation of H2O2 by Leptospira.

The enzymes responsible for reducing H2O2 were surveyed in 49 strains of Leptospira by using semiquantitative assays for catalase and peroxidase. The survey revealed a differential distribution of catalase and peroxidase activities between the two leptospiral complexes. The pathogenic Leptospira interrogans strains gave strong catalase and weak or negative peroxidase reactions. Conversely, the nonpathogenic Leptospira biflexa strains gave strong peroxidase and negative or weak catalase reactions. An intermediate group of four L. biflexa strains, which were isolated from mammals, fell into the high peroxidase, low or negative catalase group. One water isolate, H-23, gave strong reactions for both enzymes and was examined for virulence and in vitro growth parameters. Results indicate metabolic differences between pathogens and water forms in their abilities to reduce H2O2.

Catalase↗

PubMed1983

Multiple pathways for isoleucine biosynthesis in the spirochete Leptospira.

Spirochetes of the genus Leptospira have previously been shown to use an unusual pathway to synthesize isoleucine. For reasons of convenience, we assume that only one unusual pathway is found in the genus, and we refer to it as the pyruvate pathway. We determined the distribution of this pyruvate pathway in representatives of the seven Leptospira DNA hybridization groups. Our method included labeling the representative strains with radioactive carbon dioxide and other radioactive precursors, fractionating the cells, and determining the specific activities (counts detected per nanomole) of the amino acids found in the protein fractions. On the basis of isoleucine biosynthesis, we found that the genus can be classified as follows: class I primarily, if not exclusively, uses the well-known threonine pathway; class II uses mostly the pyruvate pathway, with a minor amount of isoleucine being synthesized via the threonine pathway; and class III uses the pyruvate pathway exclusively. No relationship appears to exist between the degree of DNA hybridization and the classes of isoleucine biosynthesis. Although the precise intermediates on the pyruvate pathway are unknown, the origin of the carbon skeleton of isoleucine synthesized by this pathway is consistent with a borrowing of the leucine biosynthetic enzymes. However, we found that the pyruvate pathway is not controlled by leucine and that the two isoleucine pathways are independently regulated. Finding major and highly evolved multiple biosynthetic pathways of a specific amino acid within one genus is unique, and, conceivably, represents phylogenetic diversity within Leptospira.

Isoleucine↗

PubMed1964

CATALASE ACTIVITY IN LEPTOSPIRA.

Rao, P. J. (University of Illinois, Urbana), A. D. Larson, and C. D. Cox. Catalase activity in Leptospira. J. Bacteriol. 88:1045-1048. 1964.-A number of serotypes of Leptospira were found to possess catalase activity, although considerable variation in activity existed among various serotypes. Catalase activity of L. pomona was reduced by inhibitors commonly employed for arresting catalase activity in other biological systems. Catalase activity was increased three to five times by growing cultures under conditions of oxygen availability; however, aeration had no beneficial effect on total viable cell crop. The relationship of oxygen to metabolism and future studies on virulence of the leptospirae is discussed.

Azides↗

PubMed2000

Technological advances in the molecular biology of Leptospira.

Pathogenic members of the genus Leptospira have been refractory to genetic study due to lack of known mechanisms of genetic exchange. To bypass this limitation, several techniques have been useful for Leptospira gene discovery, including heterologous complementation of Escherichia coli mutants, screening of DNA libraries with probes, and random sequence analysis. Construction of combined physical and genetic maps revealed the presence of two circular chromosomal replicons. The organization of the L. interrogans genome is quite variable, with genetically similar strains differentiated by many rearrangements. These rearrangements likely occur through recombination between repetitive DNA elements found scattered throughout the genome. Analysis of intervening sequences and genes encoding LPS biosynthetic enzymes provide evidence of lateral transfer of DNA between Leptospira spp. We have also gained insight into the biology of these bacteria by analyzing genes encoding LPS and outer membrane proteins (OMPs). Some of these OMPs are differentially expressed. Characterization of mechanisms governing the expression of the OMP genes should provide insight into host-parasite interactions. Furthermore, recent advances in heterologous expression of leptospiral OMP genes are opening new avenues of vaccine development.

Animals↗

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↗

PubMed1994

Survey on the prevalence of leptospira infections in the Italian population.

