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PubMed1982

Experimental infection with a Leptospira hardjo strain isolated from cattle of the eastern plains of Colombia.

The infectivity and pathogenicity of a strain of Leptospira hardjo isolated from the eastern plains of Colombia were evaluated. Ten pregnant heifers were artifically inoculated and monitored during 10 months. During the trial, isolation of leptospires was attempted and antibodies were detected by the microscopic agglutination test. Leptospires were recovered from the urine of six of the inoculated animals up to 6 months after infection. Eight of ten calves born from the inoculated heifers were born weak, and one of them died 12 h after parturition. Three of the weak calves had generalized jaundice of the internal surfaces. Half of the cows developed metritis and had a retained placenta. Serological reactions were seen against serotypes other than L. hardjo. A chronic infection was apparently established in the inoculated heifers with leptospiruria resulting in reinfection of the animals and a secondary rise in antibody levels.

Agglutination Tests↗
PubMed1982

Bovine IgM and IgG response to Leptospira interrogans serovar hardjo as measured by enzyme immunoassay.

The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen--antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.

Agglutination Tests↗
PubMed1985

The use of the enzyme-linked immunosorbent assay (ELISA) to detect the IgM and IgG antibody response to Leptospira interrogans serovars hardjo, pomona and tarassovi in cattle.

Leptospira interrogans serovars pomona, hardjo and tarassovi were each used to inoculate 6 cattle. Three-hundred and ninety-nine sera collected from the inoculated animals and from a control group over a 3-month period were tested using the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA). Leptospiruria was monitored by microscopic examination and culture. The ELISA detected specific IgM antibody against the serovars in all infected cattle 1 week after inoculation. This IgM antibody persisted in most of the animals for 3-5 weeks. Specific IgG antibody appeared at the same time or just after IgM, but persisted for much longer. Levels of antibody detected by the ELISA and the MAT did not correlate with each other, nor with the periods of leptospiruria found in the infected cattle.

Agglutination Tests↗
PubMed1985

Enzymatic radioimmunoassay for detecting Leptospira interrogans serovar pomona in the urine of experimentally-infected pigs.

An enzymatic radioimmunoassay (ERIA) has been developed for detecting Leptospira interrogans serovar pomona in porcine urine. Four grower pigs were experimentally infected with serovar pomona. A total of 39 urine samples was collected, and ERIA was compared with dark ground microscopy (DGM) and culture for demonstrating leptospiruria. Of 20 samples positive by at least one technique, leptospires were detected by ERIA in 14, by culture in 16 and by DGM in 13. ERIA, unlike the other 2 methods, was suitable for use with urine which had been stored frozen for several months.

Animals↗
PubMed1986

In vitro studies of haemolysis by Leptospira interrogans serovars pomona and ballum.

Washed and unwashed red blood cells (RBC) from young calves, adult cattle, hamsters and humans were incubated with Leptospira interrogans serovars pomona and ballum. Washed cells suspended in saline were always haemolysed while unwashed cells and those which were washed and resuspended in plasma were never haemolysed, despite the presence of large numbers of organisms within the culture supernatant. Pomona produced greater haemolysis of cattle and human RBC than did ballum, but with hamster RBC ballum produced greater haemolysis than did pomona. A group of 6- to 9-month-old cattle infected with pomona showed no signs of clinical disease and RBC taken from them before infection and during the development of antibodies to pomona were haemolysed by pomona only after the cells were washed. Plasma therefore appears to have a protective function. This in vitro protective function of plasma even extended to plasma from young seronegative calves.

Animals↗
PubMed1986

Pathology of acute Leptospira interrogans serotype icterohaemorrhagiae infection in the Syrian hamster.

