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PubMed1997

Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA.

Bacterial specific primers were used to amplify 23S rRNA genes from a representative strain from each of the 23 serogroups of the pathogenic Leptospira interrogans and 8 strains from 6 serogroups of the non-pathogenic Leptospira biflexa. Only regions of extreme variability, which had been identified on the basis of homology-based search of all the 23S rRNA sequences available in GenBank database, were sequenced from the amplified products. PCR primers that had the potential to distinguish L. interrogans from L. biflexa species were designed from the derived sequences and a sensitive PCR protocol developed. The PCR method enabled the differentiation of the 59 strains of the 23 serogroups of L. interrogans from the 8 strains of 6 serogroups of L. biflexa. Further investigation by 16S rDNA sequencing of two strains of L. interrogans, which gave unexpected PCR results, provided evidence that they had been misclassified and hence we propose to reassign them to L. biflexa.

Base Sequence↗
PubMed1984

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa.

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A1 was identified by the formation of 32P- and 14C-labeled lysoderivatives from 32P-phosphatidylcholine, 32P-phosphatidylethanolamine, or 1-acyl-2-[1-14C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A1 activity was independent of lipase in the microorganism since 14C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using 32P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A1 was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-14C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A1 and/or A2?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.

Hot Temperature↗
PubMed2005

Serologic responses of dogs given a commercial vaccine against Leptospira interrogans serovar pomona and Leptospira kirschneri serovar grippotyphosa.

OBJECTIVE: To evaluate serum titers obtained by use of the microscopic agglutination test (ie, MAT titers) to Leptospira interrogans serovar pomona and autumnalis and Leptospira kirschneri serovar grippotyphosa in dogs given a commercial vaccine against serovars pomona and grippotyphosa. ANIMALS: Forty 12-week-old puppies and 20 mature Beagles. PROCEDURE: Puppies received a commercial vaccine against serovars pomona and grippotyphosa at 12 weeks of age, then received a booster vaccine and 3 weeks later; mature dogs received the vaccine once. Serum MAT titers to serovars pomona, autumnalis, and grippotyphosa were measured before vaccination and at 2, 4, 6, 10, and 16 weeks after the first or only vaccination. RESULTS: Of the 40 puppies vaccinated, 40, 0, and 40 developed MAT titers of > 100 after vaccination to serovars pomona, grippotyphosa, and autumnalis, respectively. Microscopic agglutination test titers to serovar autumnalis were higher than MAT titers to serovars pomona and grippotyphosa and persisted in some dogs for 16 weeks (6 weeks longer than for titers to serovar pomona). Of the 20 mature dogs, 13, 5, and 20 developed MAT titers of > 100 at 2 weeks to serovars pomona, grippotyphosa, and autumnalis, respectively. Titers to serovar pomona were higher and persisted in some dogs beyond 16 weeks after vaccination, compared with titers to serovars pomona and grippotyphosa, which persisted for 10 and 6 weeks, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Subunit vaccines against serovars pomona and grippotyphosa induce MAT titers not only to homologous antigens but also to serovar autumnalis, which could lead to a misdiagnosis of leptospirosis caused by serovar autumnalis.

Agglutination Tests↗
PubMed1979

Isolation of antigenic variants from leptospiras grown in vitro and from heart blood of guinea pigs inoculated with a clonized strain of Leptospira.

A clonized culture of Leptospira interrogans serovar copenhageni strain Shibaura (Cl-Shibaura) was inoculated into guinea pigs. The heart blood of the guinea pigs, obtained at the febrile stage and inoculated onto the solid serum medium containing the homologous immune serum, produced large and small colonies. The serological examinations revealed that the large colonies were found mainly to be the antigenic variants, while the small colonies were mostly the parent. The antigenic variants accounted for 16.4% of all the colonies from the blood of the guinea pigs infected with Cl-Shibaura, and for 1.2% of all the colonies from the blood of the guniea pigs infected with Cl-Shibaura, and for 1.2% of all the ll colonies. The serological examinations revealed that the large colonies were found mainly to be the antigenic variants, while the small colonies were mostly the parent. The antigenic variants accounted for 16.4% of all the colonies from the blood of the guniea pigs infected with Cl-Shibaura, and for 1.2% of all the colonies from the culture of Cl-Shibaura in the normal serum medium. Antigenic variants were also isolated in vitro from the culture of 2 other serovars. The fact that the frequency of the antigenic variants of leptospiras was higher in vivo than in vitro is discussed.

Animals↗
PubMed1993

[The use of nonpathogenic leptospira as diagnostic antigens for the diagnosis of leptospira infections in cattle].

The seroprevalence of leptospira antibodies was determined in 4377 bovine sera by microagglutination assay using 11 Leptospira interrogans serovars. In 10% (439 samples) of the sera, a positive reaction was detected. These included 275 sera (62.6%) with reaction to L. grippotyphosa, 159 (36.2%) to L. saxkoebing and 5 (1.1%) sera with reactions to other serovars. Multiple reactions were found in 9.8% of the 439 positive sera, whereby the Sejroe group dominated (65%) within the possible combinations of crossreactions. To determine the suitability of nonpathogenic strains as polyvalent diagnostic antigens, two L. biflexa serovar patoc strains were compared with the above-mentioned 11 L. interrogans serovars in microagglutination testing of 1995 screen sera. The sensitivity of the test was found to be 0.3% and the specificity was 80.3%. Using the two L. patoc strains, all of the 415 positive field sera and 11 positive control sera yielded negative reactions. Based on these results, the use of L. biflexa serovar patoc cannot be recommended as polyvalent antigen in the diagnosis of bovine leptospirosis by microagglutination.