This investigation is the first nationwide survey on the circulation of leptospira infections in human beings in Italy. In nine out of twenty Italian regions, representative samples of the population were investigated for the presence of leptospira infections. Unexpectedly, leptospira infections were found to be widespread, the number of cases being much higher than the diagnosed clinical cases. There were found to be high, medium, and low risk areas. On the whole, the risk for the rural population was no higher than the risk for urban dwellers; leisure activities, contact with animals and residence on the plain versus residence in the hills were important risk factors. There was an unidentified risk factor in urbanites which was absent in the rural population. A changing pattern in infecting serovars was observed, with infections from serogroups Sejroe, Javanica and Australis prevailing over infections from the Icterohaemorrhagiae and Bataviae serogroups, which were the main agents of human leptospirosis during the 1950s. The mechanisms of these changes, the need for epidemiological surveys and improved diagnostic methods of screening are discussed.

Adult↗

PubMed2001

PCR genome walking identifies a genetic locus comprising two heat shock genes (hslV and hslU) from Leptospira borgpetersenii serovar hardjobovis.

PCR walking on genomic DNA of Leptospira borgpetersenii serovar hardjobovis has identified a genetic locus comprising two heat shock genes (hslV and hslU). This is the first molecular study on hslV and hslU in a Leptospira species. The hslV gene has an open reading frame (ORF) of 543 bp coding for a 180-amino acid polypeptide (19.476 kD) with 49-62% identity to other bacterial HslV homologs. Forty-three nucleotides downstream from the hslV stop codon was found a second heat shock gene hslU with an ORF of 1401 bp encoding a 53.139-kD polypeptide of 467 amino acids with 49-56% identity to the HslU homologs from other bacteria. An Escherichia coli-like ribosome binding site (RBS) and a final sigma32-like heat shock promoter sequence were present upstream of both heat shock genes. Identification of the hslV and hslU genes in a Leptospira species and the high degree of similarity of the encoded proteins to the E. coli homologs suggest that the encoded HslV and HslU proteins function as forming an ATP-dependent protease complex as in E. coli.

ATP-Dependent Proteases↗

PubMed1993

Diversity of ribosomal DNA fingerprints of Leptospira serovars provides a database for subtyping and species assignation.

Ribosomal DNA fingerprints from 103 pathogenic Leptospira strains were examined using EcoRI restriction and fragment length polymorphisms of rRNA genes. Sixty-nine new leptospiral ribotypes were described, in addition to 49 previously observed. Except for 5 strains, a good correlation between DNA homology data and ribotyping was observed. The genospecies of 31 reference strains could be presumed, since they shared 13 ribotypes with strain(s) previously studied by DNA homology. Furthermore, the definition of common rRNA hybridization fragments in each recognized DNA hybridization group provides information about the status of Leptospira reference strains yet to be classified. With 118 ribotypes now defined among the validated serovar reference strains, rRNA fingerprints constitute a database for subtyping Leptospira species.

DNA Fingerprinting↗

PubMed2006

Detection and differentiation between pathogenic and saprophytic Leptospira spp. by multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was developed for diagnosing leptospirosis and differentiating pathogenic and saprophytic leptospires. Specific primers were designed to amplify 23S rDNA from pathogenic Leptospira and saprophytic Leptospira spp. PCR products from 27 pathogenic and 5 (including 1 intermediate) saprophytic serovars were 615 and 316 base pairs (bp), respectively. After the restriction enzyme's digestion of PCR products, the fragments by SacI of pathogenic serovars and by PstI of saprophytic serovars were 339 and 276 bp and 202 and 114 bp, respectively. The PCR primers enabled amplification of DNA from L. meyeri serovar Ranarum as a pathogenic Leptospira spp. The PCR assay could detect 1 to 2 cells of leptospires and not amplify DNA from other 18 bacterial species. The sensitivity and specificity of this PCR in rat kidney, using isolation as gold standard, were 98.6% and 100%, respectively. The most appropriate sample preparation of blood for detecting DNA was buffy coat. Among the sample preparations from 7 laboratory-confirmed leptospirosis cases, leptospiral DNA was detected in all 7 buffy coat preparations, whereas leptospiral DNA was detected in only 3 plasma or serum samples. The PCR assay may be useful as a diagnostic tool for leptospirosis.

Animals↗

PubMed2004

Identification Leptospira santarosai serovar shermani specific sequences by suppression subtractive hybridization.

