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PubMed2004

Unique structural characteristics of peptide deformylase from pathogenic bacterium Leptospira interrogans.

Peptide deformylase (PDF), which is essential for normal growth of bacteria but not for higher organisms, is explored as an attractive target for developing novel antibiotics. Here, we present the crystal structure of Leptospira interrogans PDF (LiPDF) at 2.2A resolution. To our knowledge, this is the first crystal structure of PDF associating in a stable dimer. The key loop (named the CD-loop: amino acid residues 66-76) near the active-site pocket adopts "closed" or "open" conformations in the two monomers forming the dimer. In the closed subunit, the CD-loop and residue Arg109 block the entry of the substrate-binding pocket, while the active-site pocket of the open subunit is occupied by the C-terminal tail from the neighbouring molecule. Moreover, a formate group, as one product of deformylisation, is observed bound with the active-site zinc ion. LiPDF displays significant structural differences in the C-terminal region compared to both type-I and type-II PDFs, suggesting a new family of PDFs.

Amidohydrolases↗
PubMed2006

Spatial hierarchical variances and age covariances for seroprevalence to Leptospira interrogans serovar hardjo, BoHV-1 and BVDV for cattle in the State of Paraíba, Brazil.

We estimated spatial hierarchal variances and age-group covariances for seroprevalence to Leptospira interrogans serovar hardjo (LeptoH), bovine viral-diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1) among 2343 cattle from 72 properties sampled in the State of Paraíba, Brazil in 2000. From each property, eight animals in each of the four age categories were evaluated. The age categories studied were: pre-weaned (0-6 months), young (7-18 months), replacement (19-30 months) and mature (>30 months). Overall seroprevalence to LeptoH was 16.0% and showed clustering at all levels of the spatial hierarchy and had a high posterior probability of being negatively correlated between replacement and mature groups within herds. Seroprevalence to BoHV-1 was 46.6% and demonstrated very little clustering among levels of the spatial hierarchy and was positively correlated between young and replacement groups within herds. Seroprevalence to BVD was 22.2% and was strongly clustered within herds and was positively correlated between young and replacement age groups within herds.

Age Factors↗
PubMed2002

Enzymatic properties of a new peptide deformylase from pathogenic bacterium Leptospira interrogans.

Peptide deformylase (LiPDF), a target protein for antibacterial agents from pathogenic bacteria Leptospira interrogans was identified and purified. Enzymatic studies including kinetics and inhibition revealed new inspiring highlights. The purified active enzyme was a dimer and showed a hyperbolic progress plot when the substrate was low but an excess substrate inhibition effect in higher substrate concentration. Variants on the metal-binding ligand-Cys102 were constructed to verify the indispensable attribute. Also the variant, LiPDF with the insertion residues (R(70)Y(71)P(72)G(73)T(74) P(75)D(76)V(77)) between the conserved motif 1 and motif 2 excised, was constructed and displayed no marked changes on enzymatic features. The results of atom absorbance proved that it contains a tightly bound Zn2+ rather than Fe2+ in E. coliPDF that is an essential cofactor for its high catalytic activity.

Amidohydrolases↗
PubMed2000

Occurrence of [--> 3)-beta-D-Manp-(1 --> 4)-beta-D-Manp-(1 -->]n units in the antigenic polysaccharides from Leptospira biflexa serovar patoc strain Patoc I.

In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1-3) from cells of Leptospira biflexa serovar patoc strain Patoc I (L. biflexa patoc Patoc I) by the hot phenol-water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti-L. biflexa serum and also cross-reacted in similar extents toward most of the other leptospiral antisera tested. Such immunoreactions were specifically inhibited by a beta-(1 --> 4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1H and 13C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Manp-(1 --> in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. biflexa polysaccharides.

Animals↗
PubMed1996

Presence of antigen and antibodies in serum and genital discharges of heifers after experimental intrauterine inoculation with Leptospira interrogans serovar hardjo.

