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PubMed2005

Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohae-morrhagiae serovar Lai.

AIM: To identify and classify all potential hemolysin candidates of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. METHODS: All of the potential hemolysin encoding genes were characterized in silico. These genes were cloned and expressed in Escherichia coli. The hemolytic activities of the expressed proteins were assayed observing the hemolysis on sheep blood agar plates. Sphingomyelinase activities of the hemolysin candidates were measured by thin-layer chromatography (TLC) and HPLC for sphingomyelin-hydrolysis. Expression and secretion of the hemolysins in L interrogans were studied by reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assays. RESULTS AND CONCLUSION: The hemolytic activities of hemolysin candidates (LA0327, LA0378, LA1027, LA1029, LA1650, LA3050, LA3937, LA4004) from L interrogans strain Lai were confirmed. They were further divided into two groups, sphingomyelinase hemolysins and non-sphingomyelinase hemolysins, based on their ability to hydrolyze sphingomyelin. Most of these hemolysins were actually expressed in living L interrogans and some of them were secreted into the environment. This study establishes an essential and complete basis for further studying the contribution of hemolysins to the pathogenesis of L interrogans.

Amino Acid Sequence↗
PubMed2002

Quantitative PCR assay to evaluate ampicillin, ofloxacin, and doxycycline for treatment of experimental leptospirosis.

The susceptibility of Leptospira interrogans serovar icterohaemorrhagiae strain Verdun to selected antibiotics used in medical practice (ampicillin, doxycycline, and ofloxacin) was evaluated in a Syrian hamster model, to determine the efficacy of these antibiotics during the course of the disease. A quantitative PCR assay was used to monitor the density of leptospires in blood and in target organs (liver, kidney, lung, heart, and spleen). Our results demonstrated the ability of ampicillin at a high dose (100 mg/kg of body weight) to clear leptospires from the host, except from kidneys and heart, where 10(2) leptospires/g remained at day 6. Ofloxacin (30 mg/kg) was unable to clear bacteria from blood or kidneys. With doxycycline (10 mg/kg), the clearance of leptospires occurred in 2 days in all the target organs studied, with the exception of liver, which required 3 days. Our data demonstrate the value of monitoring the kinetics of experimental leptospiral infection in order to accurately evaluate the efficacy of antibiotics. We have demonstrated the potential value of doxycycline for the treatment of leptospirosis cases, except in circumstances where it is contraindicated. This experimental model could be used to define better therapeutic strategies for human leptospirosis, by testing associations or new formulations of antibiotics.

Ampicillin↗
PubMed2006

Genome-wide transcriptional analysis of temperature shift in L. interrogans serovar lai strain 56601.

BACKGROUND: Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptation to a range of new environmental conditions in the organs and tissues of the infected host. Several studies have shown that a shift in culture temperature from 28 degrees C to 37 degrees C, similar to that encountered during infection of a host from an environmental source, is associated with differential synthesis of several proteins of the outer membrane, periplasm and cytoplasm. The whole genome of the Leptospira interrogans serogroup Icterohaemorrhagiae serovar lai type strain #56601 was sequenced in 2003 and microarrays were constructed to compare differential transcription of the whole genome at 37 degrees C and 28 degrees C. RESULTS: DNA microarray analyses were used to investigate the influence of temperature on global gene expression in L. interrogans grown to mid-exponential phase at 28 degrees C and 37 degrees C. Expression of 106 genes differed significantly at the two temperatures. The differentially expressed genes belonged to nine functional categories: Cell wall/membrane biogenesis genes, hemolysin genes, heat shock proteins genes, intracellular trafficking and secretion genes, two-component system and transcriptional regulator genes, information storage and processing genes, chemotaxis and flagellar genes, metabolism genes and genes with no known homologue. Real-time reverse transcription-PCR assays confirmed the microarray data. CONCLUSION: Microarray analyses demonstrated that L. interrogans responds globally to temperature alteration. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many new insights into its pathogenesis.

Bacterial Proteins↗
PubMed2006

Characterization of the cheY genes from Leptospira interrogans and their effects on the behavior of Escherichia coli.

The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome. In order to verify the function of these proteins, the putative cheY genes were cloned into pQE31 vector and then expressed, respectively, in wild-type Escherichia coli strain RP437 and cheY defective strain RP5232. The results showed that all the five cheYs could restore the swarming of RP5232 strain to some extend. Overexpression of CheYs in RP437 showed inhibited swarming of RP437. To investigate the mechanism of chemotaxis signaling in L. interrogans strain Lai, certain aspartates (Asp-53, Asp-61, Asp-70, Asp-62, and Asp-66 for L. interrogans strain Lai CheY1, CheY2, CheY3, CheY4, and CheY5, respectively) were mutated. Expression of these mutated cheYs manifested neither restoration of the swarming ability of RP5232 nor inhibition on swarming ability of RP437. Multiple amino acid sequence alignment predicted ternary structures and the result of mutation experiment suggested that these conserved aspartate residues of L. interrogans were analogous to that in E. coli CheY in function and structure. So, L. interrogans and E. coli may have similar mechanisms of activation of the chemotaxis phosphorelay pathway, but there are differences in their control by signal terminator.

Amino Acid Sequence↗
PubMed2003

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Bacterial Adhesion↗
PubMed2004

A cluster of leptospirosis cases in canoeists following a competition on the River Liffey.

