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1.  Human Leptospirosis Caused by a New, Antigenically Unique Leptospira Associated with a Rattus Species Reservoir in the Peruvian Amazon 
As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.
Author Summary
Leptospirosis has emerged as a globally important infectious disease. Its impact on public health is often difficult to determine, sometimes because of low clinical suspicion, or, as is more common, difficulty in laboratory diagnosis. Gold-standard serology-based diagnosis has a number of important limitations, including the need to use live leptospires that have a sufficient diversity of antigens to be able to detect specific anti-leptospiral antibodies; such antigens vary greatly from region to region. In this paper, we report the discovery of a new species of Leptospira in the highly biodiverse region of the Peruvian Amazon, and demonstrate that the animal source of infection for humans is the domestic rat. Detailed biological characterization of this new species shows that it is antigenically unique and represents a new serogroup and serovar, proposed as Leptospira licerasiae serogroup Iquitos serovar Varillal. Incorporation of this new isolate into serological testing of patients presenting with acute febrile illness in Iquitos, Peru, showed a far higher incidence of leptospirosis than previously suspected, showing the important of using region-specific Leptospira in diagnosis. The field-to-laboratory approach presented here has general application to the discovery of other emerging pathogens and their impact on human health.
doi:10.1371/journal.pntd.0000213
PMCID: PMC2271056  PMID: 18382606
2.  Species-Specific Identification of Leptospiraceae by 16S rRNA Gene Sequencing 
Journal of Clinical Microbiology  2006;44(10):3510-3516.
The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases ≤10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.
doi:10.1128/JCM.00670-06
PMCID: PMC1594759  PMID: 17021075
3.  Genetic diversity among major endemic strains of Leptospira interrogans in China 
BMC Genomics  2007;8:204.
Background
Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars).
Results
Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification.
Conclusion
CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools.
doi:10.1186/1471-2164-8-204
PMCID: PMC1936430  PMID: 17603913
4.  Similarities in Leptospira Serogroup and Species Distribution in Animals and Humans in the Indian Ocean Island of Mayotte 
Our objective was to identify local animal reservoirs of leptospirosis to explain the unusual features of Leptospira strains recently described among patients on the island of Mayotte. By means of a microscopic agglutination test using local clinical isolates, we found that 11.2% of black rats were seropositive to Leptospira, whereas 10.2% of flying foxes, 2% of lemurs, 93.1% of domestic dogs, and 87.5% of stray dogs were seropositive. As observed in humans, Mini was the main serogroup circulating in animals, whereas serogroup Icterohaemorrhagiae was absent. Using quantitative polymerase chain reaction, we also showed that 29.8% of rats carried leptospires in their kidneys. The sequencing of 16S rRNA gene sequences of Leptospira found in black rat kidneys identified four genomospecies (Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, and L. borgpetersenii group B), which established black rats as the major source of leptospirosis transmission to humans. The origins of such a genetic diversity in Leptospira strains are discussed.
doi:10.4269/ajtmh.2012.12-0012
PMCID: PMC3391038  PMID: 22764304
5.  Rapid Leptospira identification by direct sequencing of the diagnostic PCR products in New Caledonia 
BMC Microbiology  2010;10:325.
Background
Most of the current knowledge of leptospirosis epidemiology originates from serological results obtained with the reference Microscopic Agglutination Test (MAT). However, inconsistencies and weaknesses of this diagnostic technique are evident. A growing use of PCR has improved the early diagnosis of leptospirosis but a drawback is that it cannot provide information on the infecting Leptospira strain which provides important epidemiologic data. Our work is aimed at evaluating if the sequence polymorphism of diagnostic PCR products could be used to identify the infecting Leptospira strains in the New Caledonian environment.
Results
Both the lfb1 and secY diagnostic PCR products displayed a sequence polymorphism that could prove useful in presumptively identifying the infecting leptospire. Using both this polymorphism and MLST results with New Caledonian isolates and clinical samples, we confirmed the epidemiological relevance of the sequence-based identification of Leptospira strains. Additionally, we identified one cluster of L. interrogans that contained no reference strain and one cluster of L. borgpetersenii found only in the introduced Rusa deer Cervus timorensis russa that is its probable reservoir.
