Pathogenic Leptospira Species Research Articles

Mixed Leptospira infections in domestic animals from a rural community with high leptospirosis endemicity.

Leptospirosis is one of the most common zoonoses in the world which is associated with a severe febrile disease in humans causing a variety of syndromes including meningitis, interstitial nephritis, hepatitis, and sometimes death. Leptospirosis is caused by different pathogenic Leptospira species divided into almost 30 serogroups and more than 300 serovars which are carried by some animal asymptomatic chronic infections. Humans can become infected through direct contact with animal urine or indirectly by coming into contact with fresh water or mud contaminated with urine. In this research, we looked for leptospiral DNA in urine samples from dogs living in a rural, low-income and highly endemic community in the coast of Ecuador. We used molecular biology and next-generation sequencing for the detection. Our results showed evidence of two Leptospira species, L interrogans and L. santarosai, genomes in three dogs. It has been widely known that animal carriers are typically infected with a single leptospiral strain. However, recent reports, including the present one, indicate that carrier animals may be coinfected with two or more leptospiral species.

Unveiling the impact of Leptospira TolC efflux protein on host tissue adherence, complement evasion, and diagnostic potential.

The TolC family protein of Leptospira is a type I outer membrane efflux protein. Phylogenetic analysis revealed significant sequence conservation among pathogenic Leptospira species (83%-98% identity) compared with intermediate and saprophytic species. Structural modeling indicated a composition of six β-strands and 10 α-helices arranged in two repeats, resembling bacterial outer membrane efflux proteins. Recombinant TolC (rTolC), expressed in a heterologous host and purified via Ni-NTA chromatography, maintained its secondary structural integrity, as verified by circular dichroism spectroscopy. Polyclonal antibodies against rTolC detected native TolC expression in pathogenic Leptospira but not in nonpathogenic ones. Immunoassays and detergent fractionation assays indicated surface localization of TolC. The rTolC's recognition by sera from leptospirosis-infected hosts across species suggests its utility as a diagnostic marker. Notably, rTolC demonstrated binding affinity for various extracellular matrix components, including collagen and chondroitin sulfate A, as well as plasma proteins such as factor H, C3b, and plasminogen, indicating potential roles in tissue adhesion and immune evasion. Functional assays demonstrated that rTolC-bound FH retained cofactor activity for C3b cleavage, highlighting TolC's role in complement regulation. The rTolC protein inhibited both the alternative and the classical pathway-mediated membrane attack complex (MAC) deposition in vitro. Blocking surface-expressed TolC on leptospires using specific antibodies reduced FH acquisition by Leptospira and increased MAC deposition on the spirochete. These findings indicate that TolC contributes to leptospiral virulence by promoting host tissue colonization and evading the immune response, presenting it as a potential target for diagnostic and therapeutic strategies.

Under-Reporting of Human Leptospirosis Cases in Cities of Triângulo Mineiro, Minas Gerais, Brazil.

Leptospirosis is an infectious disease caused by the pathogenic Leptospira species through direct or indirect contact with infected animals. Due to protean clinical manifestation in the early stages, leptospirosis is often difficult to distinguish from other common acute febrile illnesses, such as dengue. Thus, this study aimed to investigate the prevalence of leptospirosis in suspected dengue patients whose serological diagnosis was negative. A total of 449 serum samples from patients (negative IgM-ELISA dengue) with fever, headache, myalgia, and nausea were tested. The Dual-Path Platform (DPP) rapid test developed by the Instituto de Tecnologia em Imunobiológicos Bio-Manguinhos in the city of Rio de Janeiro, Brazil was used for screening IgM antibodies against Leptospira in blood serum, and the microscopic agglutination test (MAT) was performed on samples positive in the DPP for leptospirosis, as well as on an equal number of negative samples. Results: The data obtained from the samples analyzed with the DPP assay showed 26 positive results (5.79%), of which 38.46% were male and 61.54% female, with a mean age of 41 years. We tested 52 samples using the MAT, including 26 reactive for IgM and 26 non-reactive in the DPP assay. Nine samples (17.31%) were reactive, and among them, six also showed reactivity in the DPP assay. Of the six samples reactive in both tests, 66.67% were female, living in urban areas in the city of Uberlândia, with a mean age of 50 years, being 50% white, 33.33% brown, and 16.67% black. The findings demonstrated that leptospirosis cases are underdiagnosed and undertreated in the study population and more attention needs to be paid for ruling out leptospirosis and other pathogens causing acute febrile illness in dengue-endemic areas.