Agglutination Tests↗
PubMed2001

Rattus norvegicus acting as reservoir of leptospira interrogans in the Middle Black Sea region of Turkey, as evidenced by PCR and presence of serum antibodies to Leptospira strain.

Leptospirosis, a common form of zoonosis, especially in rainy countries, is caused by Leptospira interrogans. In our region of Turkey this type of disease has often been encountered in connection with rice harvesting and we therefore attempted to evaluate the prevalence of L. interrogans in wild rats in our region. Fifty-nine Rattus norvegicus rats were trapped alive in different areas of an approximately 100 km stretch of seashore in the Middle Black Sea region of Turkey. L. interrogans was determined by PCR in sera, kidney and brain tissue. Sixteen (27.1%) kidney samples and 10 brain tissue samples (16.9%) were positive for L. interrogans. No PCR positivity was seen in sera samples. Five sera were positive by microagglutination test. A large proportion of wild rats in our region were found to be carriers of L. interrogans. We conclude that people who are exposed to rat urine in their daily life are at risk of acquiring L. interrogans.

Agglutination Tests↗
PubMed1989

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa.

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.

Blotting, Southern↗
PubMed1999

Repetitive sequence of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1: a sensitive probe for demonstration of Leptospira interrogans strains.

A 4.8-kilobase (kb) repetitive sequence element generated with KpnI digestion was cloned from the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1. The sequence, repeated in tandem, was located on the 280-kb fragment between the FseI and AscI sites on the chromosome by hybridization using the 4.8-kb fragment as a probe. We cloned the fragment containing the element for the Ictero No. 1 strain in a lambda EMBL3 bacteriophage DNA, and one out of 5 clones was sequenced. Within the sequenced 9-kb segment that partially repeated, 9 putative open-reading frames and 2 transfer RNA genes, for alanine and isoleucine, were identified. A similarity search for the products deduced from the sequenced data revealed that the repeated sequence includes both beta-oxidation enzymes, acyl-CoA dehydrogenase and enoyl-CoA hydratase, and hydroxythiazole kinase protein homologues. Hybridization experiments against different leptospiral strains using the element as a probe showed a similar sequence in the strains of L. interrogans and L. kirschneri, but not in any strains of L. borgpetersenii, L. weillii, L. meyeri or L. biflexa. Results indicated that the highly repeated element in the Ictero No. 1 strain exists as a well conserved sequence, though at a moderate level of repetition, in certain strains of L. interrogans and L. kirschneri. PCR amplification targeting the repetitive element was successful and indicated that the procedure provides a sensitive and specific probe to detect leptospires.

Base Sequence↗
PubMed1996

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor alpha from human monocytes.

Elevated plasma concentrations of the cytokine tumor necrosis factor alpha (TNF alpha) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans, serovar copenhageni, was able to induce the release of TNF alpha from peripheral blood mononuclear cells. TNF alpha induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNF alpha release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNF alpha production.

Biological Assay↗
PubMed1999

Comparative analysis of the LPS biosynthetic loci of the genetic subtypes of serovar Hardjo: Leptospira interrogans subtype Hardjoprajitno and Leptospira borgpetersenii subtype Hardjobovis.

Although Leptospira borgpetersenii subtype Hardjobovis and L. interrogans subtype Hardjoprajitno belong to different species, they are serologically indistinguishable and are therefore classified as serovar Hardjo. Since LPS is the major antigen involved in serological classification, this implies that the LPS of these subtypes is identical. Comparison of the LPS biosynthetic loci (rfb) of the subtypes revealed remarkable similarity, with 32 and 31 origins of replication (orfs) in the Hardjoprajitno and Hardjobovis rfb loci, respectively. The order and orientation of these orfs were identical with the exception of an additional orf in Hardjoprajitno between orfs 4 and 5 and intergenic sequences differing between the subtypes. The Hardjoprajitno rfb locus has been divided into four intercalated regions based on sequence similarity to other leptospiral rfb loci. orfJ1-orfJ14 as well as orfJ21-orfJ22 are more similar to regions of the rfb locus of L. borgpetersenii subtype Hardjobovis. orfJ15-orfJ20 as well as orfJ23-orfJ31 are almost identical to the corresponding orfs in L. interrogans serovar Copenhageni. We propose that the progenitor Hardjoprajitno strain, containing an rfb locus which closely resembled the Copenhageni locus, acquired orfs 1-14 and orfs 21-22 from subtype Hardjobovis resulting in two serologically indistinguishable subtypes of serovar Hardjo which in turn constituted the main bovine-adapted leptospiral serovar.

Bacterial Proteins↗
PubMed1982

Presence of Vi antigen in a virulent strain of leptospira interrogans serovar pomona and relation of Vi antigens of leptospiras to resistance to leptospiricidal activity mediated by antiserum plus complement.

Vi antigen was found in a virulent strain of Leptospira interrogans serovar pomona. The presence of Vi antigen was shown by the agglutinin-absorption and precipitin-absorption tests. Each strain of pomona and copenhageni which possessed Vi antigen and was resistant to the leptospiricidal activity mediated by the antiserum plus complement lost Vi antigen and became susceptible to the leptospiricidal activity when cultured in the presence of 8-azaguanine. The strains thus treated with 8-azaguanine recovered the resistance and Vi antigen when cultured in medium without 8-azaguanine.

Antigens, Bacterial↗
PubMed1978

The immunoglobulins response in rabbit to single antigenic factors of leptospira. Investigations with two serovars from saprophytic leptospira.

The agglutinating antibodies formed after immunization of rabbits with viable leptospira consist of 19S and 7S immunoglobulins. IgM and IgG are formed already earlier in the course of immunization. after a lapse of one month more IgG is found than IgM. The specificity of agglutination with the main antigenic factors is due mainly to IgG.

Agglutination Tests↗