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PubMed1991

Experimental lethal infection of Leptospira interrogans in mice treated with cyclophosphamide.

After preadministration of cyclophosphamide (300 mg/kg), BALB/c mice were lethally infected with Leptospira interrogans serovar lai and a virulent strain of Leptospira interrogans serovar copenhageni, and leptospiral cells were detected in both kidneys of infected mice by indirect immunofluorescent assay. Nonpathogenic leptospirae, Leptospira biflexa serovar patoc, Leptonema illini, and an avirulent strain of L. interrogans serovar copenhageni, were not parasitic to the mice treated with cyclophosphamide. The cyclophosphamide-treated mice were protected from the homologous leptospiral infection by passive immunization with anti-leptospiral monoclonal antibody or with rabbit antiserum and by active immunization with lyophilized organisms or with protective antigen. The results of active immunization in mice treated with cyclophosphamide agreed well with those in nontreated hamsters, which were sensitive to the organisms. Furthermore, these experiments were reproducible with any lot of cyclophosphamide used. These results indicated that cyclophosphamide-treated mice can be used in the experimental infection of Leptospira in place of hamsters or guinea pigs.

Animals↗

PubMed2005

Enzyme-linked immunosorbent assay for the detection of canine Leptospira antibodies using recombinant OmpL1 protein.

OmpL1 is a 31-kDa outer membrane protein characterized in 1993 and known to be expressed only in pathogenic Leptospira spp. Recombinant OmpL1 (GST-rOmpL1) was expressed for use as an ELISA antigen for the detection of anti-Leptospira antibodies. In immunoblot analysis, the protein reacted with sera of dogs infected with three different serotypes of Leptospira interrogans, while did not react with sera of dogs both uninfected negative controls and infected with Borrelia burgdorferi, which is closely related to Leptospira spp. Moreover, in ELISA using GST-rOmpL1, the optical density (O.D.) values from the positive controls were very high (1.125 +/- 0.549). In contrast, the O.D. values from clinically healthy dogs and dogs with diseases other than leptospirosis were very low (0.109 +/- 0.046 and 0.089 +/- 0.046, respectively). These data suggest that the detection of anti-Leptospira antibodies by ELISA using the GST-rOmpL1 protein can be applied for diagnosis of canine leptospirosis.

Animals↗

PubMedDate not supplied

LipL32, an outer membrane protein of Leptospira, as an antigen in a dipstick assay for diagnosis of leptospirosis.

Microscopic agglutination test (MAT), as well as other serological assays that aimed at detecting antibodies to Leptospira, supplements the leptospirosis diagnosis based on the clinical features. Nevertheless, false positive results have been occasionally reported when the crude antigen was used in those antibody-based tests due either to the presence of antibodies stimulated by other antigenically related pathogens in the patient's serum, or the antibodies in the serum may be stimulated by a previously unrecognized Leptospira infection, especially in the disease endemic areas. Thus, the more refined antigen should improve the serodiagnostic accuracy. Among Leptospira spp., LipL32, which is a pathogenic Leptospira outer membrane protein (OMP), expressed by the bacteria grown both in vitro and in vivo. In this study, recombinant LipL32 protein was tested by a dipstick method for its potential in serodiagnosis of leptospirosis. Preliminary results suggest that the recombinant LipL32 is a good diagnostic detection reagent for specific Leptospira IgG. Diagnostic sensitivity and specificity of the Lip32 dipstick assay, when compared to those of MAT, were 100% and 98.33%, respectively.

Agglutination Tests↗

PubMed2006

[Detection of leptospira in the vitreous body of horses without ocular diseases and of horses with equine recurrent uveitis (ERU) using transmission-electron microscopy].

Equine recurrent uveitis (ERU) is caused by persistent intraocular leptospira, which appear to use the vitreous body as a refuge. The detection of leptospira in the vitreous body of horses with spontaneous ERU by histological methods has not yet been described. Thirty eight vitreous body samples from 36 horses with ERU (collected during vitrectomy), and 10 vitreous body samples obtained from 5 horses without ocular disease (control group) were examined by transmission electron microscopy. Prior to sample collection, 2 ml of a leptospira culture suspension were injected into the vitreous body of 2 eyes enucleated from horses of the control group. The detection of leptospira in samples, experimentally inoculated with these bacteria was uncomplicated; in vitreous body samples from horses with spontaneous ERU the detection was successful in only a few cases (3/38). The morphologically varying envelope of leptospira in vitreous body samples of horses which developed ERU spontaneously suggests the existence of a bacterial masquerade in vivo.

