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Chemical and biological properties of endotoxin from Leptospira interrogans serovars canicola and icterohaemorrhagiae.

1. Endotoxin-like activity was extracted with phenol-chloroform-petroleum either (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola. Chemical analysis of leptospiral cells obtained from the PCP extract indicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml: 3.0, 4.5 and 1.0 for icterohaemorrhagiae and 3.3, 5.6 and 1.5 for canicola. 2. The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorrhagiae and canicola PCP extract and 0.5 pg/ml for E. coli O111:B4 LPS, lethality for chicken embryos (LD50 45, 25 and 1.0) for icterohaemorrhagiae, canicola and E. coli O111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 degrees C, 0.9 degrees C and 2.2 degrees C for canicola, icterohaemorrhagiae and E. coli O111:B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells, 62%, 75% and 90% for 2.0 micrograms/ml of icterohaemorrhagiae, canicola PCP extract and E. coli O111:B4 LPS. The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells. 3. These results demonstrate that Leptospira endotoxin activity is similar to E. coli O111:B4 lipopolysaccharide.

Animals↗

[Assay of genomic DNA homology among strains of different virulent leptospira by DNA hybridization].

Nick translation labelling technique was applied to the preparation of two 32P labelled genomic DNA probes from L. interrogans serovar Lai strain 017 and Leptonema illini strain 3055, respectively. Dot-blotting and Southern-blotting with DNA of five strains leptospira from different genus and species were performed. The results showed that certain differences among the L. interrogans, L. biflexa and Leptonema illini could be detected by endonuclease assay. L. interrogans strain 017 with L. biflexa strain Patoc I and Leptonema illini 3055 strain exhibited very little homology. L. interrogans strains 017, 601, and 245 from different serogroup and serovar had a high degree of homology. Leptonema illini strain 3055 showed little homology with L. interrogans and L. biflexa. Therefore DNA hybridization may be used as a tool for the identification and classification of leptospira.

Blotting, Southern↗

[The serogroup and serotype distribution of Leptospira in Sichuan Province from 1958-1987].

This paper summarized the serogroup and serotype distribution of 7,560 strains Leptospira in Sichuan province from 1958 to 1987. At present, Leptospira 16 serogroup and 35 serotype have been found. The Icterohaemorrhagiae group Lai type is the major and stable serogroup and serotype in patients. We have proved that Rodents insectivores and domestic animals are the main reservoir host. The former carries Icterohaemorrhagiae group and the latter carries Pomona group. The serotype carried on the reservoir animal Apodemus agrarius is mostly Icterohaemorrhagiae Lai and is the principal infection source of Leptospirosis in Sichuan province. In addition, this paper discussed the reason of main serogroup change, the possible role of frog as an infection source of Hebdomadis Leptospirosis.

Animals↗

Characterization of trypsin extractable antigens of Leptospira interrogans serovars pomona, bataviae & L. biflexa serovar patoc.

Leptospira strains after treatment with trypsin released an antigen (Tx) which consisted of protein (4.2 mg/ml), carbohydrate (0.39 mg/ml) and hexosamine (0.025 mg/ml). Immunodiffusion, immunoelectrophoresis and indirect haemagglutination tests revealed serological cross reactions among the three strains used. Inhibition of microscopic agglutination reaction by the Tx antigen was observed in homologous system only. The antigen was partially sensitive to heat (80 degrees C for 10 min) as detected by immunodiffusion and microscopic agglutination inhibition. These data revealed the presence of serovar-specific and genus-specific antigens on Leptospira strains. It is suggested that this antigen could be useful in developing a serodiagnostic test for leptospirosis.

Agglutination Tests↗

[Human diseases caused by leptospirae of the canicola serogroup in the Krasnodar Territory].

During the period of 34 years (1957-1990) diseases caused by leptospires of the serogroup canicola were confirmed in 2.7% of the total number of patients. The analysis revealed that in the presence of epidemic outbreaks Leptospira infection caused by bacteria of the serogroup canicola occurred 1.7 times more often than at the time when such outbreaks were absent. Periodic increases of leptospirosis caused by this serogroup were registered in individual towns and regions of the territory in different years. The analysis of 33 medical histories made it possible to state that the clinical manifestations of leptospirosis caused by bacteria of the serogroup canicola were characteristic of Leptospira infections caused not only by this serovar. As a rule, this kind of leptospirosis was registered in rural and urban areas as individual isolated cases.

Adult↗

Serosurvey of Leptospira agglutinins among railway employees in the National Railway Compartments of Bologna and Venice.