The excretion of Leptospira interrogans serovar hardjo in cervico-vaginal mucus (CVM) or urine and the local and systemic immune responses to the organism were monitored in eight susceptible heifers after intrauterine inoculation while six similar heifers served as controls. All the heifers were inseminated at the subsequent oestrous periods. The overall percentage pregnancy rate (the number of pregnancies divided by the total number of inseminations) was lower in the infected heifers than in the controls though not significantly (33.3 v 50.0 per cent). Leptospires were detected, in either the urine or the CVM of six of the eight infected heifers during the study period of 15 weeks, either by direct immunofluorescence or dark ground microscopy; the bacteria did not grow in culture from any of the CVM samples. The control heifers remained free from evidence of infection. In the infected heifers, mean titres of at least 1:100 in a microscopic agglutination test were maintained for one to two weeks before declining to 1:10 to 1:30, whereas in serum IgG-ELISA tests (developed by using either protein or carbohydrate antigens), antibody titres of at least 1:100 were maintained throughout the study. During oestrous periods, IgA antibodies were detected more frequently in CVM with titres which were usually higher than the titres of IgG.

Agglutination Tests↗
PubMed1996

Presence of antigen and antibodies in serum and genital discharges of cows from dairy herds naturally infected with Leptospira interrogans serovar hardjo.

Samples of cervico-vaginal mucus from 163 bulling cows (group 1) and post calving discharges from 59 newly calved cows (group 2) in five dairy herds naturally infected with Leptospira interrogans serovar hardjo were examined for the presence of antigen and IgG and IgA antibodies by using two ELISA systems which were protein or carbohydrate based. Corresponding serum samples were examined for systemic immune responses by using a microscopic agglutination test (MAT) and IgG-ELISA tests. Antigen was detected by direct immunofluorescence in six of the 163 samples of cervico-vaginal mucus. Both IgG and IgA antibodies were detected by ELISA in the genital discharges with a prevalence much higher than that obtained by the MAT but lower than that observed with the serum IgG-ELISA. Combining both groups, none of the MAT-positive cattle was negative by serum-ELISA. By using the protein or carbohydrate fraction serum IgG-ELISA assays, respectively, 29 or 41 per cent of the MAT-negative cows were positive at a titre of at least 1:40. Similarly, eight or 23 samples (10 or 27 per cent) had titres of at least 1:20 in the genital discharge ELISA for IgG and IgA antibodies, respectively. The serum IgG-ELISA was the most efficient in detecting hardjo antibodies, but in group 2 the IgG- and IgA-ELISA of the post calving discharge proved to be equally effective.

Animals↗
PubMed1986

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay.

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.

Adenosine Triphosphate↗
PubMed1998

Epidemiologic features of equine Leptospira interrogans of human significance.

Leptospirosis is a zoonotic bacterial disease caused by Leptospira interrogans. There is a serologic evidence that horses are exposed to L. interrogans and, as a shedder of these organisms, can be a threat to humans. We examined risk factors associated with the risk of testing seropositive to three L. interrogans serovars (L. icterohaemorrhagiae, L. grippotyphosa, and L. canicola) in the horses of New York State, in order to understand the epidemiology of the disease and suggest strategies to control and prevent equine leptospirosis. To carry out this study, blood samples were collected from a random sample of 2551 horses and tested for the presence of antibodies to the above serovars using the microscopic agglutination test. Samples with a titer $100 were considered positive. Clinical and demographic data were collected on each horse, the farms' management practices and ecology. Logistic regression analysis was used to develop a multivariate indexing system and to identify factors significantly associated with the risk of leptospirosis. Four indices were developed based on the possible sources of exposure: rodent exposure index; wildlife exposure index; soil and water index; and management index. The soil and water index was significantly associated with the risk of exposure to all three serovars. Management was positively associated with L. icterohaemorrhagiae and L. canicola. Density of horses turned out together was positively associated with the risk of exposure to L. grippotyphosa. We concluded that indirect exposure of horses to L. interrogans through contaminated soil and water appears to be significantly associated with the risk of exposure to all three serovars. Management appears to play an important role in the exposure to L. interrogans. Modification of management practices might reduce the horses' risk of exposure and hopefully minimize the human hazards.

Animals↗
PubMed1984

The immunoglobulin response of swine following experimental infection with Leptospira interrogans serovar pomona.

The antibody response of pigs following experimental infection with Leptospira interrogans serovar pomona was examined using enzyme immunoassay (EIA) and the microscopic agglutination test (MAT). Leptospires elicited the production of both IgM and IgG classes of antibody, with IgG levels persisting for much longer than IgM. A comparison of MAT and EIA indicated that the detection of specific IgM by EIA was potentially useful in distinguishing between past and recent infection in pigs. Agglutinins were also detected in the urine of infected animals but these antibodies could not be detected by EIA.