The pathology of acute Leptospira interrogans serotype icterohaemorrhagiae infection in the Syrian hamster was investigated up to 7 days after infection using histology, electron microscopy and an indirect fluorescence test for leptospires. The disease was characterized by the presence of many leptospires in the tissues, jaundice, leukocytosis, haemorrhages, endothelial alteration and thrombotic glomerulopathy. The leptospires were present intravascularly, in the interstitium penetrating between liver cells and tubular epithelial cells and in the tubular lumina. The presence of leptospires was not necessarily associated with lesions. These findings support both pathogenetic mechanisms suggested in the literature, namely: the ability of leptospires to penetrate actively between cells with detachment of tight junctions, without obvious lesions to the cells, and an immune-mediated process with immune complex formation and binding and activation of complement resulting in leukocytosis, thrombotic glomerulopathy, endothelial alteration and haemorrhages.

Acute Disease↗
PubMed1988

Opsonic effect of monoclonal antibodies against Leptospira interrogans serovar copenhageni.

The opsonic effect of monoclonal antibodies (McAbs) against Leptospira interrogans serovar copenhageni strain Shibaura was examined in vitro using radiolabeled organisms and mouse peritoneal exudate macrophages. Four IgG McAbs (all IgG3) and 2 IgM McAbs were used, all showed different reactivities and were bound to homologous lipopolysaccharide (LPS). IgG3 McAbs at the sub-agglutinating concentration were opsonic, but the degree of the opsonic effect varied among IgG3 McAbs. Opsonization with IgG3 McAbs at the concentration showing the same level of ELISA binding showed that the highest opsonic effect was exerted by Sw-6 antibody. The other IgG3 McAbs showed a similar but lower opsonic effect. IgM McAbs, which were not opsonic at the sub-agglutinating concentration even in the presence of complement, showed opsonic effect at the agglutinating concentration.

Agglutination Tests↗
PubMed1990

Nucleic acid probe characterizes Leptospira interrogans serovars by restriction fragment length polymorphisms.

Restriction endonuclease analysis (REA) of genomic DNA can discriminate between many Leptospira interrogans serovars. However, several serovars have similar restriction endonuclease digestion patterns which prohibits accurate identification. This investigation expands previous REA studies of L. interrogans to include serovars in serogroup Tarassovi. Most serovars in this serogroup had characteristic digestion patterns by which they could be identified. However, four of the serovars in this serogroup had similar digestion patterns, thus preventing serovar identification by REA alone. To discriminate between these serovars REA was supplemented with Southern blot analysis. The DNA from each serovar showed similar but unique patterns when hybridized with a probe synthesized from a repetitive sequence element cloned from L. interrogans serovar hardjo type hardjo-bovis. The applicability of this technique to characterize other serogroups was assessed. One hundred sixty six of 190 serovars screened by Southern blot analysis contained sequences which hybridized with the repetitive element probe under conditions of relaxed stringency. These results suggest that Southern blot analysis using this probe will be a valuable supplement for typing L. interrogans.

Animals↗
PubMed1991

The serological response of calves to Leptospira interrogans serovar hardjo vaccines and infection as measured by the microscopic agglutination test and anti-IgM and anti-IgG enzyme-linked immunosorbent assay.

The microscopic agglutination test (MAT) and the anti-IgM and anti-IgG enzyme-linked immunosorbent assays (ELISA) were used to examine sera taken over the course of 16 weeks from 35 calves vaccinated and/or infected with Leptospira interrogans serovar hardjo. The relationship between the IgM and IgG responses to vaccination and infection were determined. The rapid and high rise in IgM levels following challenge made the anti-IgM ELISA a potentially good indicator of recently established infection although some transitory high levels were seen where infection did not become established. The slow IgG response to infection made the anti-IgG ELISA of limited diagnostic use.

Agglutination Tests↗
PubMed1993

Outer membrane proteins of three pathogenic Leptospira species.