An epidemiological investigation was performed to evaluate the prevalence of anti-leptospiral agglutinins within a selected population: the railway workers of the Italian Railway Compartments of Bologna and Venice. Leptospira infection prevalence was found to be 10.38%, which represents the highest frequency ever reported in Italy. There was a significantly higher prevalence of agglutinins among the staff of the Railway Compartment of Venice. A clear prevalence of the bratislava serovar was registered, followed by the australis serovar and the icterohaemorrhagiae serovar. The influence of different kinds of railway work and that of supposed risk factors, such as hobbies and housing conditions, together with the influence of the ABO blood groups on the distribution of Leptospira agglutinins, are discussed.

ABO Blood-Group System↗

Chemical and biological properties of a phenol-water extract from Leptospira interrogans. Evidence for the absence of lipopolysaccharide.

Leptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.

Animals↗

Aseptic meningitis caused by Leptospira australis.

Meningeal involvement in leptospiral infection is quite common, usually mild and often overlooked. In contrast, cases of isolated involvement of the central nervous system, including aseptic meningitis, have been reported only rarely. A case of a patient with acute aseptic meningitis caused by Leptospira australis serovar bratislava is reported. This is believed to be the first report of aseptic meningitis due to Leptospira australis. This case indicates the need to consider human leptospirosis in the differential diagnosis of aseptic meningitis.

Adult↗

Identification of a second flagellin gene and functional characterization of a sigma70-like promoter upstream of a Leptospira borgpetersenii flaB gene.

Leptospira borgpetersenii, one of the causative agents of leptospirosis in both animals and humans, is a bacterial pathogen with characteristic motility that is mediated by the rotation of two periplasmic flagella (PF). The flaB gene coding for a core polypeptide subunit of PF was previously characterized by sequence analysis of its open reading frame (ORF) (M. Lin, J Biochem Mol Biol Biophys 2:181-187, 1999). The present study was undertaken to isolate and clone the uncharacterized sequence upstream of the flaB gene by using a PCR-based genome walking procedure. This has resulted in a 1470-bp genomic DNA sequence in which an 846-bp ORF coding for a 281-amino acid polypeptide (31.3 kDa) is identified 455 bp upstream from the flaB start codon. The encoded protein exhibits 72% amino acid identity to the deduced FlaB protein sequence of L. borgpetersenii and a high degree of sequence homology to the FlaB proteins of other spirochaetes. This has demonstrated for the first time that a second flaB gene homolog is present in a Leptospira species. The newly identified gene is designated flaB1, and the previously cloned flaB renamed flaB2. Within the intergenic sequence between flaB1 and flaB2, a potential stem-loop structure (12-bp inverted repeats) was identified 25 bp downstream of the flaB1 stop codon; this could serve as a transcription terminator for the flaB1 mRNA. Three E. coli-like promoter regions (I, II, and III) for binding Esigma(70), a regulatory sequence uncommonly found in flagellar genes, were predicted upstream of the flaB2 ORF. Only promoter region II contains a promoter that is functional in E. coli, as revealed at phenotypic and transcriptional levels by its capability of directing the expression of the chloramphenicol acetyltransferase (CAT) gene in the promoter probe vector pKK232-8. These observations may suggest that flaB1 and flaB2 are transcribed separately and do not form a transcriptional operon controlled by a single promoter.

Bacterial Proteins↗

Cloning and sequence analysis of the gene encoding LipL32 of Leptospira interrogans serovar Sejroe.

Leptospira, a member of the order Spirochaetales, is the causative agent of leptospirosis, an important zoonosis encountered worldwide. The Leptospira interrogans serovar Sejroe was grown in EMJH medium and its DNA was isolated using standard techniques. The LipL32 gene was amplified using the reported primer of Kirschneri of LipL32. The amplified product was found to comprise 756 base pairs. This amplified gene fragment of LipL32 lipoprotein was cloned in E. coli (DH5 alpha) cells using pDrive plasmid as a vector. The recombinant cells were selected on LB agar medium containing ampicillin, X-gal and isopropyl-beta-D-thiogalactopyranoside. Plasmid was extracted from the recombinant white colonies, and restriction endonuclease (RE) analysis was carried out using PstI and SalI. On partial sequence analysis, the product exhibited 756 base pairs, corresponding to 251 amino acids. The cloned gene could be further used for expression of recombinant protein for serodiagnosis of leptospirosis.

Bacterial Outer Membrane Proteins↗

Experimental demonstration of an antigenic relationship between Leptospira and equine cornea.