Isolation of Virulent Leptospira Serogroup Australis Field Strains from Symptomatic Dogs for Canine Leptospiral Vaccine Development.

Leptospirosis is a widespread zoonosis caused by spirochaetes belonging to the pathogenic species of Leptospira, which are classified into more than 25 serogroups and 250 serovars. Vaccination can prevent the disease in dogs but offers incomplete efficacy because of a lack of cross-protection between serogroups. The aim of this study was to validate a robust recruitment and sampling process, with the objectives of isolating and typing circulating Leptospira pathogenic strains and then selecting those of proven virulence and pathogenicity for vaccine development. Blood and urine samples from dogs with clinical syndromes compatible with acute leptospirosis were sterilely collected and transported to a reference laboratory for a micro-agglutination test (MAT), PCR, and bacterial isolation. Isolated strains underwent molecular typing using RNA16S, variable-number tandem repeat (VNTR), and pulsed-field gel electrophoresis (PFGE). Subtyping was performed using core genome multilocus sequence typing (CgMLST). Among 64 included dogs, 41 had MAT and/or PCR results compatible with Leptospira infection, and 14 Leptospira strains were isolated. Based on molecular typing, 11 isolates were classified as L. interrogans serogroup Australis, serovar Bratislava, and 3 as serogroup Icterohaemorrhagiae, serovar Icterohaemorrhagiae. CgMLST subtyping revealed a diversity of clonal groups (CGs) distributed in several regional clusters. Besides validating a robust recruitment and sampling process, this study outlines the value of combining PCR and serological testing when suspecting leptospirosis and the usefulness of implementing molecular typing methods to identify circulating field strains. It also confirms the epidemiological importance of the Australis serogroup and allows for the collection of different highly pathogenic strains for vaccine development.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding

ObjectiveLeptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. MethodsForty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. ResultsWe revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. ConclusionWe repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

Molecular Characterization of Leptospira Species among Patients with Acute Undifferentiated Febrile Illness from the Municipality of Villeta, Colombia.

Leptospira is a bacterial genus that includes several pathogenic species related to leptospirosis. In Colombia, leptospirosis is a mandatorily reported disease, widely distributed across the country. In the Villeta municipality, leptospirosis has been identified as an important cause of febrile illness; however, to date, no studies have been performed to identify the circulating species. A genus-specific qualitative qPCR was performed on DNA extracted from febrile patients' acute-phase whole-blood samples targeting a fragment of the rrs gene. Positive qPCR samples were further amplified for the adk, icdA, LipL32, LipL41, rrs, and secY genes through conventional PCR for sequencing. All high-quality obtained sequences were further assessed through concatenated phylogenetic analysis. A total of 25% (14/56) of febrile patients' acute blood samples were positive for Leptospira spp. High-quality sequences were obtained for only five genes, and analysis through concatenated phylogeny identified that all sequences clustered within the P1/pathogenic clade; some of them formed a robustly supported clade with Leptospira santarosai, and others were closely related with other Leptospira species but exhibited considerable genetic divergence. We describe the presence of pathogenic Leptospira species among febrile patients from the Villeta municipality and identify L. santarosai and other Leptospira species as causative agents of leptospirosis in the region.

Epidemiological Features of Leptospirosis and Identification of Leptospira wolffii as a Persistently Prevailing Species in North-Central Bangladesh.

