Lipopolysaccharide biosynthesis in Leptospira

Lipopolysaccharide is the major surface antigen of Leptospira. Variation in LPS structure is the basis for the more than 200 serovars that have been identified. Despite the importance of this antigen in immunity and diagnostics, there is relatively little known about the genetics and chemistry of leptospiral LPS, as compared to some members of the Enterobacteriaceae. The nucleotide sequence of the locus encoding enzymes for the biosynthesis of the O-antigen component of leptospiral LPS (rfb locus) has been determined for three serovars namely, L. interrogans serovar Pomona, L. interrogans serovar Hardjo subtype Hardjoprajitno and L. borgpetersenii serovar Hardjo subtype Hardjobovis. In the absence of data relating to the chemical structure or genetic tools to construct isogenic mutants in Leptospira, similarity analysis has been used to provide insight into the mechanisms by which the leptospiral O-antigen is assembled by comparison with characterized systems from other bacteria. In addition, comparison of the gene layout in each of the serovars provides an indication of the genetic basis for serovar diversity.     

Exposure to Leptospira in stray dogs in the city of Cali

In Colombia, little information is available concerning the epidemiology of leptospirosis in urban environments. Furthermore, the role of dogs in the transmission cycle of leptospirosis in the urban setting is unclear. To explore the potential role of canines in the transmission of leptospirosis in Cali, a serological study was conducted with 197 serum samples collected from stray dogs during 2001 and 2003. Serum specimens were screened with the Microscopic Agglutination Test (MAT) and 7 serovars--Icterohaemorrhagiae, Canicola, Gryppotyphosa, Hardjo strain Hardjobovis, Pomona, Hardjo strain Hardjoprajitno and Bratislava. All serovars were provided by the Instituto Colombiano Agropecuario (ICA), Tuluá, Colombia. The MAT was considered positive when 50% or more leptospiras were agglutinated with one or more serovars in a serum dilution of 1:100. At least one serovar showed evidence of infection in 41.1% of the dogs. The most prevalent serovar was Icterohaemorrhagiae, found in 55.6% of the seropositive dogs. 48.1% were co-agglutinations. No reactions against the serovars Pomona, Hardjo strain Hardjoprajitno and Bratislava were observed. These findings suggested that stray dogs are potential reservoirs of Leptospira in Cali and underscored the need to study the epidemiology of this disease in Colombia.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona, were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona. In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Abstract Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona , were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona . In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and se- quenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indi- cated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and vali- dated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a pro- tein. The superposition of the Cα traces of cpn60 structure and predicted structure of leptospiral GroEL indi- cates good agreement of secondary structure elements with an RMSD value of 1.5 . Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.     

Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California

One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona.     

Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C

The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10, and 1% survival were determined for representative serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay, L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.     

Leptospira interrogans is recognized through DC-SIGN and induces maturation and cytokine production by human dendritic cells

Leptospirosis is a global zoonotic disease, caused by pathogenic Leptospira species including Leptospira interrogans, that causes public health and livestock problems. Pathogenesis, immune response and cellular receptors for Leptospira are not well understood. Interaction of dendritic cells (DCs) with L. interrogans serovar Autumnalis L-643 and BL-6 isolated from leptospirosis patients, and both virulent and avirulent serovar Pyrogenes 2317 strains isolated from animal were investigated. Carbohydrate analysis using lectins showed that all of these leptospires contained high mannose components as a common backbone and DC-SIGN was involved in leptospires' attachment. Interaction of the L. interrogans strains with DCs induced maturation, but had different effects on IL-10, IL-12p70 and tumor necrosis factor (TNF)-alpha production. Both virulent and avirulent Pyrogenes 2317 and Autumnalis BL-6 but not L-643 strains induced IL-12p70 and TNF-alpha production, but minimal IL-10 secretion. These data demonstrated that L. interrogans binds DC-SIGN and induces DCs maturation and cytokine production, which should provide new insights into cellular immune processes during leptospirosis.     