In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar shermani. Suppression subtractive hybridization was employed to isolate DNA fragments present in pathogenic L. santarosai serovar shermani but absent in non-pathogenic L. biflexa serovar patoc. Analysis of 23 subtracted DNA clones revealed 25 gene fragments by BLASTX program. Eight clones showed similarity to transposase genes and three clones displayed homology with either translation or metabolism related genes. Four clones were similar to outer membrane protein, penicillin-binding protein, CreD-like protein and the protein of two-component signal transduction system, respectively. One clone had TPR repeat domain and five clones had significant similarity with hypothetical proteins of unknown functions. The remaining four clones exhibited no homology with any known genes. These results indicate that subtractive hybridization can successfully identify genes that are absent from the non-pathogenic Leptospira and provide a starting point for clarifying the differential genes expression between pathogenic and non-pathogenic Leptospira species.

Base Sequence↗

PubMed2005

Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1.

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.

Animals↗

PubMed2006

NF-kB activation and p38 phosphorilation in microglial cells infected with Leptospira or exposed to partially purified leptospiral lipoproteins.

Recently, we have shown a differential susceptibility of non-pathogenic vs. pathogenic leptospires to phagocytosis and killing by microglial cells. Although all ingested to some extent, only the pathogenic strains survived intracellularly while the non-pathogenic ones were killed in a time-dependent manner. By the same infection model, here we demonstrate that microglial cells respond to Leptospira infection with a time- and dose-dependent induction of molecular signals (p38 phosphorilation and NF-kB activation) and the production of soluble factors (cytokines and nitric oxide). Such bio-molecular response is predominantly observed against the pathogenic Leptospira; the phenomenon is reproduced by leptospiral lipoproteins and, to a lower extent, by leptospiral-derived LPS. These data provide initial evidence that Leptospira affects microglial cell response in a different manner depending upon the virulence of the infecting strain; specific bacterial components happen to be involved in the induction of such pathogen-induced immune response.

Animals↗

PubMed2006

Mimotope of Leptospira from phage-displayed random peptide library is reactive with both monoclonal antibodies and patients' sera.

The study aim was to use random heptapeptide library displayed by bacteriophage T7 for identifying mimotopes from 15 monoclonal antibodies (MAbs) specific to Leptospira spp., and from four leptospirosis patient sera, respectively. The bound phages, selected from fourth round of bio-panning with each antibody, were cloned by plaque isolation and the binding specificity of individual clones were confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. All together 150 phages were selected, mimotope from 86 phages (56.6%) were found to match with protein sequences of Leptospira from GenBank database. The predominant mimotopes were mimotope with sequence LTPCD that found in 27.3%, followed by TPCSK (16%), KSKKSS (4%), KTKRXAS (4%), SSKSYR (3.3%), DPNXNSF (3.3%), KSGRC (2.6%), TLINIF (2%), TPCI (2%), 1.33% each with mimotopes PKKS, PCNTKXTA, and CTKKK, and one phage each (0.66%) with mimotopes PTFGS, TNSKRK, SKSSRC, RSKRIR, VTNNTP, and CSNXSKR. Interestingly, mimotopes LTPCD, TPCSK, and TPCI were found to react with both MAb and patient's sera. The matched proteins from GenBank namely, leptospiral putative outer membrane protein (matched with mimotope PTFGS), thermolysin precursor protein (matched with mimotope TPCIXXGSAS), and hypothetical protein LIC12228 (matched with mimotope CSNXSKR), were found to locate at outer membrane of Leptospira. These phage mimotopes and matched proteins may have potential for further use as diagnostic reagent and immunogen against leptospirosis in the future. The results demonstrate that phage display technique has potential for rapidly identifying phage mimotopes that interact with leptospiral MAbs and patient's sera.

Amino Acid Sequence↗

PubMed2006

Evaluation of LSSP-PCR for identification of Leptospira spp. in urine samples of cattle with clinical suspicion of leptospirosis.

We evaluated the use of low-stringency single specific primer PCR (LSSP-PCR) for genetically typing Leptospira directly from urine samples of cattle with clinical suspicion of leptospirosis. Urine samples obtained from 40 cattle with clinical suspicion of leptospirosis were amplified by specific PCR using the following primers: Internal 1/Internal 2 and G1/G2. The internal primers were designed from the gene sequence of the outer membrane lipoprotein Lip32 from Leptospira kirschneri, strain RM52. The PCR products were amplified with these two pairs of primers, which had approximately 497 and 285bp, respectively, and were subsequently used as a template for LSSP-PCR analysis. The genetic signatures from the leptospires which were present in the urine samples allowed us to make a preliminary identification of the leptospires by comparing the LSSP-PCR profiles obtained directly from urine samples with those from reference leptospires. The LSSP-PCR profiles obtained with the Internal 1 primer or with the G1 primer allowed the grouping of the leptospires into serogroups. LSSP-PCR was found to be a useful and sensitive approach capable of identifying leptospires directly from biological samples without the need for prior bacterial isolation. In conclusion, the LSSP-PCR technique may still be helpful in discriminating serogroups of Leptospira from different animal reservoirs, since the early identification of carrier animals and information on the shedding state are crucial to prevent the spread of leptospiral infection to other animals and humans.