Leptospirosis is considered to be the most widespread, yet neglected, re-emerging zoonotic disease caused by infection with a pathogenic species of the genus Leptospira. Although this disease is prevalent in Bangladesh, the recent epidemiological status has not yet been well documented. In this study, we aimed to determine the prevalence of leptospirosis among febrile patients using different diagnostic methods and to characterize the epidemiological features and species of Leptospira in Mymensingh, north-central Bangladesh. Among the blood samples of 186 patients with suspected leptospirosis who met the inclusion criteria, including having a fever for more than 5 days (November 2021-June 2022), 88 samples (47%) were Leptospira-positive according to IgM LAT, IgM ELISA, or nested PCR (positivity rates: 38%, 37%, and 42%, respectively). Nested PCR showed a significantly higher positivity rate (54%) in patients with a short fever (5-10 day) than the other methods did, with lower rates among those with a longer fever. Leptospirosis cases were more common in males (68%), those 16-45 years of age (70%), residents of rural areas (81%), and farmers (41%). In addition to a fever, myalgia and jaundice were found in more than 70% of the patients, while variable symptoms were observed. The 16S rRNA sequencing analysis revealed that the Leptospira species in all the 22 samples tested were L. wolffii, belonging to the pathogenic subclade P2. This study showed the recent epidemiological features of leptospirosis in Bangladesh, indicating the presumptive predominance of L. wolffii since 2019.

A New Beginning to the Existing Medicines; Repurposing FDA-Approved Drugs for the Neglected Re-Emerging Disease Leptospirosis.

Leptospirosis is one of the re-emerging zoonotic diseases, especially in tropical regions. Many antibiotics are used to treat leptospirosis, but there are no scientific evidence-based guidelines or systematic clinical trials for using these drugs. A bioinformatics approach was made to shortlist some Food and Drug Administration (FDA) of the United States of America-approved and currently used drugs for leptospirosis. The existing drugs from the Drug Bank database, which are currently not used for leptospirosis, were selected to identify their target proteins and binding sites using bioinformatics methods. Orthologues of these target proteins were selected from the proteome database of Leptospira. The similar sites and their interactions with the drugs were validated and recommended for use in leptospirosis. Further, the sensitivity of recommended drugs was also validated in vitro. The sequences and structures of these proteins were compared under strictly controlled parameters and shortlisted Gatifloxacin, Imipenem, Latamoxef, Doripenem, Tigecycline, and Lactams as repurposable drugs for leptospirosis. An in vitro validation of the drugs showed significant antileptospiral activity in 12 serovars with low IC50 concentrations and also showed that the IC50 values varied across Leptospira serovars. Further, suitable proteins under the concept of "One Target, Many Drugs" identified DNA gyrase subunit A (Q72WD1), 30S ribosomal protein S9 (Q72U99), and 30S ribosomal protein S12 (Q72UA6), and these proteins were found across the pathogenic, saprophytic, and intermediate species of Leptospira. We describe a method to find repurposable drugs from the approved list that are not currently used to treat leptospirosis and validate them to be taken forward for systematic clinical trials specific to leptospirosis for recommendations in clinical use.

Identification of Leptospira interrogans in Ototylomys phyllotis (Rodentia: Cricetidae) from Yucatan, Mexico

Small rodents are the most relevant natural reservoirs of pathogenic Leptospira species. Several species of these small rodents have been described as carriers in Yucatan, Mexico. It is recognized that identifying the carriers and knowing their distribution is relevant to reducing the transmission risk of Leptospira spp. to susceptible hosts, including humans. The aim is to evidence the presence of pathogenic Leptospira spp. in small rodents captured in Tinum, Yucatan, Mexico. Forty–seven rodents of the species Heteromys gaumeri, Ototylomys phyllotis, Peromyscus yucatanicus, Sigmodon hispidus and Mus musculus were captured in four sites from the study municipality. A kidney fragment was collected and used in the extraction of total DNA. Two endpoint polymerase chain reactions (PCR) were used to detect Leptospira spp. DNA. The bioinformatic analysis and the construction of a phylogenetic tree determined the bacteria species. The reactions showed a total infection frequency of 8.5 % (95 % CI 3.3 – 19.9 %). All positive specimens were O. phyllotis. The species identified in the bioinformatic analysis and the phylogenetic tree was L. interrogans. This Leptospira species is relevant for public health because it is responsible for most severe cases of leptospirosis in humans. Likewise, it has been previously identified in small rodents from Mexico and Yucatan. The study shows that O. phyllotis carries L. interrogans. More research is needed to determine the risk of transmission to other hosts, including humans.

