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Immunizing effects of structural components of Leptospira icterohaemorrhagiae.

The protective attributes of the structural components of Leptospira icterohaemorrhagiae strain Shibaura were examined. Guinea pigs were immunized with the outer envelope, the cell wall, the type-specific main antigen, and the protein constituent of the outer envelope respectively. Of the 4 preparations, the outer envelope, the cell wall and the type-specific main antigen showed a considerable protective effect in this order. The protein constituent of the outer envelope was less effective. Immunization with 200 mug of the outer envelope protected guinea pigs from lethal infection but no completely from the renal carrier state. The rate of protection from renal leptospirosis by the preparations of leptospiral components and lyophilized whole cells was always inferior to the survivor rate.

Animals↗

Leptospira fort-bragg isolated from a rat in Barbados.

Leptospira fort-bragg was isolated from the kidney tissue of a rat (Rattus rattus) trapped on a plantation in St. Philip Parish, Barbados. This constitutes the first reported isolation of this Autumnalis group serotype outside of the United States and its first known isolation from an animal species. Serologic evidence is presented which indicates that this serotype may be the cause of leptospiral infections in Barbadian cattle.

Animals↗

[Analysis of rRNA gene restriction fragments length polymorphism of Leptospira in China].

Sixty-four Leptospira international and domestic reference strains, which belonging to fifty-four serovars, and twenty-seven field strains were examined by using EcoR I restriction endonuclease analysis of genomic DNA and restriction fragments length polymorphism of rRNA gene, fifty-six Leptospiral ribotypes(RTs) were described. Most serovars gave specific patterns. Serovars in the same serogroup possess common core-segments, but we found RTs of reference strains from China and other countries are different. Most field strains have RTs with correspond of reference strains, only a few bands were shown different if RTs were different. A notable result was that the field strains of serovar pomona have the same Rt as the international reference strain but different from the domestic reference strain.

China↗

[Construction of DNA vaccine including a chimeric gene encoding flagellin and outer membrane protein antigen from Leptospira interrogons serovar lai].

OBJECTIVE: To construct a fusion expression vector for DNA vaccine, including a flagellin gene (flaB2) and an outer membrane protein gene (ompL1) to enhance the protective immunity of Leptospira interrogans serova Lai (Stain 017). METHODS: The DNA fragments encoding flaB2 and ompL1 were amplified respectively by PCR and then fused. The obtained chimeric gene ompL1-flaB2 contained a synthetic linker of ten amino acids to sustain its space conformation. RESULTS: Identified by restriction enzyme analysis, an insert fragment of 1.8 kb was demonstrated. FlaB2 and ompL1 respectively had the same sequence as reported by DNA sequencing analysis. CONCLUSION: A fusion expression plasmid containing flaB2 and ompL1 was constructed.

Bacterial Outer Membrane Proteins↗

[A new methodological approach for leptospira persistence studies in case of mixed leptospirosis].

A new methodical approach for Leptospira persistence studies in case of mixed leptospirosis, based on the use of PCR test systems with different taxonomic specificity for the indication and identification of leptospires, was developed. Two PCR test systems (G and B) were used in experiments on BALB/c white mice to study patterns of the development of mixed infection caused by leptospires of serovar poi (genomospecies L. borgpeterseni) and grippotyphosa (genomospecies L. kirschneri). The conclusion was made of good prospects of this method application in studies on symbiotic relationships of leptospires both in vivo and in vitro.

Animals↗

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars.

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G1 that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay. At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation.

Animals↗

[Infection with Leptospira interrogans, serovar mozdok, in cattle].

In East Germany the same serovar, Leptospira mozdok, of the Pomona serogroup is found in cattle as well as in swine populations (Zieris 1989). Nowadays cases of bovine leptospirosis caused by infection with L. pomona have no significance. There are marked epizoological differences between infection with L. mozdok and L. pomona. The main source of infection with L. mozdok for cattle is the black striped field mouse (Apodemus agrarius). Secondary homonomous transmission occurs among the cattle. The clinical course of both infections is the same--including peracute, acute and chronic forms. Important prophylactic measures are effective rodent control and optimising hygiene conditions both in housing and on pasture. Veterinarians in abattoirs must consider leptospirosis in the differential diagnosis in cases with icteric signs, especially when the meat is derived from emergency slaughter.

Abortion, Veterinary↗

Susceptibility of Leptospira serovars to antimalarial agents.