Animals↗

PubMed1987

Isolation of Leptospira hardjo from kidneys of Ontario cattle at slaughter.

Kidneys from 117 cattle from 110 Ontario farms were examined at slaughter for leptospires. Leptospira hardjo (hardjo-bovis A) was isolated from 11 kidneys and L. kennewicki from one. The isolations were all made (12/89, 13.5%) from beef cattle from feedlots, no isolates being obtained from dairy or beef cattle from extensive farms (0/28). Isolations were only made from cattle with antibody titers (greater than or equal to 20) against the serovars recovered. Isolation was more sensitive than immunofluorescence in identifying leptospira, particularly in animals with low antibody titers against L. hardjo. Leptospira were isolated from two kidneys with multiple gross lesions of focal nephritis, but there was no correlation between the presence of scanty kidney lesions and isolations of leptospira. Leptospira hardjo infection appears to be common in Ontario feedlot cattle.

Abattoirs↗

PubMed1988

Investigation of hemolytic activity of leptospirae on solid culture media.

Results of investigation of hemolytic activity of leptospirae against red blood cells of various animal species on solid culture media using the technique of agar layers are presented. It has been shown that hemolytic properties of pathogenic and saprophytic leptospirae differ with respect to red blood cells of the sheep, the rabbit, the hamster, the albino rat and the albino mouse. Hemolytic activity of leptospirae regarding the erythrocytes of the cattle, the horse and the fowl depends on the strain of leptospirae. The advantage of using solid nutrient media for the determination of hemolytic properties of leptospirae in comparison with liquid media has been demonstrated.

Animals↗

PubMedDate not supplied

[Antibiograms of Leptospira on solid media in Petri dishes].

The sensitivity of fowl leptospirae to antibiotics was tested by diffusiometry on solid media in Petri dishes. The method includes two stages: 1) visible leptospirae cultures are obtained by central seeding and incubation during 3--5 days at 28 or 37 degrees C; 2) antibiotic tablets are disposed at 2 cm from the visible edge of the multiplication zone of the leptospirae. The edge is marked with a pencil at the moment in which the antibiotics are applied. Incubation is continued at the same temperatures. The results are read after five days taking as reference the position of the marginal multiplication line. The sensitivity of leptospirae to antibiotics is expressed by arrested multiplication and resistance by advance of the marginal multiplication line up to or even beyond the antibiotic tablets. Fowl leptospirae are sensitive to penicillin, tetracyclin, chloramphenicol, streptomycin, novobiocin, polymixin, pristinamycin, neomycin, erythromycin, and resistant to mycostatin, septrin, rifampicin, optoquine, sulphatiazol.

Anti-Bacterial Agents↗

PubMed1994

[Transcript expression of the CPL 5x, BMD-3A, BMD-10 Interrogans leptospira].

Total RNA of Leptospira interrogans SV Lai strain 017 was prepared by the method of Licl-Urea, and was used in dot hybridization with biotin-labelled DNA probes. The probes included BMD-3A, BMD-10, which were the leptospirial protective antigen genes, and CPL 5x, which was the genus specific gene of interrogans Leptospira. All of the three probes have shown various degrees of hybridization signs, proving that they all have transcript expression in leptospira. The transcript expression is the main way of regulating the gene expression in procaryotae, and it is significant in molecular genetics of Leptospira. The results indicate that the antigens encoded by the BMD-3A, BMD-10 may play an important role in immune reaction against leptospiral infection and provide a clue to the development of gene-engineering vaccine. The results also suggest that the antibody against the antigen encoded by CPL 5x is a useful tool in the classification of Leptospira interrogans.

Antigens, Bacterial↗

PubMed1993

[Antigen analysis of McAb E4B7D5 directed against outer envelope of Leptospira interrogans serovar lai by SDS-PAGE and immunoblot].

