Look and you will find—a literature review of new strains of Leptospira spp., 2000–2025

Leptospirosis is a zoonosis of worldwide distribution with major incidence in the tropics. In New Caledonia, it has been studied for years and is regarded as a major public health concern. It is characterized by an endemic pattern with seasonal epidemics arising during rainy periods, notably during “la Nina” periods. Both the endemic and epidemics have a rural epidemiology and result from the infection by a variety of Leptospira strains. The Institute Pasteur in New Caledonia (IPNC) is the reference laboratory for human leptospirosis and has been studying this disease for years, notably developing and implementing efficient molecular diagnostic tools. At the same time, an expertise on this disease has led to numerous studies on the epidemiology and physiopathology of leptospirosis. IPNC recently started a study aiming at identifying the Leptospira strains currently circulating in New Caledonia and their corresponding animal reservoirs. Because leptospirosis is a complex pathology involving reservoir hosts, environmental sources of infection and susceptible mammals including Man, the control of human leptospirosis has to take these animal reservoirs into account. In any region, a limited number of pathogenic Leptospira strains are circulating which can cause disease in humans. Every single Leptospira strain is supposed to be maintained by one or several reservoir animal host species. Current molecular methods allow identifying the infecting Leptospira strain at a sub-species level, possibly by directly using these molecular tools with DNA extracted from animal and human clinical specimens. We evaluated the usefulness of the sequence polymorphism of diagnostic PCR products and used this molecular tool for studying the prevalence and identification of the different Leptospira strains in both rodent and non-rodent Mammals. Because rodents are known to play a pivotal role in the maintenance of major Leptospira strains, an ecological study of rodent dynamics and their Leptospira prevalence is conducted in an area of highest incidence, taking seasonal and meteorological variability into account. Preliminary results confirm their role as reservoirs for L. interrogans Icterohaemorragiae and L. borgpetersenii Ballum and highlight a joint effect of temperature and pluviometry on rodents’ populations and their Leptospira carriage. Additionally, the introduced deer Cervus timorensis russa was shown to be a reservoir for L. borgpetersenii Hardjo.

BackgroundIn 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.ResultsDefined 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.ConclusionsMALDI-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.

Leptospirosis is a zoonotic disease found virtually worldwide. Microscopic Agglutination Test with live leptospira (MAT) is the reference method for the serological diagnosis of leptospirosis. MAT is based on assessing serum potential to agglutinate live reference serovar Leptospira maintained at a reference laboratory. At some laboratories having own collections of isolated and reference Leptospira strains applicable for serological diagnosis, those microorganisms are maintained for many years by repeated subculturing, that increases markedly a chance of strain cross-contamination. The lack of adequate quality control for reference strains may affect data of epidemiological studies. Control of Leptospira spp. reference strains purity and stability of their antigenic composition is very important for diagnosis of leptospirosis. The study objective was to compare the 16S rRNA gene nucleotide sequences of some Leptospira strains from the collection of the St. Petersburg Pasteur Institute to with relevant sequences uploaded to GenBank. In this study, 38 Leptospira strains were investigated. Nucleotide sequences of 36 strains were deposited in the international GenBank database, inconsistencies were revealed in two strains. The study found that the control Leptospira strains from the collection of the St. Petersburg Pasteur Institute had minimal dissimilarities from international control strains. The analysis of the resultant 16S rRNA sequences has shown the presence of point mutations, transitions, deletions and insertions, regardless of the strain species. The open leptospira pan-genome demonstrates high genomic variability in species due to the capability of leptospira for lateral gene transfer in order to adapt to changing environmental conditions. The massive acquisition and loss of genes give rise to an increased species diversity. The 16S rRNA gene is suitable for screening diagnostics; however, high level of the fragment similarity and close phylogenetic relationship between different species put bounds to its use in genotyping. The presence of point nucleotide mutations is most likely associated with the evolutionary mechanisms of leptospira, their ability to horizontal gene transfer and crossing-over, including ribosomal genes, but this assumption necessitates additional research. For specimen genotyping it is necessary to select alternative genes with high specificity and sufficient level of nucleotide divergence. The study shows a need for genetic analysis of collection strains in order to control the purity of cultures.