Animals↗

PubMed2001

Herd-level risk factors associated with Leptospira spp. seroprevalence in dairy and beef cattle in Spain.

Our aim in this cross-sectional study was to investigate the seroprevalence of Leptospira spp. infection in herds and cattle and the relationships between seroprevalence and beef versus dairy, size, replacement policy and grazing management in a representative area of beef- and dairy-cattle production in Spain. Herds were the initial sampling unit. Blood samples were collected from 762 dairy cattle belonging to 81 herds and 1238 beef cattle from 134 herds; sera were tested for antibodies against 11 serovars of Leptospira (autumnalis, ballum, bratislava, canicola, castellonis, copenhagheni, grippotyphosa, hardjo, louisiana, pomona and tarassovi) using the microagglutination test. Forty-three percent (36.2-49.5%) of the herds and 8% (6.4-8.8%) of the individuals were seropositive against one or more of the serovars studied. Bratislava was the most-prevalent serovar (24% of the herds and 4% of the individuals) followed by hardjo (11 and 1%, respectively). Grippotyphosa, copenhagheni and tarassovi were more prevalent in dairy than in beef herds (P<0.001, P<0.05, P<0.05, respectively) -- but no significant association was found between herd-size and Leptospira seroprevalence for any of the serovars considered.

Animal Husbandry↗

PubMed1986

Decreased lipopolysaccharide content and enhanced susceptibility of leptospiras to serum leptospiricidal action and phagocytosis after treatment with diphenylamine.

Growth of leptospiras in the presence of diphenylamine (DPA) caused a decrease in the content of leptospiral lipopolysaccharide (LPS). In association with this decrease of LPS, leptospiras became susceptible to anti-leptospiral action of normal rabbit serum (NRS), leptospiricidal action of antibody and complement, and killing by phagocytes. DPA-treated leptospiras were eliminated rapidly from the blood of infected mice and could not grow in the animals.

Aniline Compounds↗

PubMed2000

Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai.

It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis. We have isolated an Escherichia coli clone carrying the 7.8kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L. borgpetersenii. The clone showed a clear beta-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays. The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively. The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases. Interestingly, however, E. coli cells harboring sphH did not show sphingomyelinase or phospholipase activities. Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane. The SphH was successfully expressed in E. coli as a histidine (His)-SphH fusion protein. Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L. biflexa serovar patoc. We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.

Amino Acid Sequence↗

PubMed1998

Physical and genetic maps of the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero no.1 chromosome and sequencing of a 19-kb region of the genome containing the 5S rRNA gene.

We report the construction of physical and genetic maps of the chromosome of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No.1 using pulsed-field gel electrophoresis of DNA fragments generated by digestion with enzymes SrfI, AscI, FseI, and NotI and using reciprocal hybridization. We also sequenced the 19-kilobase (kb) DNA segment including the one gene for 5S rRNA (rrf) of pathogenic Leptospira. The size of the chromosome of the strain Ictero No.1 was estimated to be 4673kb and was found to be similar to those of the chromosomes of the leptospira strains Verdun (serovar icterohaemorrhagiae) and RZ11 (serovar pomona). The strains Verdun and RZ11 carry a small 350-kb replicon (minichromosome), and the strain Ictero No.1 also contained the same kind of molecule together with the chromosome. The physical maps of the strains Ictero No.1 and Verdun were almost identical, as were the locations of the selected genes, except for the location of one of the 16S rRNA genes. Overall, the genetic organization appeared to be conserved within the serovar icterohaemorrhagiae strains. In the sequenced region, we identified 10 putative ORFs and one rrf sequence, and the transcription orientations were all the same. A homology search for the products deduced from the sequenced data revealed that the orf H exhibited high similarity to malic acid enzyme of Haemophilus influenzae and fumarate hydratase of Escherichia coli (orf J). The rest of the putative products encoded by ORFs in the sequenced region showed little similarity with the proteins contained in the databases and were considered to be unknown proteins.

Bacterial Proteins↗