McAb E4B7D5 was prepared by hybridoma technology in BALB/c mice immunized to outer envelope of Leptospira interrogans serovar lai. This McAb agglutinated specifically with all the 13 serovars of Icterohaemorrhagiae serogroup in MAT test at high titres and protected the guinea pigs against the attack of virulent strain (017) of serovar lai. SDS-PAGE and immunoblot were used to analyse the reaction of the outer envelopes of the five strains of Leptospira (Leptospira interrogans Icterohaemorrhagiae serogroup, serovar lai, 017 and 601 stains; L. interrogans Hebdomadis serogroup, hebdomadis serovar strain 156; L. interrogans Australis serogroup australis serovar strain 620; biflexa leptospira patoc serovar strain Patoc I; with McAb E4B7D5. Results indicated that this McAb E4B7D5 recognized specifically 34.5 kd and 39.5 kd outer envelope proteins of serovar lai, strain 017 and 601, so it might be an immunoprotective McAb directed against Leptospira interrogans serovar lai. It was suggested that 34.5 kd and 39.5 kd outer envelope proteins of serovar lai recognized specifically by McAb E4B7D5 might be immunoprotective antigens.

Animals↗

PubMed1997

A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen.

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.

Animals↗

PubMed1998

[Intraocular and serum antibody titers to Leptospira in 150 horses with equine recurrent uveitis (ERU) subjected to vitrectomy].

Between February 1993 and July 1997, 150 horses suffering from recurrent uveitis were subjected to parsplana vitrectomy. In these horses, antibody titers to Leptospira serovars were determined in serum samples and in samples from diluted vitreous collected during vitrectomy. Although the vitreous samples were diluted with 250 ml of balanced salt solution, in 86 of the 150 vitreous samples (= 57%) the antibody titers were higher than in the serum samples. Additionally, serum samples from 77 horses suffering from ERU, but which were not subjected to vitrectomy, and serum samples from 97 horses with clinically normal eyes were analyzed for antibodies to Leptospira serovars. Among the 227 horses with ERU (150 treated surgically, 77 treated conservatively) 50 horses (50 of 227 = 22%) had serum antibody titers to Leptospira serovars of > or = 1:800. Among the 97 horses with clinically normal eyes, 24 horses (24 of 97 = 25%) had serum antibody titers to Leptospira serovars of > or = 1:800. In undiluted vitreous samples from 20 horses with clinically normal eyes, no antibody titers to Leptospira serovars could be detected. Among the 150 horses with ERU, 90 animals (90 of 150 = 60%) had antibody titers of > or = 1:100 in the diluted vitreous samples, the difference being highly significant (p < 0.001). The findings are discussed in relation to the etiology of recurrent uveitis in horses.

Animals↗

PubMed1994

Rapid and specific detection of pathogenic Leptospira species by amplification of ribosomal sequences.

We have developed an assay for the detection of pathogenic Leptospira that is based on the polymerase chain reaction. With the combination of agarose gel electrophoresis and blotting, pathogenic Leptospira can be discriminated specifically from nonpathogenic Leptospira and other bacterial species. This method, based on the amplification of 16S ribosomal RNA sequences, is able to detect 10 leptospiral cells/mL in cattle urine samples and 100 leptospiral cells/mL in pig urine samples. Using this assay leptospires were detected in urine samples from cattle that were experimentally infected with Leptospira interrogans serovar hardjo type hardjobovis.

Animals↗

PubMed1986

What is the correct specific epithet for pathogenic Leptospirae?

The underlying documentation of the first descriptions of members of the genus Leptospira is carefully revised. Stimson's publication of? Spirochaeta interrogans (1907) is to be inadequate and not suitable to be recognized as the first valid description of a new genus being only a description of a spirochete-like microorganism without any documentation. The naming of the species of pathogenic leptospira interrogans is therefore not in confirmity with the requirements of the International Code of Nomenclature. The first valid description of saprophytic leptospira is given by Wolbach and Binger (1914) and that of pathogenic ones by Inada and Ido (1915). The correct name for the species of pathogenic leptospira is therefore icterohaemorrhagiae and for the saprophytic ones biflexa.

Humans↗

PubMed1988

Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C.

The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10, and 1% survival were determined for representative serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay, L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.