Circulating Foamy Macrophages in the Golden Syrian Hamster (Mesocricetus auratus) Model of Leptospirosis

El objetivo de este estudio fue determinar la diversidad molecular de las proteínas OmpL1, LipL32, LipL41, LigA y LigB y de los genes que las codifican mediante análisis bioinformáticos en diferentes cepas patógenas de Leptospira spp., a partir de la información disponible en las bases de datos. Se utilizaron las secuencias de aminoácidos de las proteínas OmpL1, LipL32, LipL41, LigA y LigB, así como las de los genes que las codifican en las cepas de Leptospira spp. registradas en The National Center for Biotechnology Information (NCBI). Los análisis de las proteínas y los genes se realizaron mediante los recursos Protein, Nucleotide y Gene del NCBI. La alineación de las secuencias consenso se realizó con las herramientas PSI-BLAST y BLASTn. El porcentaje de cobertura de las secuencias seleccionadas de los genes ompL1, lipL32, lipL41, ligA y ligB en cepas patógenas de Leptospira spp. es de 100 % para ompL1, lipL32 y lipL41, 75 % para ligA y 99 % para ligB con porcentajes de identidad de 85, 98, 88, 90 y 80 % respectivamente; el porcentaje de cobertura de las secuencias seleccionadas de las proteínas es de 100, 77, 99, 100 y 100 % con porcentajes de identidad de 90, 99, 92, 63 y 60 % respectivamente, lo cual indica que los genes y las proteínas, excepto las proteínas LigA y LigB, son bastante conservadas en los diferentes serovares patógenos de Leptospira spp. Según dichos resultados, se recomienda realizar análisis complementarios de estas proteínas con el fin de determinar si es viable su uso como candidatos vacunales.

The general goal of reference centres is to support the community, from diagnostic laboratories to research institutions, in the execution of their work by providing reference strains and reagents and giving instructions and recommendations to individual colleagues and national and international organisations on a wide variety of issues. There are different levels of reference centres, from local to international, with an increasing package of tasks and responsibilities. Local reference centres might limit activities to diagnostic confirmation by applying standard testing, while international reference centres cover a wider range of activities from design, validation and harmonisation of diagnostic and reference technologies to international monitoring associated with recommendations on the global burden and distribution of leptospirosis and its prevention and control to national and international health decision makers. This chapter focusses on four major pillars constituting reference tasks in addition to the obvious provision of reference substances, i.e. Research and training, Diagnosis, Identification of Leptospira and Surveillance. Due to financial and organisational constraints, reference centres are restricted in their capacity for basic research and consequently focus on applied research into various aspects of leptospirosis. They offer training, either individually or groupwise, that might vary from standard technologies to novel sophisticated methodologies, depending on the need and requests of the trainee. Most reference centres are involved in the confirmation of preliminary diagnosis obtained at peripheral levels, such as local hospitals and health centres, while other major activities involve the design and validation of diagnostics, their international harmonisation and quality assurance. Identification of causative Leptospira strains (or serovars) is key to the identification of infection sources and is critical for surveillance. Hence, reference centres also focus on the development, application and provision of methods that are required for unambiguous characterisation of new and recognised Leptospira strains and the maintenance of the integrity of strain collections. In line with their central role, reference centres are frequently associated with local, national and/or international surveillance activities linked to an advisory role and the production of guidelines. Such surveillance activities usually comprise collation of morbidity and mortality data, signalling of outbreaks and the investigation of infection sources and risks.

Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. This disease is common in tropical and subtropical areas, constituting a serious public health problem. Pathogenic Leptospira have the ability to escape the human Complement System, being able to survive when in contact with normal human serum. In a previous study, our group demonstrated that supernatants of pathogenic Leptospira (SPL) inhibit the three activation pathways of the Complement System. This inhibition can be directly correlated with the activity of secreted proteases, which cleave the Complement molecules C3, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Terminal Pathway of Complement, called the membrane attack complex (MAC). We observed that proteases present in SPL from different Leptospira strains were able to cleave the purified proteins C5, C6, C7, C8, and C9, while culture supernatant from non-pathogenic Leptospira strains (SNPL) had no significant proteolytic activity on these substrates. The cleavages occurred in a time-dependent and specificity manner. No cleavage was observed when we used whole serum as a source of C5–C9 proteins, probably because of the abundant presence of plasma protease inhibitors such as α2-macroglobulin. Complement protein cleavage by SPL was inhibited by 1,10-phenanthroline, indicating the involvement of metalloproteases. Furthermore, 1,10-phenanthroline- treated normal human serum diminished pathogenic leptospira survival. We also analyzed the proteolytic activity of thermolysin (LIC13322) a metalloprotease expressed exclusively by pathogenic Leptospira strains. Recombinant thermolysin was capable of cleaving the component C6, either purified or as part of the SC5b-9 complex. Furthermore, we found that the MAC proteins C6–C9 interact with thermolysin, indicating that this metalloprotease may have an additional inhibitory effect on these molecules by direct interactions. Finally, a functional assay demonstrated that thermolysin was able to inhibit MAC-dependent erythrocytes lysis. We conclude that proteases secreted exclusively by pathogenic Leptospira strains are capable of degrading several Complement effector molecules, representing potential targets for the development of new therapies and prophylactic approaches in leptospirosis.

BackgroundLeptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software.ResultsThe spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data.ConclusionsIn conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for epidemiological purposes in order to enrich our knowledge about the epidemiology of the infection in different areas and generate control strategies.

Leptospirosis, a zoonosis caused by Leptospira, is recognized as an emergent infectious disease. In currently, the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. In this experiment, a 550 bp fragment of large ribosomal RNA gene (16S rRNA) was sequenced and constructed phylogenetic tree from a panel of six Vietnamese pathogenic strains of Leptospira spirochetes (e.g., Pomona, Canicola, Mitis, Ictero haemohagiae, Bataviae, and Grippotyphosa). The results showed a close relationship of L.Pomona_VN and L.Hardjo (bootstrap: 99%). L.Canicola_VN and L.Ictero haemohagiae_VN appeared to be weak related to the classic L.Canicola, L. Grippotyphosa, these assemblage have a bootstrap support of 62%. The other strains (L.Mitis_VN and L.Grippotyphosa_VN) were appeared monophyletic, while their sister group (L.Bataviae_VN) relationship found only weak support (bootstrap: 62%). We also selected six genes [e.g. the immunoglobulin like proteins A and B (LigA and LigB genes), outer membrane protein (OmpL1 gene), and lipopolysaccharide (LipL32, LipL41, and LipL21 genes)] and checked gene expression in these Leptospira strains by polymerase chain reaction (PCR) method. There were three genes (e.g., LipL32, LipL21, and LigA genes) expressed in all strains, OmpL1 gene occured in 4 strains (L.Bataviae_VN, L.Canicola_VN, L.Grippotyphosa_VN and L.Mitis_VN), whereas LipL41 and LigB genes did not appear in any Leptospira strains. A multi-antigenic epitope potential of two gene (Lip L21 and Lip L32) was predicted by bioinformatic tools for designing a recombinant vaccine against leptospirosis. There were 3 multi-epitope regions (1 region and 95 antigenic epitope for B and T cells of LipL21 peptide; 2 regions and 124 antigenic epitope for both B and T cells of LipL32 peptide). It should be more of the deeply molecular biology studies to confirm the level agglutinating, antigen cleavage, peptide specificity matrices as well as neutralizing antibodies in the immune responses of DNA vaccine of these genes.

Leptospirosis is an important worldwide zoonosis, and it has also been reported in Slovenia. The cultivation of Leptospira from human material is difficult. Despite that, we successfully isolated 12 human Leptospira strains isolated from patients between 2002 and 2020 and used various methods for the phenotypic and genotypic characterization of the strains, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using our own MALDI-TOF data library, melting temperature analysis of the amplified lfb1 gene, determination of Leptospira serogroups using rabbit immune sera, NotI-RFLP of the whole Leptospira genome, multilocus sequence typing (MLST) of seven housekeeping genes, and whole-genome sequencing (WGS)-based typing. We confirmed the presence of four pathogenic Leptospira species (L. kirschneri, L. interrogans, L. borgpetersenii, and L. santarosai) and three serogroups: Grippotyphosa, Icterohaemorrhagiae, and Sejroe. MALDI-TOF identified three of seven isolates at the species level and four isolates at the genus level. Serovars of 8 of the 10 strains were determined using NotI-RFLP. MLST showed that the clinical isolates belonged to sequence types ST17, ST110, and ST155. WGS confirmed the analysis of Leptospira strains using conventional methods. In addition, WGS provided better taxonomic resolution for isolate DDA 10944/10.