Conclusions
The sequence polymorphism of diagnostic PCR products proved useful in presumptively identifying the infecting Leptospira strains. This could contribute to a better understanding of leptospirosis epidemiology by providing epidemiological information that cannot be directly attained from the use of PCR as an early diagnostic test for leptospirosis.
doi:10.1186/1471-2180-10-325
PMCID: PMC3022709  PMID: 21176235
6.  Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST) 
BMC Microbiology  2012;12:185.
Background
In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis.
Results
Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering.
Conclusions
MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.
doi:10.1186/1471-2180-12-185
PMCID: PMC3460781  PMID: 22925589
MALDI-TOF MS; Leptospira interrogans; Leptospira kirschneri; Leptospira borgpetersenii; multi locus sequence typing; LipL32; LipL41; rrs2; 16S rRNA; ClinProTools
7.  Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene 
BMC Microbiology  2006;6:95.
Background
Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing.
Results
A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results.
Conclusion
This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.
doi:10.1186/1471-2180-6-95
PMCID: PMC1630700  PMID: 17067399
8.  A Dominant Clone of Leptospira interrogans Associated with an Outbreak of Human Leptospirosis in Thailand 
Background
A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown.
Methods and Findings
A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection.
Conclusions
Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.
Author Summary
A sustained outbreak of human leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. Leptospirosis is a potentially serious infection cause by bacteria known as Leptospira; infection usually occurs following environmental exposure to pathogenic Leptospira shed in the urine of an infected animal. The purpose of this study was to obtain bacterial isolates from humans with leptospirosis around the time of the Thai outbreak for genotyping, and to relate these to the maintenance host animal. To achieve this, a bacterial typing scheme (multilocus sequence typing, MLST) was developed for L. interrogans, the major cause of human disease. This approach has the advantage over existing typing schemes in that the data generated are amenable to detailed evolutionary analysis, and are readily comparable via the internet. Our results demonstrated the emergence of a dominant clone of L. interrogans serovar Autumnalis; this was the major cause of human disease during the outbreak, and was found in a maintenance host which was defined as the bandicoot rat.
doi:10.1371/journal.pntd.0000056
PMCID: PMC2041815  PMID: 17989782
9.  Characterization of the ompL1 gene of pathogenic Leptospira species in China and cross-immunogenicity of the OmpL1 protein 
BMC Microbiology  2008;8:223.
Background
The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.
Results
PCR analysis demonstrated that the ompL1 gene existed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China. In the standard strains, the ompL1 gene could be divided into three groups (ompL1/1, ompL1/2 and ompL1/3) according to their sequence identities. Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires. The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1 (rOmpL1) and leptospiral strains belonging to different ompL1 gene types. These cross-immunoreactions were further verified by ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients. All the antisera against rOmpL1 proteins could inhibit L. interrogans strain Lai from adhering to J774A.1 cells. Furthermore, immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.
Conclusion
Three types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.
doi:10.1186/1471-2180-8-223
PMCID: PMC2632671  PMID: 19087358
10.  Human Leptospira Isolates Circulating in Mayotte (Indian Ocean) Have Unique Serological and Molecular Features 
Journal of Clinical Microbiology  2012;50(2):307-311.
Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.
doi:10.1128/JCM.05931-11
PMCID: PMC3264139  PMID: 22162544
11.  Bioinformatics Describes Novel Loci for High Resolution Discrimination of Leptospira Isolates 
PLoS ONE  2010;5(10):e15335.
Background
Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.
