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PubMed1999

Low prevalence of antibodies against the zoonotic agents Brucella abortus, Leptospira spp., Streptococcus suis serotype II, hantavirus, and lymphocytic choriomeningitis virus among veterinarians and pig farmers in the southern part of The Netherlands.

Serum samples from 102 veterinarians and 191 pig farmers from the southern part of the Netherlands were investigated for antibodies against Brucella abortus, Leptospira spp, Streptococcus suis serotype II, Hantavirus (HV), and lymphocytic choriomeningitis virus (LCMV). All samples were collected in 1993 and stored until this study was performed. The prevalence of antibodies against B.abortus in veterinarians (4.5%) was significantly higher (P = 0.01) than in pig farmers (0%). None of the veterinarians (0%) and only one pig farmer (0.5%) had antibodies against Leptospira spp. Furthermore, significantly (P = 0.015) more veterinarians (6%) than pig farmers (1%) had antibody titres against muramidase-released protein (MRP),a protein of pathogenic S. suis serotype II strains. None of the veterinarians and a total of 3 (1.6%) pig farmers had antibody titres against HV. The prevalence of antibodies against LCMV tended to be higher in pig farmers (2.6%) than in veterinarians (0%) (P = 0.10). It can be concluded that the prevalence of antibodies against the investigated zoonotic agents in veterinarians and pig farmers is low.

Agriculture↗
PubMed1990

Leptospira interrogans serovar unipertama isolated in Malaysia.

A leptospiral isolate from a bovine kidney was found to be antigenically different from all previously recognized serovars of Leptospira interrogans based on the cross-agglutinin absorption test. The new serovar belongs to the Sejroe serogroup, and the name Leptospira interrogans serovar unipertama is proposed for it, with strain K2-1 as the reference strain.

Agglutination Tests↗
PubMed1990

DNA relatedness among strains of Leptospira biflexa.

The slot blot method of DNA hybridization was used to study 38 strains of Leptospira biflexa belonging to 38 serovars. Fifteen of these serovars were placed into six groups. The remaining 23 serovars were generally too diverse to show significant DNA relatedness either to these groups or to one another. Serovar thracia was related to Group 5, but it was not included in this group because its percent relatedness was too low. We found that genetically related organisms were antigenically dissimilar. The absence of any significant genetic relationship between Leptonema illini and the Leptospira biflexa serovars tested supports the placement of the former species in a separate genus.

DNA, Bacterial↗
PubMed1992

Genetic characterization of pathogenic Leptospira species by DNA hybridization.

A total of 66 serovars of potentially pathogenic Leptospira species were examined by slot blot hybridization, and 57 of these serovars were classified in six DNA homology groups. In cases in which common serovars were studied, the results were in general agreement with the results of previous workers, who used different DNA homology methods. However, we propose a new species, Leptospira kirschneri, comprising the following serovars: bulgarica, butembo, cynopteri, dania, grippotyphosa, kabura, kambale, ramisi, and tsaratsovo. Seven of these serovars have not had their DNAs studied by other workers.

DNA, Bacterial↗
PubMed1986

Heterogeneity of lipopolysaccharide banding patterns in Leptospira spp.

Strains of Leptospira interrogans and Leptospira biflexa, examined by electrophoresis after whole cell lysis and protein digestion, revealed the presence of 2-keto-3-deoxyoctonate and an heterogeneous lipopolysaccharide electrophoretic banding pattern, which was characteristic of the species.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Purification, characterization and serological properties of a glycolipid antigen reactive with a serovar-specific monoclonal antibody against Leptospira interrogans serovar canicola.

A glycolipid antigen possessing a serovar-specific antigenic determinant of Leptospira interrogans serovar canicola was purified from a chloroform/methanol extract of the organism. The purification procedures included silicic acid column chromatography and preparative thin-layer chromatography (TLC). Antigenic activity was detected by a TLC-enzyme immunostaining technique using monoclonal antibody CT3, which specifically agglutinates serovar canicola and only weakly serovar sumneri but no other serovars of Leptospira. The purified glycolipid reacted with CT3 antibody, indicating that the glycolipid possessed a serovar-specific antigenic determinant. Infrared spectrum and proton nuclear magnetic resonance analyses showed that the glycolipid contained sugar and lipid moieties, which possessed amide linkages and an acetyl group. Gas-liquid chromatography-mass spectrometry analysis showed that the glycolipid contained two unknown sugars, one of which (unknown sugar II) appeared to be associated with the antigenic determinant specific for canicola. The serovar-specific antigenic determinant was destroyed by mild alkali treatment of the glycolipid. These findings suggested that the antigenic determinant was an alkali-labile moiety which may be related to the unknown sugar II.