Dose-Response Relationship, Radiation↗

PubMed2006

Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp.

The spirochetes of the Leptospira genus contain saprophytic and pathogenic members, the latter being responsible for leptospirosis. Despite the recent sequencing of the genome of the pathogen L. interrogans, the slow growth of these bacteria, their virulence in humans, and a lack of genetic tools make it difficult to work with these pathogens. In contrast, the development of numerous genetic tools for the saprophyte L. biflexa enables its use as a model bacterium. Leptospira spp. require iron for growth. In this work, we show that Leptospira spp. can acquire iron from different sources, including siderophores. A comparative genome analysis of iron uptake systems and their regulation in the saprophyte L. biflexa and the pathogen L. interrogans is presented in this study. Our data indicated that, for instance, L. biflexa and L. interrogans contain 8 and 12 genes, respectively, whose products share homology with proteins that have been shown to be TonB-dependent receptors. We show that some genes involved in iron uptake were differentially expressed in response to iron. In addition, we were able to disrupt several putative genes involved in iron acquisition systems or iron regulation in L. biflexa. Comparative genomics, in combination with gene inactivation, gives us significant functional information on iron homeostasis in Leptospira spp.

Amino Acid Sequence↗

PubMed1962

A new pathogenic Leptospira, not readily cultivated.

Alexander, Aaron D. (Walter Reed Army Institute of Research, Washington, D.C.), Herbert G. Stoenner, Garnett E. Wood, and Robert J. Byrne. A new pathogenic Leptospira, not readily cultivated. J. Bacteriol. 83:754-760. 1962.-A pathogenic Leptospira was isolated from water of the Grand River, (S.D.) that differed significantly from other known leptospirae in that it could not be cultivated in conventional leptospiral media. Growth was promoted in Fletcher's medium modified to contain 20% rabbit serum. The isolate, after several serial passages, was lethal for hamsters. It could not be adapted to grow in the chick embryo. Guinea pigs and calves inoculated with the isolate developed febrile and antibody responses but showed no other overt signs of disease. The strain was identified on the basis of cross-agglutination and agglutinin-adsorption tests as a new subserotype of Leptospira naam and was therefore designated as L. naam, subserotype dakotii.

Animals↗

PubMed1988

Motility of the spirochete Leptospira.

Spirochetes are a group of bacteria with a unique ultrastructure and a fascinating swimming behavior. This article reviews the hydrodynamics of spirochete motility, and examines the motility of the spirochete Leptospira in detail. Models of Leptospira motility are discussed, and future experiments are proposed. The outermost structure of Leptospira is a membrane sheath, and within this sheath are a helically shaped cell cylinder and two periplasmic flagella. One periplasmic flagellum is attached subterminally at either end of the cell cylinder and extends partway down the length of the cell. In swimming cells, each end of the cell may assume either a spiral or a hook shape. Translational cells have the anterior end spiral shaped, and the posterior end hook shaped. In the model of Berg et al., the periplasmic flagella are believed to rotate between the sheath and the cell cylinder. Rotation of the anterior periplasmic flagellum causes the generation of a gyrating spiral-shaped wave. This wave is believed sufficient to propel the cells forward in a low-viscosity medium. The cell cylinder concomitantly rolls around the periplasmic flagella in the opposite direction--which allows the cell to literally screw through a gel-like viscous medium without slippage. This model is presented, and it is contrasted to previous models of Leptospira motility.

Cell Movement↗

PubMed1987

Tears and aqueous humor from horses inoculated with Leptospira contain antibodies which bind to cornea.

An antigenic relationship between Leptospira interrogans and equine cornea was previously described by us. An enzyme-linked immunosorbent assay was employed in the present work to investigate the existence of anti-leptospira and anti-cornea antibodies in tears, aqueous humor and serum from horses inoculated i.m. with those antigens. Ten days after a booster by the same route, antibodies that bind to microtiter plates, coated with an homogenate of either equine cornea or leptospira, were detected in those fluids and in the sera. At the same time, the corneas of the horses began to develop a diffuse opacity. This finding of anti-leptospira antibodies in equine tears and aqueous humor shows the pathway along which they arrive at the cornea and bind to it.

Animals↗