Horses inoculated with either equine cornea or killed Leptospira interrogans serovars pomona, tarassovi, icterohaemorrhagiae, wolffi and hardjo, developed corneal opacity and produced antibodies which made it possible to demonstrate partial antigenic identity between equine cornea and four of those serovars employed. These antibodies were isolated by means of immunoadsorptions, purified by ion-exchange chromatography (DEAE-Sephadex A-50) and run by immuno-electrophoresis in agar gel. Both antibodies, anti-equine cornea and anti-leptospira, showed that they corresponded to the IgGb subclass. They bound themselves to equine cornea in vivo and in vitro as was proved by immunofluorescence. This antigenic relationship may be in part responsible for pathogenesis of corneal opacity in leptospirosis of horses.

Animals↗

Biochemical analysis by SDS-PAGE and western blotting of the antigenic relationship between Leptospira and equine ocular tissues.

The antigenic relationship between Leptospira interrogans, equine cornea and lens was previously noted in our studies. Serum antibodies from horses inoculated with serovars wolffi, pomona, icterohaemorrhagiae, and tarassovi, were able to bind to five antigenic fractions from both cornea and lens, as demonstrated by immunoblotting. These antigens seem to be made up of protein and carbohydrates. After treatment with periodate for cleavage of glycoside ring structures, those fractions kept their condition of target for anti-Leptospira antibodies. Nevertheless, all fractions lost that condition after being subjected to peptide hydrolysis.

Animals↗

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans.

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti-Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

Animals↗

Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira hardjo infections in cattle.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Leptospira interrogans serovar hardjo (hardjo) infection in cattle was compared with the microscopic agglutination test (MAT). Glutardialdehyde was used in the ELISA to couple sonicated hardjo antigen to the microtiter plate. Mouse monoclonal anti-bovine IgG1 coupled to peroxidase was used as conjugate. Sera from calves experimentally inoculated with hardjo reacted positively in the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Positive and negative field sera from 704 adult cattle on 90 farms were examined by the MAT and the ELISA; a 90% correlation between the two tests was demonstrated. Eighty-six sera from calves inoculated with four Leptospira serogroups other than hardjo and 227 field sera from adult cattle with naturally occurring leptospirosis other than hardjo were examined by the ELISA. Fewer than 1% of these heterologous sera reacted with hardjo antigen in the ELISA. We concluded that the ELISA described in this report is an advantageous alternative to the MAT for diagnosing leptospirosis.

Agglutination Tests↗

The role of the common vole (Microtus arvalis) in the epidemiology of bovine infection with Leptospira interrogans serovar hardjo.

Control of leptospirosis in cattle depends on the presence of other possible maintenance hosts, with which cattle may have contact. Twenty-seven common voles (Microtus arvalis) were trapped on a dairy farm where the cattle were infected with Leptospira interrogans serovar hardjo (hardjo). In the sera of 11 voles, titres greater than or equal to 100 against serogroup Grippotyphosa were measured with the microscopical agglutination test (MAT). From 8 of these 11 voles, which also showed interstitial lymphoplasmacellular nephritis, Leptospira interrogans serovar grippotyphosa was isolated. We found no evidence that the common vole is a maintenance host for hardjo in this biotope.

Agglutination Tests↗

Effect of streptomycin treatment on the shedding of and the serologic responses to Leptospira interrogans serovar hardjo subtype hardjobovis in experimentally infected cows.

Shedding patterns of and serologic responses to Leptospira interrogans serovar hardjo subtype hardjobovis (L. hardjobovis) have been studied in experimentally infected cows treated with streptomycin in comparison to experimentally infected cows receiving no such treatment. Fourteen cows were experimentally infected with L. hardjobovis, and blood and urine samples were collected weekly for 24 weeks. The microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. The polymerase chain reaction (PCR) was used to determine bacterial shedding in urine. Six weeks after infection six cows were treated with the antibiotic streptomycin (25 mg/kg body weight/day); three cows were treated only once, and the remaining three were treated for five consecutive days. After treatment all six cows had lower serologic responses compared to the untreated cows. The treated cows became also PCR-negative two days after the first treatment, whereas the eight untreated cows remained PCR-positive for at least 70 days. Cows that stopped shedding did not resume shedding within the observation period. Since streptomycin treatment reduces the period of shedding, transmission of leptospira via contaminated urine might be prevented by a single treatment of an infected herd.

Agglutination Tests↗

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Bacterial Typing Techniques↗