On 6 November 2001, a possible case of leptospirosis was notified in a canoeist following a white-water event on the River Liffey. It emerged that a second race participant was also a possible case. An outbreak control team coordinated the epidemiological investigation, laboratory investigation, environmental assessment, communication and control measures. A cluster of six laboratory-confirmed cases of leptospirosis, serologically Leptospira interrogans serogroup Icterohaemorrhagiae was found. The attack rate was 9.2% (6/65). Fever, chills, red eyes and shortness of breath were significantly associated with being a confirmed case. Five cases were hospitalized. Swallowing more than one mouthful of water was associated with an increased risk of developing leptospirosis. Increased rainfall and release of hydroelectric water may have contributed to this outbreak. A multidisciplinary approach and use of the Internet and e-mail facilitated rapid and effective communication.

Adolescent↗
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↗
PubMed1970

Enhancement of glutamic-pyruvic transaminase activity in leptospirae by addition of rabbit serum.

Glutamic-pyruvic transaminase (GPT) in leptospirae was shown to have an optimal temperature of 30 C and pH 8.0. It was activated by cofactor pyridoxal phosphate, similar to GPT from other sources. However, GPT in all leptospirae, such as Leptospira canicola, L. icterohaemorrhagiae, L. autumnalis, and L. biflexa, evidently had less activity even when pyridoxal phosphate was present, in comparison with the cultivable spirochete Reiter treponeme and Streptococcus faecalis. The low level of activity of leptospiral GPT was enhanced more than 2.5-fold by the addition of about 10% rabbit serum, whereas GPT of S. faecalis did not show such enhancement. Enhancement of leptospiral GPT was greater with rabbit serum than with bovine or horse serum.

Journal Article↗
PubMed1978

Leptospirosis in zoo workers associated with bears.

Leptospirosis due to Leptospira interrogans serovar icterohaemorrhagiae was diagnosed in two zoo animal keepers. The implicated source of infection was bear cubs that had microscopic agglutination antibody titers to leptospires of the Icterohaemorrhagiae serogroup.

Adult↗
PubMed1992

Leptospirosis in the Seychelles.

OBJECTIVE: To ascertain the incidence of leptospirosis in the Seychelles, identify its sources, review diagnostic features and assess complications. DESIGN: A prospective survey over a two-year period 1988-1990 of all cases diagnosed as having leptospirosis at Victoria Hospital, the main referral hospital for the Islands. RESULTS: Eighty cases were diagnosed on clinical grounds with serological confirmation in 58 (73%). Leptospira interrogans serovar icterohaemorrhagiae was identified in 27 (33%) and serovar autumnalis in 2 (3%), there was broad cross-reactivity in the other specimens. In a further 7 (9%), the diagnosis was confirmed at autopsy. Useful diagnostic aids were the finding of myalgia in 68 (85%) and a raised creatine phosphokinase (CPK) level in 61 (76%). CPK elevation was found to be a more reliable indicator of disease the earlier in the illness it was measured. Other important clinical signs are reviewed. Sixty (76%) were regular drinkers suggesting a relationship with home-brewed alcohol, 48 (60%) were in at-risk occupations; 13 (16%) deaths resulted. CONCLUSIONS: Leptospirosis in the Seychelles is likely to be rat borne. An annual incidence of 60 per 100,000 was found with significant mortality. Muscle tenderness and raised CPK levels were the most reliable early diagnostic aids.

Adult↗
PubMed1976

[Leptospirosis in rodents in the old natural focus of Eastern Slovakia].

A total of 770 rodents (40 rats, 100 hamsters and 630 rodents) were serologicall examined in the old natural foci in some of the Eastern Slovakia regions; there were 10.6% of positive reactions to Leptospirosis in dilutions ranging from 1 : 100 to 1 : 1600. In rats 17 sers (40%) proved to be positive to leptospira, mostly reacting with the L. icterohaemorrhagiae. A positive reaction with the leptospira antigen was found in 13 sera (15%) of hamsters; the sera reacted with the Leptospira grypposa more frequently than with the L. serjoe. In the group of small rodents the greatest number of positive reactions to leptospirosis was obtained in microtus arvalis (10.7%); the sera reacted most frequently with L. grippotyphosa and L. serjoe. As many as 9 serological types were revealed in the group examined.

Animals↗
PubMedDate not supplied

[High resolution light microscopy: adaptation of the method and its use in the study of experimental leptospirosis in guinea pigs].

Morphological lesions in parenchimal and mesenchimal structures of liver and kidney were studied in guinea-pigs experimentally infected with Leptospira interrogans serogroup icterohaemorrhagiae in comparison with a group of non-infected guinea-pigs. All specimens were submitted to conventional light microscopy as well as to high resolution light microscopy, in one micrometer sections of tissue embedded in glycolmethacrylate. High resolution light microscopy, applied for the first time in leptospirosis, was proved very useful, since it enabled us to visualize cellular structures in the same slide used for panoramic view. Cell cohesion, brush borders, pynocytotic vesicles and organellae distributions were parameters especially suitable for analysis at this low-cost, highly precise procedure in microscopy.

Animals↗
PubMed1994

Pathologic-clinical characterization of leptospirosis in a golden Syrian hamster model.

Experimental leptospirosis was reproduced in golden Syrian hamsters (Mesocricetus aureatus). Evaluation of three Leptospira serogroups (canicola, icterohaemorrhagiae and pomona) was carried out regarding their virulence, production of symptoms and lethal activity. Macroscopic and microscopic studies of tissue lesions were also made. Animals were inoculated with 3.75 x 10(6) to 6 x 10(7) bacteria through the intraperitoneal (IP) route. The clinical development of the disease in Syrian hamsters was characterized by the presence of general symptoms between the third and fifth day after inoculation. With highly virulent strains, the distinctive clinical picture occurred and death followed before the seventh day. The strain of the pomona serogroup proved to be more virulent than those of the other serogroups and produced the most florid picture of the disease. Pathogenic aspects of this process were also studied.

Animals↗