The outer membrane proteins of seven reference strains of pathogenic Leptospira (L. alstoni serovar grippotyphosa, L. borgpetersenii serovar hardjo, and L. interrogans serovars autumnalis, bratislava, canicola, icterohaemorrhagiae, and pomona) were investigated to identify common surface-exposed outer membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sodium-N-lauroylsarcosinate-insoluble outer membrane enriched fractions of the reference serovars and two field isolates of serovars hardjo and pomona revealed six common protein bands with approximate molecular masses of 77, 66, 42, 35.5, 24, and 18 kDa. At times the 35.5 kDa endoflagellar band resolved into two distinct bands, 35.5 kDa and 34 kDa. Immunoblotting of the same fractions using rabbit leptospiral antibodies showed six bands to be common (66, 59.5, 44, 42, 35.5, and 18 kDa). The 44 kDa band stained poorly with Coomassie blue but prominently by immunoblotting. Four reference strains (serovars bratislava, canicola, icterohaemorrhagiae, pomona), and two field isolates of serovar pomona and one of serovar bratislava were grown in low iron media to which the iron chelators 2,2'-dipyridyl or ethylenediaminehydroxyphenylacetic acid were added. No iron-dependent expression of outer membrane proteins was observed. The only difference observed between the outer membrane proteins when reference serovars of canicola or pomona were grown in dialysis bags in the peritoneum of swine or in vitro was the loss of the 77 kDa band from in vivo grown organisms. Treatment of whole leptospires with proteinase K did not remove the 77, 66, 59.5, or 42 kDa protein; these proteins may not be surface expressed or are inaccessible to the proteinase K. The 44 kDa band could not be evaluated by this method and the 18 kDa band was proteinase K resistant.

Animals↗
PubMed1994

Deoxynucleotide sequence conservation of the endoflagellin subunit protein gene, flaB, within the genus Leptospira.

A Polymerase chain reaction was developed to amplify the entire open reading frame of flaB, the gene encoding the endoflagellin subunit protein. The 852 bp amplified products from 23 serovars of the genus Leptospira were subjected to restriction endonuclease analysis and the profiles correlated well with phylogenetic relationships between these serovars. The flaB deoxynucleotide sequences of L. hardjo-bovis, L. hardjo-prajitno and L. grippotyphosa were determined. The deduced primary amino acid sequences of each were highly conserved with only three amino acid residue differences observed. The deoxynucleotide sequences showed genetic drift with alternative bases in the third position of codons. The PCR product derived by amplification of flaB from L. grippotyphosa was cloned into the expression vector pGEX-2T and a recombinant FlaB fusion protein made. As predicted from the deduced amino acid sequences, the recombinant FlaB cross-reacted with heterologous antiserum derived from a rabbit infected with L. hardjo-bovis.

Amino Acid Sequence↗
PubMed1994

Protective effects of serum thymic factor to Leptospira interrogans serovar Copenhageni infection in Mongolian gerbils.

The susceptibility to Leptospira interrogans serovar copenhageni in Mongolian gerbils treated with 10 micrograms of serum thymic factor (FTS) 1 day before infection was examined. Susceptibility of gerbils treated 5 times with 10 micrograms of FTS was also investigated. Mortality of FTS-treated gerbils was significantly lower than that of controls when small challenge doses were used. To analyse the FTS-induced resistance to leptospiral infection, natural killer (NK) cell activity and macrophage activity were studied. Macrophage activity was unaltered but NK cell activity was enhanced in FTS-treated gerbils, with or without leptospiral infection. Since no side-effects of FTS were observed, this compound should be considered for the treatment of leptospirosis.

Animals↗
PubMed1996

The development of a ligase mediated PCR with potential for the differentiation of serovars within Leptospira interrogans.

A ligase mediated polymerase chain reaction (LMPCR) was developed to amplify between the repetitive element, IS1533, of Leptospira and adjacent chromosomally located Bg/II restriction endonuclease enzyme sites. To do this, complimentary oligonucleotide linkers designed to anneal together with an overhanging Bg/II end were ligated to Bg/II digested DNA from 35 leptospiral reference strains and field isolates. This ligated DNA was used as template for PCR with oligonucleotide primers specific for the linker and for the repetitive element IS1533. The resultant amplicon profile hybridised a 102 bp region derived from the terminus of IS1533 thus confirming that amplicons generated by LMPCR contained part of IS1533. The number of fragments generated containing IS1533 was significantly fewer than that generated by RFLP but the LMPCR method has the potential to use far less template DNA and be quicker than standard RFLP. Obvious and reproducible interserovar differences were demonstrated by LMPCR whereas for 20 of 21 L. hardjo-bovis isolates tested no intraserovar differences were observed. Of those serovars known to possess IS1533 homologues and tested here by LMPCR, each produced a unique amplicon profile which hybridised the IS1533 terminus probe. The limited heterogeneity amongst hardjo-bovis isolates is discussed as is the potential contribution of this method to diagnosis, differentiation and the phylogenetics of the Leptospires.