Antibodies, Monoclonal↗
PubMed1990

Protective activity of glycolipid antigen against infection by Leptospira interrogans serovar canicola.

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23-30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.

Animals↗
PubMed1990

Cloning of genes required for amino acid biosynthesis from Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.

Amino Acids↗
PubMed1990

Purification and characterization of a protein antigen from Leptospira interrogans serovar hardjo, common to a wide range of bacteria.

A protein with a molecular mass of 64 kDa (P64) from Leptospira interrogans serovar hardjo was partially purified by using successively, phase partitioning with Triton X-114, ion-exchange chromatography and sucrose gradient centrifugation. Purification to homogeneity was obtained by electroelution of P64 from SDS-polyacrylamide gels. Monospecific rabbit antiserum (R alpha P64) was prepared using the purified protein preparation. P64 had a native molecular mass of greater than 670 kDa and was recognized by R alpha P64 as well as by human antisera. Western blotting of leptospiral serovars and 18 other bacterial species with R alpha P64 showed that P64 was cross-reactive with an equivalent antigen in a wide range of bacteria, indicating that it belongs to a family of antigens previously designated 'common antigen'. This putative common antigen from Leptospira appears to have a sub-surface location, but its function is not yet known.

Animals↗
PubMed1988

Reaction of monoclonal antibodies with species specific determinants in Leptospira interrogans outer envelope.

A set of 24 monoclonal antibodies (MABs) was produced against an outer envelope preparation from Leptospira interrogans serovar copenhageni. The MABs reacted in enzyme immunoassay with species-specific determinants of an antigen in the leptospiral outer envelope (OE) of pathogenic but not of saprophytic species of Leptospira. The MABs did not agglutinate whole leptospires, nor could they opsonise homologous leptospires for phagocytosis by mouse macrophages or protect new-born guinea-pigs against lethal infection. The MABs reacted by Western blotting with a 35 x 10(3)-mol-wt band in OE separated on SDS-polyacrylamide gels, and also reacted with other bands to a lesser extent. The determinants to which the MABs were directed were localised in the leptospiral OE by immunogold labelling techniques.

Agglutination Tests↗
PubMed1995

Random amplified polymorphic DNA fingerprinting for rapid identification of leptospiras of serogroup Sejroe.

Eight leptospiral and three non-leptospiral primers were examined in the development of a random amplified polymorphic DNA (RAPD) fingerprinting method for identification of leptospiral serovars within the serogroup Sejroe. The strains examined comprised 19 reference strains of serogroup Sejroe, five field strains and 17 Leptospira borgpetersenii serovar hardjo type hardjobovis strains. Unique DNA banding patterns were obtained for each of the 17 reference strains with L. hardjo type hardjobovis-derived primers, and all clinical isolates were identified as L. hardjo type hardjobovis. Although the profiles were specific for each serovar, most serovars within serogroup Sejroe clustered into previously recognised genetic groups. RAPD fingerprinting is a simple, rapid method suitable for the identification of some Leptospira serovars.

Animals↗
PubMed2004

Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA.

The search for novel antigens suitable for improved vaccines and diagnostic reagents against leptospirosis led to the identification of LigA and LigB. LigA and LigB expression were not detectable at the translation level but were detectable at the transcription level in leptospires grown in vitro. Lig genes were present in pathogenic serovars of Leptospira, but not in non-pathogenic Leptospira biflexa. The conserved and variable regions of LigA and LigB (Con, VarA and VarB) were cloned, expressed and purified as GST-fusion proteins. Purified recombinant LigA and LigB were evaluated for their diagnostic potential in a kinetic ELISA (KELA) using sera from vaccinated and microscopic agglutination test (MAT)-positive dogs. Sera from vaccinated dogs showed reactivity to whole-cell antigens of leptospires but did not show reactivity in the KELA assay with recombinant antigens, suggesting a lack of antibodies to Lig proteins in the vaccinated animals. The diagnostic potential of recombinant Lig antigens in the KELA assay was evaluated by using 67 serum samples with MAT > or =1600, which showed reactivity of 76, 41 and 35% to rConA, rVarA and rVarB, respectively. These findings suggest that recombinant antigen to the conserved region of LigA and LigB can differentiate between vaccinated and naturally infected animals.

Amino Acid Sequence↗
PubMed1987

Biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton.

The biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method were studied in mice. The addition of 12.5 micrograms/ml or more of LLS fraction increased the incorporation of [3H]thymidine into in vitro cultured spleen cells of C57BL/6 mice, while the activity of the LLS fraction was about 20 times weaker than that of Salmonella typhimurium lipopolysaccharide (LPS). Pretreatment of murine spleen cells with rabbit anti-mouse thymocyte antiserum did not diminish the mitogenic activity of leptospiral LLS, and the LLS could not increase the incorporation of [3H]thymidine into thymocytes, suggesting that LLS acts on a B-lymphocyte population of lymphocytes. When sheep erythrocytes and LLS fraction were injected intraperitoneally into BALB/c mice, LLS exhibited an enhancing effect on antibody response in vivo. However, lethal toxicity of the LLS fraction was about 500 times lower than that of LPS in C57BL/6 mice loaded with galactosamine. No antitumor activity of leptospiral LLS (250-1,000 micrograms/mouse) against the ascites form of Ehrlich carcinoma in ddY mice was observed. The biological activities of the LLS fraction from the organism were weaker than those of gram-negative bacterial LPS, suggesting that Leptospira possesses no typical LPS.

Adjuvants, Immunologic↗
PubMed1988

Comparative studies of strains Ictero No. I and RGA as the type strain of Leptospira interrogans: agglutinin absorption test, protein and antigen profiles, and enzyme activities.

Strain Ictero No. I, the first isolate of Leptospira, isolated by Inada and Ido in 1914, was found to be sufficiently qualified to be the type strain of Leptospira interrogans rather than strain RGA. In an agglutinin absorption test, anti-Ictero No. I serum was not absorbed completely with strain RGA, and 25% of the homologous titer remained unabsorbed, while anti-RGA serum was completely absorbed with strain Ictero No. I. Thus, strain Ictero No. I was not serologically identical with strain RGA, and the two strains were considered to be different serovars. A protein band with a molecular weight of approximately 33,000 daltons was detected in strain Ictero No. I but not in strain RGA by SDS-PAGE. By Western blotting, this protein band was detectable with anti-Ictero No. I serum but not with anti-RGA serum. The presence of the 33K protein in strain Ictero No. I, but not in strain RGA, was confirmed by radioimmunoprecipitation using [125I]-labeled antigens, indicating that the protein antigen was surface-exposed. Only 8 of the 89 enzymes activities were different between strains Ictero No. I and RGA (line Sapporo). From the above results, we propose that strain Ictero No. I should be designated as the type strain of L. interrogans instead of strain RGA.

Agglutination Tests↗
PubMed1990

Cloning of genes for a hemolytic factor of Leptospira interrogans serovar autumnalis strain Congo 21-543.

A DNA fragment encoding a hemolytic factor was cloned from the parasitic spirochete Leptospira interrogans serovar autumnalis strain Congo 21-543. Initial clones were isolated by screening a genomic library in pBR322 in Escherichia coli for hemolytic activity. Hemolytic activity was coded by a 4.5 kilobase BamHI-HindIII fragment. Southern hybridization with DNAs from other strains of Leptospira using this gene as a probe showed that DNAs from non-parasitic strains failed to hybridize with the probe, whereas those from all parasitic strains tested had the sequence which hybridize to the probe.

Cloning, Molecular↗
PubMed2006

First record of Leptospira borgpetersenii isolation in the Amami Islands, Japan.

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI-long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.

Animals↗
PubMed1993

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni.

A chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with R alpha P64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4 kb EcoRI-Bam HI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5 kDa (Hsp10) and 58 kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an E sigma 70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat-shock conditions as determined by [35S]-methionine pulse labelling experiments. As no functional heat-shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat-shock conditions, similar to mechanisms described for several Gram-positive organisms.

Amino Acid Sequence↗
PubMed1992

The isolation and identification of Leptospira interrogans serovar bratislava from a pig in Germany.

During a study on the improvement of selective media for the isolation of leptospires from clinical material, leptospires were isolated from the kidneys of a pig. Test material was cultured in EMJH-medium containing 0.4% rabbit serum and 3 inhibitory substances (Rifampicin 10 micrograms/ml, Amphotericin B 2 micrograms/ml, 5-Fluorouracil 100 micrograms/ml). The cultured leptospira-strain (Berlin 406) was identified to serogroup level using the cross agglutination method and it was further typed to serovar level by MAB's, factor analysis and restriction endonuclease analysis (REA). MAB typing of Berlin 406 gave an agglutination pattern profile typical of serovar bratislava. Factor serum identification clearly identified this strain as serovar bratislava. On REA examination, Berlin 406 gave a profile identical to that of the serovar bratislava type strain Jez Bratislava i.e. genotype B1. This is the first report of a confirmed isolate of Leptospira interrogans serovar bratislava from domestic animals in Germany. There exist a number of possibly important implications as this agent is being incriminated in the cause of clinical disease not only in pigs, but also in horses and dogs in other countries.

Animals↗