Leptospirosis has recently been described to cause concomitant infection with malaria. Only doxycycline has proven to have chemoprophylactic and therapeutic efficacy for both malaria and leptospirosis. To assess whether other traditional antimalarial agents have antileptospiral activity, we performed broth microdilution susceptibility testing of 16 Leptospira serovars (6 species/14 serogroups) to various agents. Artemisinin, atovaquone, chloroquine, mefloquine, primaquine, proguanil, pyrimethamine, sulfadoxine, quinine, quinidine, and combinations of atovaquone/proguanil and pyrimethamine/sulfadoxine all had a 90% minimum inhibitory concentration (MIC(90)) > 25 microg/mL (the upper limit of testing). The only agents identified with the potential to treat both infections other than doxycycline (MIC(90) = 1.56 microg/mL) were azithromycin (MIC(90) = 0.002 microg/mL) and clindamycin (MIC(90) = 0.2 microg/mL).

Antimalarials↗

[Adaptation of Leptospira interrograns (sensu stricto) to fresh water].

The contact with polluted waters is one of the main risk factors to catch leptospirosis. A study is presented about the adaptation of Leptospira interrograns to nutrient-lacking water media. For this end, leptospires were incubated in distilled water and tampon saline solution for an undetermined period of time. Leptospires kept viable in water for 98 days whereas the incubated ones in tampon saline solution survived 3 weeks only. Protein cellular and external membrane components were analyzed with electrophoresis in acrylamide gel (SDS-PAGE). When OM protein profiles of leptospires kept in water were compared to those OM profiles of cells cultured in ENJA medium, some differences were observed. A 56 kDa protein was present in leptospires kept in water for a week. This protein was identified as GroEL through Western Blot test.

Adaptation, Physiological↗

[Growth, virulence and antigenecity of Leptospira interrogans serovar mozdok in modified EMJH medium].

The effect of higher Tween 80 concentrations in EMJH synthetic medium on the growth, virulence and antigenecity of Leptospira interrogans serovar mozdok was evaluated for increasing the performances and making a full use of the detoxifying capacity of bovine serum albumin. The growth was spectrophotographically evaluated by the analysis of the bacterial growth kinetics; the obtained biomass performance and the consumption of the carbon source. The virulence was estimated in Syrian Hamster model whereas antigenecity was determined through the microagglutination technique in rabbit's polyclonal antiserum. Under controlled culture conditions, the increase of Tween 80 concentration up to 3.25 mg/ml brought about an acceleration in bacterial metabolism that managed to double cell performances with a full consumption of the carbon source, without affecting virulence and antigenecity for a number of successive subcultures.

Animals↗

[Identification of Leptospira isolates by serological and genetic methods].

Serological and genetic methods were used to study 18 leptospiral strains isolated from patients with leptospirosis in 3 Cuban provinces. The strains were grouped by microscopic agglutination with 8 polyclonal antisera. Nine monoclonal antibodies served to determine the strain serovars. Alternatively, DNA was extracted from these strains and applied by a polymerase chain reaction system described for pathogenic leptospiras. Amplified DNA was digested by restriction enzymes Alu I and Hae III. MAT grouped the strains among serogroups Ballum, Ponoma, Canicula and Icterohaemorrhagiae. Monoclonal antibodies determined the serovars of seven of the studied strains. For all the strains, an amplified fragment of 631 pb was obtained by polymerase chain reaction. The analysis of the amplified product with restriction enzymes revealed similar patterns of restriction in all the strains. The used methods made it possible to identify all the strains at different levels. It is also pointed out that the use of monoclonal antibodies allowed to typify seven strains up to the serovar level.

Agglutination Tests↗

[Microbiological characterization of candidate vaccine strains of Ballum serogroup Leptospira interrogans].

Two candidate vaccines of Ballum serogroup Leptospira interrogans were microbiologically characterized as part of the work directed to the obtention of new antileptospirosis vaccine formulations for human use. The growth kynetics of both strains was evaluated in EMJH protein medium and in 3 protein free media. The virulence was estimated in hamsters by the calculation of the mean lethal dose. The cellular and extracellular antigenic profiles were analyzed by unidimensional SDS-PAGE and compared with those from strains of Canicola, Icterohaemorrhagiae and Pomona serogroups. The antigenic homology among heterologous groups was analyzed by western blotting with serun from hasterms vaccinated with vax-SPIRAL. The results obtained showed a fastidious growth of both strains of Ballum in the studied media, a high virulence in the animal model and a large antigenic homology with strains from other serogroups of Lepstospira prevailing in Cuba.

Animals↗

Epitope mapping of monoclonal antibodies specific to serovar of Leptospira, using phage display technique.

Random heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of five monoclonal antibodies that were specific to L. australis, L. bangkok, and L. bratislava. Phages selected by biopanning were cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Almost all of the peptide epitopes were continuous or linear. Interestingly, in phages reacting with the monoclonal antibody (MAb) clones F11, F20, 2C3D4, and 8C6C4A12, the deduced amino acid sequence of the displayed peptides corresponded to a segment of hypothetical protein of the Leptospira genome (L. interrogans serovar Lai and Copenhageni). Considering the deduced amino acid sequences of phages reacting with the MAb clones F11, F20, 2C3D4, and 8C6C4A12, the consensus motif -SKSSRC-, -TLINIF-, -SSKSYR- and -CTPKKSGRC- appeared respectively. No similarity was observed among phage reacting with the MAb clone F21. The results demonstrate that T7 phage display technique has potential for epitope mapping of leptospiral MAbs, and for rapid analysis of the interactions between phage display peptides with the MAb. The finding of a phage peptide that binds to MAb with protective activity can be further tested as a candidate for leptospirosis vaccine in the future.