Animals↗
PubMed1985

The classification of Sejroe group serovars of Leptospira interrogans with monoclonal antibodies.

Using the hybridoma technique we produced monoclonal antibodies to serovars of Leptospira interrogans. We focussed on serovar hardjo which is an important pathogen for humans and animals, and on other serovars of the Sejroe group. With combinations of monoclonals, characteristic patterns of agglutination were observed according to the specific pattern of antigenic determinants of that serovar. A set of 13 monoclonal antibodies was composed which allowed the classification of almost all serovars of the Sejroe group.

Agglutination Tests↗
PubMed1987

Comparative classification of Leptospira serovars of the Pomona group by monoclonal antibodies and restriction-endonuclease analysis.

The serovars of the Pomona group of Leptospira interrogans are antigenically closely related and can be classified only with difficulty by conventional typing methods. Monoclonal antibodies (MCAs) were prepared to serovars of the Pomona group. The MCAs were directed against antigens of polysaccharide nature. A battery of six MCAs was selected for the classification of Pomona group reference strains. These MCAs could be used for the typing of all Pomona group strains and unknown isolates. Alternatively, DNA was extracted from the same strains and isolates and digested with restriction enzymes. The patterns that were obtained after gel separation of the DNA digests were characteristic and also allowed classification. Restriction enzyme analysis was complicated but gave detailed information. Classification with MCAs could be easily and rapidly performed.

Antibodies, Monoclonal↗
PubMed1987

Molecular comparison of antigens and proteins of virulent and avirulent clones of Leptospira interrogans serovar copenhageni, strain Shibaura.

The antigens and the protein profiles of virulent and avirulent clones of Leptospira interrogans serovar copenhageni, strain Shibaura were compared by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and radioimmunoprecipitation. An antigen specific to the virulent clone was detectable only in the virulent clone by Western blotting using anti-virulent clone but not anti-avirulent clone antisera. The molecular weight of the antigen was estimated to be approximately 32000 by SDS-PAGE, and the antigen was found to be thermostable. A 43000 dalton antigenic molecule was found to be specific for the virulent clone, and the 41000 dalton antigenic molecule was found to be specific for the avirulent clone by Western blotting using both anti-virulent and anti-avirulent clones antisera. Therefore, these antigenic molecules seemed to contain at least one antigenic determinant which is common to the virulent and the avirulent clones. No difference between the virulent and avirulent clones was observed in the comparison of protein profiles on SDS-PAGE and surface exposed antigenic protein profiles on radioimmunoprecipitation using radioiodinated leptospiral surface exposed proteins. Therefore, the antigens which were different between the virulent and avirulent clones were suspected not to be protein antigens.

Antigens, Bacterial↗
PubMed1987

Selection of antigenic variants from Leptospira interrogans serovar canicola by means of anti-canicola monoclonal antibody.

Antigenic variants were isolated from canicola by a single selection with anti-canicola monoclonal antibody CT-3. The variants were not identical to any serovars of serogroup Canicola and thought to be a new serovar. Variation frequency was calculated at 5.9 X 10(-4). The usefulness of monoclonal antibodies for selection of antigenic variants of leptospiras is discussed.

Agglutination Tests↗
PubMed1988

Presence of a variety of antigenic variants belonging to at least 4 different serovars in a population of Leptospira interrogans serovar hebdomadis.

The presence of antigenic variants belonging to at least 4 different serovars, jules, kremastos and 2 new serovars, was demonstrated in a population of cloned Leptospira interrogans serovar hebdomadis. Thirteen antigenic variants were isolated from the clone of serovar hebdomadis by culturing in the presence of 13 monoclonal antibodies against 5 serovars, hebdomadis, worsfoldi, kambale, jules and kremastos Kyoto, respectively. These variants were divided into 4 groups, based on the reactivity with the above monoclonal antibodies. Four antigenic variants, one each from the 4 groups, were identified as jules, kremastos and 2 different new serovars. The variation frequency of the 13 antigenic variants ranged from 10(-5.48) to 10(-7.70) except for one, whose frequency was as high as 10(-1.31).

Agglutination Tests↗
PubMed1988

Broad reacting surface antigens in Leptospira biflexa serovar andamana.