Methodology and Principal Findings
We used bioinformatics to evaluate pre-existing loci with the potential to increase the discrimination of outbreak strains. Previously deposited sequence data were evaluated by phylogenetic analyses using either single or concatenated sequences. We identified and evaluated the applicability of the ligB, secY, rpoB and lipL41 loci, individually and in combination, to discriminate between 38 pathogenic Leptospira strains and to cluster them according to the species they belonged to. Pairwise identity among the loci ranged from 82.0–92.0%, while interspecies identity was 97.7–98.5%. Using the ligB-secY-rpoB-lipL41 superlocus it was possible to discriminate 34/38 strains, which belong to six pathogenic Leptospira species. In addition, the sequences were concatenated with the superloci from 16 sequence types from a previous MLST scheme employed to study the association of a leptospiral clone with an outbreak of human leptospirosis in Thailand. Their use enhanced the discriminative power of the existing scheme. The lipL41 and rpoB loci raised the resolution from 81.0–100%, but the enhanced scheme still remains limited to the L. interrogans and L. kirschneri species.
Conclusions
As the first aim of our study, the ligB-secY-rpoB-lipL41 superlocus demonstrated a satisfactory level of discrimination among the strains evaluated. Second, the inclusion of the rpoB and lipL41 loci to a MLST scheme provided high resolution for discrimination of strains within L. interrogans and L. kirschneri and might be useful in future epidemiological studies.
doi:10.1371/journal.pone.0015335
PMCID: PMC2955542  PMID: 21124728
12.  Isolation and Characterization of New Leptospira Genotypes from Patients in Mayotte (Indian Ocean) 
Background
Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean). To date, Leptospira isolates have never been isolated in this endemic region.
Methods and Findings
Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA), and pulsed field gel electrophoresis (PFGE). Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars.
Conclusions
Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.
Author Summary
Leptospirosis has been recognized as an increasing public health problem affecting poor people from developing countries and tropical regions. However, the epidemiology of leptospirosis remains poorly understood in remote parts of the world. In this study of patients from the island of Mayotte, we isolated 22 strains from the blood of patients during the acute phase of illness. The pathogenic Leptospira strains were characterized by serology and various molecular typing methods. Based on serological data, serogroup Mini appears to be the dominant cause of leptospirosis in Mayotte. Further molecular characterization of these isolates allowed the identification of 10 pathogenic Leptospira genotypes that could correspond to previously unknown serovars. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes in highly endemic regions should contribute to the development of novel strategies for the diagnosis and prevention of this neglected emerging disease.
doi:10.1371/journal.pntd.0000724
PMCID: PMC2889827  PMID: 20582311
13.  Evaluation of pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR  
Iranian Journal of Microbiology  2011;3(3):135-139.
Background and objectives
Leptospirosis is an important zoonotic disease caused by Leptospira interrogans. Leptospirosis leads to economical losses in dairy farm industry. The objective of this study was to evaluate the pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR.
Materials and Methods
Two hundred samples (100 urine and 100 blood) were collected from 100 cows randomly and delivered to the laboratory. Samples were stored at -20 °C. DNA was extracted and purified from the plasma and urine samples and concentrated on diatoms in the presence of guanidine thiocyanate (GuSCN). PCR products were detected and identified as Leptospira by ilumination of the expected size of DNA bands after staining of the agarose gel with ethidium bromide gels. PCR products were purified and sequenced.
Results
The results showed that 28% of urine samples and 23% of plasma samples were contaminated. The major serotypes were Icterohaemorrhagiae (50%) and Pomona (37.5%). The urine samples of 17 cows were positive for Leptospira without positive plasma samples. This indicated that these cows are reservoirs in dairy herds of Shahrekord and dangerous for human health. The plasma samples of twelve cows were positive for Leptospira without positive urine samples.
Conclusions
Leptospira serotypes can be maintained in relatively dry regions and must be considered when dealing with leptospirosis in dairy farms of Shahrekord and human health.
PMCID: PMC3279815  PMID: 22347596
Leptospira; Shahrekord; Cattle; PCR
14.  Characterization of leptospira borgpetersenii isolates from field rats (rattus norvegicus) by 16s rrna and lipl32 gene sequencing 
Brazilian Journal of Microbiology  2010;41(1):150-157.