Antibodies, Monoclonal↗

Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle.

Effectiveness of 2 concentrations of a monovalent vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis was evaluated for protection of heifers from infection with type hardjo-bovis. Nine heifers were given 2 doses of low-dose vaccine (8.32 x 10(8) cells/dose); 9 heifers were given 2 doses of high-dose vaccine (8.32 x 10(9) cells/dose); and 1 steer and 1 heifer were maintained as nonvaccinated controls. Groups of vaccinated cattle were challenge-exposed with serovar hardjo type hardjo-bovis at 7 (n = 6), 11 (n = 6), or 15 (n = 6) weeks after completion of vaccination. All cattle were challenge-exposed by conjunctival instillation of 1 x 10(5) hardjo-bovis cells on 3 consecutive days. Both control and all vaccinated cattle became infected and shed serovar hardjo type hardjo-bovis in their urine. Leptospires were detected in 15 of 16 (94%) urine samples from control cattle and in 124 of 143 (87%) samples from vaccinated cattle. Leptospires were detected in kidneys of 17 of 18 vaccinated cattle and 2 of 2 control cattle and in the uterus or oviducts of 13 of 18 vaccinates and the 1 control heifer.

Agglutination Tests↗

Survey to estimate prevalence of Leptospira interrogans infection in mature cattle in the United States.

A total of 5,142 kidney tissue samples and 5,111 serum samples from mature cattle in 49 states and Puerto Rico were collected at slaughter. Age of cattle ranged from 1 to 16 years (mean, 6.6 years). Leptospires were isolated from 88 (1.7%) kidney tissues, and 2,493 (49%) sera contained antibodies against 1 or more of 12 Leptospira interrogans serovars. Leptospires were observed by immunofluorescence in 41 (0.8%) kidney tissues. Using agglutinin-absorption tests, 73 (83%) isolates were identified as serovar hardjo, 11 (12.5%) as serovar pomona, and 4 (4.5%) as serovar grippotyphosa. By use of restriction endonuclease analysis studies of chromosomal DNA, all isolates differed from reference serovars but were identical to strains previously isolated from cattle or swine in the United States. Of the serovar hardjo isolates, 85% were identical to restriction endonuclease analysis type (genotype) hardjo-bovis A and 11 (15%) were identical to genotype hardjo-bovis B. Serovar pomona isolates were identical to genotypes kennewicki A (64%) or kennewicki B (36%), and serovar grippotyphosa isolates were identical to the RM 52 strain. Isolation rates were significantly (P less than 0.001) higher for beef cattle than for dairy cattle and were higher (P less than 0.001) for bulls than for cows. Combined culture and immunofluorescence results indicated that 2% of mature cattle were renal carriers of leptospires.

Agglutination Tests↗

Relationships between prevalence of Leptospira interrogans in cattle, and regional, climatic, and seasonal factors.

On the basis of serologic test results and isolation of leptospires from mature cattle, distribution and prevalence of Leptospira interrogans serovars and genotypes were compared by state and region of the United States. Relationships between isolation rate and month of sample collection, mean regional temperature, and mean regional precipitation were examined. Isolation rate and seroprevalence were significantly (P less than 0.001) higher for southeastern, south central, and Pacific coastal regions than for other regions of the United States. Isolates of genotypes hardjo-bovis A and kennewicki A and B, and of serovar grippotyphosa appeared to be randomly distributed. Genotype hardjo-bovis B isolates came from a southern area of the country that extends from Georgia to New Mexico. To the authors' knowledge, this is the first recorded isolation of serovar hardjo from Hawaii. Although significant relationship was not documented between isolation rate and month or season of the year, seroprevalence for summer, fall, and winter was significantly (P less than 0.001) higher than that for spring. Regional isolation rate was related more to mean temperature (r = 0.83; P less than 0.05) than to mean precipitation amount (r = 0.34; P greater than 0.50).

Animals↗

Leptospira interrogans serovar bratislava infection in two dogs.

Two dogs with clinical histories suggestive of leptospirosis were examined serologically and culturally for evidence of leptospiral infection. Antibodies to Leptospira interrogans serovar bratislava were detected in serum from one dog, and the organism was isolated from urine of that dog. In a serologic survey of dogs in the state of Illinois, reactor rates to bratislava were higher than those to canicola or icterohaemorrhagiae. In cases of suspect canine leptospirosis, serovars such as bratislava, not contained in canine vaccines, should be considered in a differential diagnosis.

Animals↗