Animals↗
PubMed1991

Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona.

Outer membrane and secreted proteins were isolated from Leptospira interrogans serovar pomona and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblot and radioimmunoprecipitation techniques. The L. interrogans outer membranes were extracted with Triton X-114 and contained several proteins. The major cellular protein with a molecular mass of 31 kDa was associated exclusively with the L. interrogans outer membrane. Using a whole cell immunoprecipitation method, five hydrophobic, Triton X-114 extractable proteins (22, 26, 31, 36 and 42 kDa) were exposed on the surface of L. interrogans. The 31 kDa protein was heat labile and was a potent antigen in animals experimentally infected with L. interrogans serovar pomona. Several proteins were secreted by L. interrogans including a 60 kDa protein tentatively identified as the L. interrogans hemolysin.

Animals↗
PubMed2006

Characterization of the cheY genes from Leptospira interrogans and their effects on the behavior of Escherichia coli.

The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome. In order to verify the function of these proteins, the putative cheY genes were cloned into pQE31 vector and then expressed, respectively, in wild-type Escherichia coli strain RP437 and cheY defective strain RP5232. The results showed that all the five cheYs could restore the swarming of RP5232 strain to some extend. Overexpression of CheYs in RP437 showed inhibited swarming of RP437. To investigate the mechanism of chemotaxis signaling in L. interrogans strain Lai, certain aspartates (Asp-53, Asp-61, Asp-70, Asp-62, and Asp-66 for L. interrogans strain Lai CheY1, CheY2, CheY3, CheY4, and CheY5, respectively) were mutated. Expression of these mutated cheYs manifested neither restoration of the swarming ability of RP5232 nor inhibition on swarming ability of RP437. Multiple amino acid sequence alignment predicted ternary structures and the result of mutation experiment suggested that these conserved aspartate residues of L. interrogans were analogous to that in E. coli CheY in function and structure. So, L. interrogans and E. coli may have similar mechanisms of activation of the chemotaxis phosphorelay pathway, but there are differences in their control by signal terminator.

Amino Acid Sequence↗
PubMed2004

Serological and molecular characterization of leptospira serovar Kenya from captive African giant pouched rats (Cricetomys gambianus) from Morogoro Tanzania.

Two identical leptospiral isolates coded Sh9 and Sh25 obtained from the urine of captive African giant pouched rats (Cricetomys gambianus), destined for use as biodetector of antipersonnel landmines were typed as serovar Kenya using cross-agglutination absorption test and DNA fingerprinting with the insertion element sequences IS1533 and IS1500 derived primers. The two isolates were previously characterized using cultural and serological-microagglutination test as pathogenic leptospires of the serogroup Ballum, closely related to serovars Kenya and Peru. To our knowledge, this is the first reported in-depth characterization of leptospira isolates from Tanzania.

Agglutination Tests↗
PubMed2005

Omp52 is a growth-phase-regulated outer membrane protein of Leptospira santarosai serovar Shermani.

We report the expression and characterization of the omp52 gene of Leptospira santarosai serovar Shermani strain CCF that is isolated in Taiwan. omp52 was identified among pathogenic leptospires but not among non-pathogenic leptospires by using suppression subtractive hybridization in our previous study. With an open reading frame of 1371 bp that encodes 456 amino acids and a predicted molecular mass of 52.6 kDa, Omp52 was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. Furthermore, Omp52 increases dramatically during the stationary phase, indicating that the expression of Omp52 is environmentally regulated. By using immunoblotting analysis, we proved that Omp52 was expressed in human patients infected with leptospires. These observations suggest that Omp52 may play roles in the interaction of host cells and pathogens during infection.

Amino Acid Sequence↗
PubMed2005

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis.

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.

Adolescent↗