The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 %, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.
doi:10.1590/S1517-838220100001000022
PMCID: PMC3768625  PMID: 24031475
Leptospirosis; Leptospira borgpetersenii; lipL32; 16S rRNA
15.  Identification of the Hemolysis-Associated Protein 1 as a Cross-Protective Immunogen of Leptospira interrogans by Adenovirus-Mediated Vaccination 
Infection and Immunity  2001;69(11):6831-6838.
New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.
doi:10.1128/IAI.69.11.6831-6838.2001
PMCID: PMC100061  PMID: 11598056
16.  Repetitive sequence element cloned from Leptospira interrogans serovar hardjo type hardjo-bovis provides a sensitive diagnostic probe for bovine leptospirosis. 
Journal of Clinical Microbiology  1988;26(12):2495-2500.
A repetitive sequence element was cloned from the primary etiological agent causing bovine leptospirosis in North America, Leptospira interrogans serovar hardjo type hardjo-bovis. This element was used to design a sensitive diagnostic probe which distinguishes hardjo-bovis from other pathogenic leptospires which commonly infect domestic animals in North America and discriminates between hardjo-bovis and the reference strain for serovar hardjo, hardjoprajitno. By using this probe, it was possible to identify infected cattle shedding hardjo-bovis in their urine. This is the first practical demonstration of a cloned DNA probe for leptospirosis, and it provides a sensitive method for studying the transmission and pathogenesis of L. interrogans infections. Control measures for L. interrogans infections may now be improved by rapidly and efficiently identifying infected animals.
Images
PMCID: PMC266932  PMID: 3230128
17.  Development of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia 
Background
Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism.
Methods
In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats [1]. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland.
Results
The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines.
Conclusion
The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.
doi:10.1186/1476-0711-4-10
PMCID: PMC1185519  PMID: 15987533
18.  Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of the LigB Gene Does Not Affect Virulence in Animal Models of Leptospirosis▿  
Infection and Immunity  2008;76(12):5826-5833.
The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spcr) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.
doi:10.1128/IAI.00989-08
PMCID: PMC2583567  PMID: 18809657
19.  Development of O-antigen gene cluster-specific PCRs for rapid typing six epidemic serogroups of Leptospira in China 
BMC Microbiology  2010;10:67.
Background
Leptospira is the causative agent of leptospirosis. The O-antigen is the distal part of the lipopolysaccharide, which is a key component of outer membrane of Gram-negative bacteria and confers serological specificity. The epidemiology and clinical characteristics of leptospirosis are relative to the serology based taxonomic unit. Identification of Leptospira strains by serotyping is laborious and has several drawbacks.
Results
In this study, the O-antigen gene clusters of four epidemic Leptospira serogroups (serogroup Canicola, Autumnalis, Grippotyphosa and Hebdomadis) in China were sequenced and all genes were predicted in silico. Adding published sequences of two serogroups, Icterohaemorrhagiae (strain Lai and Fiocruz L1-130) and Sejroe (strain JB197 and L550), we identified six O-antigen-specific genes for six epidemic serogroups in China. PCR assays using these genes were developed and tested on 75 reference strains and 40 clinical isolates.
Conclusion
The results show that the PCR-based assays can be reliable and alternative means for rapid typing of these six serogroups of Leptospira.
doi:10.1186/1471-2180-10-67
PMCID: PMC2843611  PMID: 20196873
20.  Application and Validation of PFGE for Serovar Identification of Leptospira Clinical Isolates 
Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars.
Author Summary
Leptospirosis is an infection caused by Leptospira bacteria, and is probably the most widespread zoonosis in the world. It is carried by a wide range of animals that contaminate the environment by shedding organisms in their urine. Humans become infected when they come into contact with contaminated urine or water in the environment that has been contaminated with the urine of infected animals. Despite its ubiquity, isolates are rarely identified to the serovar level due to the cumbersome, complicated serological methods that are involved. Serovar identification is important for epidemiology and enabling public health interventions. In this study, we employed a molecular method of serovar identification using pulsed-field gel electrophoresis to identify 175 clinical isolates of Leptospira. In order to validate this method for serovar identification, we also performed complex serological testing on a subset of the isolates. The results indicated that pulsed-field gel electrophoresis is an appropriate alternative to serological tests for serovar identification. Serovar identities of the clinical isolates are also discussed. Fifteen percent of the clinical isolates were identified as potentially new serovars and demonstrates the utility of a more rapid, standardized molecular method in order to keep up with the changing taxonomy and epidemiology of Leptospira.