Previous investigations had demonstrated that genus-specific and species-specific antigens of leptospires are deep-seated within the leptospiral cell. Conversely, the present study has shown that strain CH11, serovar andamana of the non-pathogenic species of Leptospira biflexa is endowed on its surface with two cross-reacting antigens; a newly recognized antigen common to L. interrogans and L. biflexa spp. and an antigenic determinant common to a previously described genus-specific protein antigen (GP-Ag). The serovar andamana, when thimerosal-treated, behaved like an interspecies-specific antigen, cross-reacting in the complement fixation test with sera from rabbits immunized with L. interrogans and L. biflexa spp. and with sera from subjects with leptospirosis from various serovars. In the immuno electron microscopic test, human leptospirotic sera bound to the surface of thimerosal-treated andamana and not to the surface of untreated andamana showing that an interspecies-specific antigen was located underneath the outermost layer of this serovar. In the same test, the monoclonal antibody GP-7 against the GP-Ag bound to the surface of untreated andamana and not to the surface of thimerosal-treated andamana, showing that an antigenic determinant, common to GP-Ag and different from the first one, was located on the outer membrane of andamana. In human leptospirotic sera, antibodies against the cross-reacting antigen of thimerosal-treated andamana demonstrated by the complement fixation test were formed earlier and in a higher percentage of sera than the serovar-specific antibodies against 16 L. interrogans serovars demonstrated by the microagglutination test.

Agglutination Tests↗
PubMed2001

Effects of hydrostatic pressure on the Leptospira interrogans: high immunogenicity of the pressure-inactivated serovar hardjo.

The Hardjoprajitno strain of Leptospira interrogans serovar hardjo was subjected to different hydrostatic pressures. Complete inactivation occurred when the leptospires were treated with 2 kbar for 60 min. Electron microscopy showed dislocation of the outer membrane, partial loss of the helical shape and extrusion of the axial filament from the cytoplasmic cylinder of the pressurized leptospires. When the pressure-treated leptospires were inoculated into rabbits they were highly immunogenic. The sera of these animals presented a titer of 2048 in the microscopic serum agglutination reaction. Fluorescence measurements indicated that the action of pressure on the leptospires might have resulted from perturbation on membrane protein components, permitting the binding of the fluorescent probe bis (8-anilinonaphthalene-1-sulfonate) (Bis-ANS). This is the first report of the use of hydrostatic pressure to inactivate pathogenic bacteria with the potential to lead to a vaccine.

Anilino Naphthalenesulfonates↗
PubMedDate not supplied

Enhanced antibody response in cattle against Leptospira hardjo by intradermal vaccination.

A commercial killed Leptospira hardjo vaccine (with adjuvant) and non-adjuvanted preparation of the same vaccine were used in comparison of the effectiveness of the intradermal (i.d.) and subcutaneous (s.c.) routes for these vaccines. The tests were conducted in 50 females aged 6-14 months. After the first vaccination, both types of vaccine elicited a very poor antibody response by both routes of vaccination. However, after booster vaccination, the commercial vaccine (with adjuvant) elicited a remarkable immune response which was twice as high by i.d. compared with s.c. vaccination. No local or general adverse reactions were observed after i.d. vaccination with the adjuvanted commercial vaccine (potassium aluminium sulphate).

Administration, Cutaneous↗
PubMed1976

[Study of a specific polyoside and a group antigen extracted from Leptospira biflexa patoc, patoc I strain].

We extracted from L. biflexa patoc a fraction F, reacting in hemagglutination and ring tests with sera prepared against more than ten different serogroups. This fraction contains mainly a polysaccharide (65 per cent), the role of which was clearly demonstrated in the precipitation reaction with homologous antisera, through periodic oxidation; it also contains lipids (20 per cent) and proteins (10 per cent). We isolated from this fraction F, by Biogel column chromatography, 2 distinct antigens, one, F2, carrying the patoc-type specificity, the other, F 1B, a group specificity shared by many leptospira. These antigens differ not only immunogically, but also in their chemical composition. The type-specific antigen F2 contains mainly a polysaccharide composed of arabinose and glucosamine (possibly an immunodominant sugar). As for the group-specific fraction F 1B, its composition is more complex since lipids and proteins are also found with the polysaccharide. This antigen could therefore be a lipoglycoprotein.

Animals↗