Application of pulsed-field gel electrophoresis for molecular identification of pathogenic Leptospira species in Iran: a rapid and reliable method.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira serovars. The genus Leptospira cannot differentiated by conventional techniques. However, identity determination of pathogenic serovar is precious of public health problems and epidemiological studies. Pulsed-field gel electrophoresis facilitates rapid identification of Leptospires to the serovar levels. In this study, we employed PFGE to evaluate 28 Leptospira isolates, with animal, human and environmental origin, obtained from Razi Vaccine and Serum Research Institute of Karaj, Iran. PFGE patterns of 28 Leptospira serovars were generated using the Not I restriction enzyme in comparison with the lambda ladder. Out of 28 serovars evaluated, we identified 22 different pulsed types, designated P1-P22. Out of 22 pulse groups, 3 were found to be a common type, but others were a single Type. Groups consisting of the common type were P3, P9, P14, and P16. The results showed that the discriminatory index of PFGE by Not I enzyme was 0.99, demonstrating heterogeneous differentiation among serovar members. The PFGE methodology used in this study showed excellent interlaboratory report usability, rapid, reliable, enabling standardization and data sharing between laboratories.

Are Pathogenic Leptospira Species Ubiquitous in Urban Recreational Parks in Sydney, Australia?

Leptospirosis is a zoonotic disease caused by the spirochete bacteria Leptospira spp. From December 2017 to December 2023, a total of 34 canine leptospirosis cases were reported in urban Sydney, Australia. During the same spatio-temporal frame, one locally acquired human case was also reported. As it was hypothesised that human residents and companion dogs might both be exposed to pathogenic Leptospira in community green spaces in Sydney, an environmental survey was conducted from December 2023 to January 2024 to detect the presence of pathogenic Leptospira DNA in multipurpose, recreational public parks in the council areas of the Inner West and City of Sydney, Australia. A total of 75 environmental samples were collected from 20 public parks that were easily accessible by human and canine visitors. Quantitative PCR (qPCR) testing targeting pathogenic and intermediate Leptospira spp. was performed, and differences in detection of Leptospira spp. between dog-allowed and dog-prohibited areas were statistically examined. The global Moran's Index was calculated to identify any spatial autocorrelation in the qPCR results. Pathogenic leptospires were detected in all 20 parks, either in water or soil samples (35/75 samples). Cycle threshold (Ct) values were slightly lower for water samples (Ct 28.52-39.10) compared to soil samples (Ct 33.78-39.77). The chi-squared test and Fisher's exact test results were statistically non-significant (p > 0.05 for both water and soil samples), and there was no spatial autocorrelation detected in the qPCR results (p > 0.05 for both sample types). Although further research is now required, our preliminary results indicate the presence of pathogenic Leptospira DNA and its potential ubiquity in recreational parks in Sydney.

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Prevalence, genetic diversity and eco-epidemiology of pathogenic Leptospira species in small mammal communities in urban parks Lyon city, France.