doi:10.1371/journal.pntd.0000824
PMCID: PMC2939049  PMID: 20856859
21.  In silico and microarray-based genomic approaches to identifying potential vaccine candidates against Leptospira interrogans 
BMC Genomics  2006;7:293.
Background
Currently available vaccines against leptospirosis are of low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, and provide no cross-protection against the different serovars of pathogenic leptospira. The current major focus in leptospirosis research is to discover conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. There is a need to screen vaccine candidate genes in the genome of Leptospira interrogans.
Results
Bioinformatics, comparative genomic hybridization (CGH) analysis and transcriptional analysis were used to identify vaccine candidates in the genome of L. interrogans serovar Lai strain #56601. Of a total of 4727 open reading frames (ORFs), 616 genes were predicted to encode surface-exposed proteins by P-CLASSIFIER combined with signal peptide prediction, α-helix transmembrane topology prediction, integral β-barrel outer membrane protein and lipoprotein prediction, as well as by retaining the genes shared by the two sequenced L. interrogans genomes and by subtracting genes with human homologues. A DNA microarray of L. interrogans strain #56601 was constructed for CGH analysis and transcriptome analysis in vitro. Three hundred and seven differential genes were identified in ten pathogenic serovars by CGH; 1427 genes had high transcriptional levels (Cy3 signal ≥ 342 and Cy5 signal ≥ 363.5, respectively). There were 565 genes in the intersection between the set encoding surface-exposed proteins and the set of 307 differential genes. The number of genes in the intersection between this set of 565 and the set of 1427 highly transcriptionally active genes was 226. These 226 genes were thus identified as putative vaccine candidates. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro.
Conclusion
Of the 4727 ORFs in the genome of L. interrogans, 226 genes were identified as vaccine candidates by bioinformatics, CGH and transcriptional analysis on the basis of the theory of reverse vaccinology. The proteins encoded by these genes might be useful as vaccine candidates as well as for diagnosis of leptospirosis.
doi:10.1186/1471-2164-7-293
PMCID: PMC1664576  PMID: 17109759
22.  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.
doi:10.1128/AEM.07465-11
PMCID: PMC3346439  PMID: 22344655
23.  Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool 
Brazilian Journal of Microbiology  2013;44(1):165-170.
Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.
doi:10.1590/S1517-83822013005000029
PMCID: PMC3804194  PMID: 24159300
Leptospira; Leptospirosis; Virulent; VNTR; rpoB
24.  Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR. 
Journal of Clinical Microbiology  1997;35(12):3140-3146.
Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.
PMCID: PMC230137  PMID: 9399509
25.  Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans 
Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.
Author Summary
Leptospirosis is one of the most common diseases transmitted by animals worldwide. It is important because it causes an often lethal febrile illnesses in tropical and subtropical areas associated with poor sanitation and agriculture. Leptospirosis may be epidemic, associated with natural disasters and flooding, or endemic in tropical regions. It is unknown how Leptospira cause disease and why different strains cause different severity of illness. In this study we attenuated (weakened) a highly virulent strain of L. interrogans by culturing it in vitro over several months. Comparison of the whole genome sequence before and after the attenuation process revealed a small set of genes that were mutated, and therefore associated with virulence. We discovered a putative soluble adenylate cyclase with host cell cAMP elevating activity, with implications for immune evasion and a new gene family that is upregulated in vivo during acute hamster infection. Interestingly, both Bartonella bacilliformis and Bartonella australis also have this unique gene family we describe in pathogenic Leptospira. This information aids in our understanding of Leptospira evolution and pathogenesis.
doi:10.1371/journal.pntd.0002468
PMCID: PMC3789758  PMID: 24098822

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