Prevalence of antibody to six Leptospira servovars in Swedish wild boars

Zoonotic Leptospira bacteria are pathogens that may increase in importance with climate change. We investigated the prevalence of antibody to six Leptospira serovars (sv) in the Swedish wild boar (Sus scrofa) population, which is increasing in number and geographic distribution. The serovars we selected cause disease in pigs or may be of use as sentinel serovars to measure the potential spread in Swedish fauna. In total, 386 serum samples from wild boars collected between 2005 and 2007 were investigated using a microscopic agglutination test for Leptospira interrogans sv Bratislava strain Jez Bratislava, sv Icterohaemorrhagiae strain Kantorowicz, sv Pomona strain Pomona, Leptospira kirschneri sv Grippotyphosa strain Duyster, and Leptospira borgpetersenii sv Tarassovi strain Perepelitsin, and a domestic strain closely related to sv Sejroe. Twelve (3.1%) of the analyzed samples were antibody-positive. Of those, nine (2.3%) were positive for sv Bratislava and 0.8% for sv Icterohaemorrhagiae. All antibody-positive samples originated from areas where wild boars are reported to be common. We conclude that Leptospira infection is less common in Swedish wild boar than in continental Europe. However, we recommend continuous surveillance to follow the effects of climate change and an increasing wild boar population.     

Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C.

The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10, and 1% survival were determined for representative serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay, L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.     

Molecular typing of Leptospira spp. based on putative O-antigen polymerase gene (wzy), the benefit over 16S rRNA gene sequence

Molecular typing of leptospiral strains based on variation within putative O-antigen polymerase gene (wzy) was determined among reference strains and those isolated from patients. Using the PCR primers designed from the flanking gene of wzy derived from Leptospira interrogans serovar Copenhageni, all L. interrogans serovars as well as human and rodent leptospiral isolates from Thailand could be amplified. The size of PCR product ranged from 1 to 1.5 kb. The limitation of these primer pairs was the inability to amplify those strains whose sequences differ in the region of the primers, these included Leptospira biflexa (serovar Patoc), Leptospira borgpetersenii (serovar Tarassovi) and Leptospira kirschneri (serovar Bim, Bulgarica, Butembo). Notably, amplification was not limited to L. interrogans as demonstrated by the amplification of some strains from L. kirschneri, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, L. borgpetersenii and Leptospira weilii. The phylogenetic tree of wzy sequence, inferred by posterior probability of the Bayesian, enabled the categorization of leptospiral serovars into seven genetically related group, of which its differentiation power was better than that of the more highly conserved 16S rRNA gene, which is used extensively for genotyping.     

Expansion of the in vitro assay for Leptospira potency testing to other serovars: Case study with Leptospira Hardjo

Evaluation of leptospiral vaccines for potency against Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, and Grippotyphosa is accomplished using the hamster potency test method described in 9 CFR 113.101-104. Applicability of this method to evaluation of bacterins developed for immunization against infection with L. interrogans serovar Hardjo or Leptospira borgpetersenii serovar Hardjo is complicated by several issues. Information from research on target host animal efficacy studies and evaluation of the immune response elicited using effective whole-cell bacterin formulations have revealed problems in relating these studies to either hamster-based or other potency testing methods. Future work on serovar Hardjo vaccines employing recombinant proteins will require preliminary testing methods in models other than the host animal. These models may also prove applicable to evaluation of potency for protein-based vaccines. Both an acute lethal infection model and a chronic infection model have been developed using two different strains of serovar Hardjo and will be described.     

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel‐based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non‐pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non‐pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.     

Partial rpoB gene sequencing for identification of Leptospira species

The usual target for sequence-based identification of Leptospira species is the 16S rRNA gene. However, because the 16S rRNA gene is not polymorphic enough, it is necessary to sequence a 1500 bp segment of this gene for accurate identification. Based on the alignment of previously determined rpoB of three Leptospira strains, we designed and tested a primer pair that enabled us to amplify and sequence a 600 bp segment of Leptospira rpoB. This segment was species-specific for the 16 species tested, but was unable to separate Leptospira interrogans serovars accurately. For the 11 L. interrogans serovars tested, only seven genotypes could be determined. We thus think that analysis of partial rpoB may be useful as an initial screening test for the identification of a new isolate of Leptospira and detection or identification of Leptospira in clinical or environmental samples, but not for serovar determination.     

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.     

Analysis of Leptospira isolates from mainland Portugal and the Azores islands

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.     

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.     

Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization

Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.     

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in S?o Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.