Rodents are recognized as the main reservoirs of Leptospira spp. Rats, in particular, serve as hosts for the widely predominant Leptospira interrogans serovar Icterohaemorrhagiae, found worldwide. Several studies have shown the importance of other reservoirs, such as mice or hedgehogs, which harbor other leptospires' serovars. Nevertheless, our knowledge of circulating Leptospira spp. in reservoirs other than rats remains limited. In this context, we proposed an eco-health approach to assess the health hazard associated with leptospires in urban green spaces, where contacts between human/small mammals and domestic animals are likely. We studied the prevalence, the diversity of circulating strains, and epidemiology of pathogenic Leptospira species in small terrestrial mammal communities (rodents and shrews), between 2020-2022, in two parks in Lyon metropolis, France. Our study showed a significant carriage of Leptospira spp. in small terrestrial mammals in these parks and unveiled a global prevalence rate of 11.4%. Significant variations of prevalence were observed among the small mammal species (from 0 to 26.1%), with Rattus norvegicus exhibiting the highest infection levels (26.1%). We also observed strong spatio-temporal variations in Leptospira spp. circulation in its reservoirs. Prevalence seems to be higher in the peri-urban park and in autumn in 2021 and 2022. This is potentially due to differences in landscape, abiotic conditions and small mammal communities' composition. Our study suggests an important public health relevance of rats and in a lesser extent of other rodents (Apodemus spp., Clethrionomys glareolus and Mus musculus) as reservoirs of L. interrogans, with rodent species carrying specific serogroups/serovars. We also emphasize the potential hazard associated between the shrew Crocidura russula and L. kirschneri. Altogether, these results improve our knowledge about the prevalence of leptospirosis in an urban environment, which is an essential prerequisite for the implementation of prevention of associated risks.

Inter-species Transcriptomic Analysis Reveals a Constitutive Adaptation Against Oxidative Stress for the Highly Virulent Leptospira Species.

Transcriptomic analyses across large scales of evolutionary distance have great potential to shed light on regulatory evolution but are complicated by difficulties in establishing orthology and limited availability of accessible software. We introduce here a method and a graphical user interface wrapper, called Annotator-RNAtor, for performing interspecies transcriptomic analysis and studying intragenus evolution. The pipeline uses third-party software to infer homologous genes in various species and highlight differences in the expression of the core-genes. To illustrate the methodology and demonstrate its usefulness, we focus on the emergence of the highly virulent Leptospira subclade known as P1+, which includes the causative agents of leptospirosis. Here, we expand on the genomic study through the comparison of transcriptomes between species from P1+ and their related P1- counterparts (low-virulent pathogens). In doing so, we shed light on differentially expressed pathways and focused on describing a specific example of adaptation based on a differential expression of PerRA-controlled genes. We showed that P1+ species exhibit higher expression of the katE gene, a well-known virulence determinant in pathogenic Leptospira species correlated with greater tolerance to peroxide. Switching PerRA alleles between P1+ and P1- species demonstrated that the lower repression of katE and greater tolerance to peroxide in P1+ species was solely controlled by PerRA and partly caused by a PerRA amino-acid permutation. Overall, these results demonstrate the strategic fit of the methodology and its ability to decipher adaptive transcriptomic changes, not observable by comparative genome analysis, that may have been implicated in the emergence of these pathogens.

Genetic structure and diversity of the rfb locus of pathogenic species of the genus Leptospira.

Leptospirosis is caused by pathogenic strains of the genus Leptospira and is considered the most widespread zoonotic bacterial disease. The genus is characterized by the large number of serology variants, which challenges developing effective serotyping methods and vaccines with a broad spectrum. Because knowledge on the genetic basis of the serological diversity among leptospires is still limited, we aimed to explore the genetic structure and patterns of the rfb locus, which is involved in the biosynthesis of lipopolysaccharides, the major surface antigen that defines the serovar in leptospires. Here, we used genomic data of 722 pathogenic samples and compared the gene composition of their rfb locus by hierarchical clustering. Clustering analysis showed that the rfb locus gene composition is species-independent and strongly associated with the serological classification. The samples were grouped into four well-defined classes, which cluster together samples either belonging to the same serogroup or from different serogroups but sharing serological affinity. Our findings can assist in the development of new strategies based on molecular methods, which can lead to better tools for serological identification in this zoonosis.