Leptospira interrogans serogroup Canicola is one of the most important pathogens causing leptospirosis and is used as a vaccine strain of the current Chinese human leptospirosis vaccine. To characterize leptospiral pathogens, L. interrogans serogroup Canicola vaccine strain 611 and circulating isolate LJ178 from different hosts at different periods were sequenced using a combined strategy of Illumina X10 and PacBio technologies, and a comprehensive comparative analysis with other published Leptospira strains was conducted in this study. High levels of genomic similarities were observed between vaccine strain 611 and circulating isolate LJ178; both had two circular chromosomes and two circular extrachromosomal replicons. Compared with the strain 611 genome, 132 single nucleotide polymorphisms and 92 indels were found in strain LJ178. The larger lipopolysaccharide biosynthesis locus of serogroup Canicola was identified in both genomes. The phylogenetic analysis based on whole-genome sequences revealed that serogroup Canicola was not restricted to a specific host or geographic location, suggesting adaptive evolution associated with the ecologic diversity. In summary, our findings provide insights into a better molecular understanding of the component strains of human leptospirosis vaccine in China. Furthermore, these data detail the genetic composition and evolutionary relatedness of Leptospira strains that pose a health risk to humans.

BackgroundAlthough Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease.Methodology/Principal findingsWe used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017.Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains.Conclusions/SignificanceThe present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.

Objectives: This study investigated the presence of Leptospira spp. strains in the Carioca River, Rio de Janeiro, whose waters have been impacted by urbanization in the city. Theoretical Framework: Leptospirosis is a global zoonosis caused by bacteria of the genus Leptospira, which infect humans and animals, usually through contact with contaminated water and soil. Environments under inadequate sanitation conditions are the main source of infection. Methods: Samples were collected at five points in the Carioca River and, after consecutive filtrations (0.45 µm and 0.2 µm), were plated on EMJH and Fletcher media containing 5-fluorouracil. Serotyping was performed using the microscopic agglutination test (MAT), and the pathogenicity genes (flaB and lipL41) and the genus marker (rrs) were analyzed using polymerase chain reaction (PCR). Antimicrobial susceptibility and Minimum Inhibitory Concentration (MIC) were determined using the microdilution technique. Results and Discussion: Four strains of Leptospira sp. were isolated, and the genus was confirmed. Only strain CLEP (Leptospira Collection) 00368 showed MAT reactivity with a titer of 800 for serogroup Icterohaemorragiae. Strains CLEP00366 and CLEP00370 presented the highest MIC values ​​for most of the antimicrobials tested, reinforcing the hypothesis of anthropogenic impact on the environment. Research Implications: The results reinforce the role of the environment as a source of infection and the importance of investigating antimicrobial resistance in the genus Leptospira. Originality/Value: The information from this pioneering study can encourage preventive measures regarding infection risks in both wild and urban areas.

Leptospirosis is a major public health problem in Andaman Islands. Several strains of Leptospira have been isolated from the Andamans over the years. Leptospires isolated recently from human cases were compared with one of the earliest available isolates from these islands, dating back to 1929, to study their serological and genetic relatedness. Randomly amplified polymorphic DNA (RAPD) fingerprints of the isolates, generated with a primer used previously to differentiate between Leptospira species and serovars, revealed that some of the recent isolates were genetically identical to the 1929 isolate. The antigenic properties of these strains, as revealed by microscopic agglutination tests with group-specific rabbit antisera and mAbs, were also similar. These findings suggest that a Leptospira strain originally isolated in 1929 has possibly persisted in these islands for over 70 years and continues to cause acute leptospirosis in humans.

There are still many people who are less concerned with environmental cleanliness, where this can lead to the accumulation of bad bacteria that cause disease. One of them is Leptospirosis, a disease caused by a bacterial infection, namely the Leptospira strain. Leptospirosis is a disease caused by the laying of bacteria in animals that is transmitted to humans. This disease is often ignored by the public due to lack of understanding about this disease and the high cost of conducting examinations and consulting a doctor or hospital. So that in overcoming this we need a way that is able to help the community in knowing and diagnosing Leptospirosis, one of which is by using an expert system. The expert system used in solving the problem of Leptospirosis is by using the Naïve Bayes method. The application of the Naïve Bayes method in diagnosing Leptospirosis is carried out by collecting data about Leptospirosis where this process aims to find out what symptoms are caused by Leptospirosis. The process of collecting data on this disease is done by interviewing an expert or doctor who handles the problem of Leptospirosis. The results of the diagnosis of Leptospirosis based on the calculation of the Naïve Bayes method with new user data samples get results with a definite level of accuracy where the user experiences Leptospirosis disease with mild symptoms of 63% and the results of the user experiencing Leptospirosis disease with severe symptoms of 37%. Naïve Bayes is able to diagnose with 100% accuracy seen from the total severe and mild symptoms