Seroprevalence of pathogenic Leptospira serogroups in asymptomatic domestic dogs and cats: systematic review and meta-analysis.

Leptospirosis is a neglected zoonotic disease transmitted by contact with the urine of animals infected with pathogenic species of the bacteria Leptospira or by contact with environments contaminated with the bacteria. Domestic dogs and cats may act as reservoirs or as sentinels of environmental contamination with leptospires, posing a public health concern. There is a great diversity of leptospires, and one common way to classify them is into serogroups that provide some information on the host species they are associated with. The aims of this study were: (1) to quantitatively summarize the overall prevalence and serogroup-specific prevalence of antibodies against pathogenic leptospires in asymptomatic dogs and cats and (2) to identify environmental and host characteristics that may affect the prevalence. Three electronic databases and the reference lists of eligible articles were screened, for epidemiological studies conducted between the years 2012-2022. We estimated overall and serogroup-specific prevalence using three-level meta-analysis models and assessed potential sources of heterogeneity by moderator analysis and meta-regression. Eighty-four studies met the inclusion criteria (dog studies 66.7%, cat studies 26.2%, and both species 7.1%). There were significant differences between dogs and cats in the overall prevalence model (P < 0.001), but not in the serogroup-specific model (P>0.05). In dogs, the prevalence of Leptospira interrogans serogroup Canicola was significantly higher than the other pathogenic serogroups (P < 0.001), while in cats there were no significant differences among serogroups (P = 0.373). Moderator analysis showed that the prevalence of L. kirschneri serogroup Grippotyphosa was significantly higher in stray/sheltered dogs than in domiciled dogs (P = 0.028). These results suggest that pathogenic serogroups associated with small mammals are circulating among asymptomatic pets and should be taken into account in the transmission cycle of leptospires, as well as in the standard MAT panel for diagnosis in dogs and cats. It also highlights the importance of including both dogs and cats as potential reservoirs when conducting eco-epidemiological studies in different geographical and ecological areas.

Signal-sensing triggers the shutdown of HemKR, regulating heme and iron metabolism in the spirochete Leptospira biflexa.

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS present in both saprophytic and pathogenic Leptospira species, involved in the control of heme metabolism. However, the molecular means by which HemKR is switched on/off in a signal-dependent way, are still unknown. Moreover, a comprehensive list of HemKR-regulated genes, potentially overlapped with iron-responsive targets, is also missing. Using the saprophytic species Leptospira biflexa as a model, we now show that 5-aminolevulinic acid (ALA) triggers the shutdown of the HemKR pathway in live cells, and does so by stimulating the phosphatase activity of HemK towards phosphorylated HemR. Phospho~HemR dephosphorylation leads to differential expression of multiple genes, including of heme metabolism and transport systems. Besides the heme-biosynthetic genes hemA and the catabolic hmuO, which we had previously reported as phospho~HemR targets, we now extend the regulon identifying additional genes. Finally, we discover that HemR inactivation brings about an iron-deficit tolerant phenotype, synergistically with iron-responsive signaling systems. Future studies with pathogenic Leptospira will be able to confirm whether such tolerance to iron deprivation is conserved among Leptospira spp., in which case HemKR could play a vital role during infection where available iron is scarce. In sum, HemKR responds to abundance of porphyrin metabolites by shutting down and controlling heme homeostasis, while also contributing to integrate the regulation of heme and iron metabolism in the L. biflexa spirochete model.

First Report on Leptospira Species Isolated from Patients in Slovenia.

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.

Phylogenomics of Leptospira santarosai, a prevalent pathogenic species in the Americas.

Leptospirosis is a complex zoonotic disease mostly caused by a group of eight pathogenic species (L. interrogans, L. borgpetersenii, L. kirschneri, L. mayottensis, L. noguchii, L. santarosai, L. weilii, L. alexanderi), with a wide spectrum of animal reservoirs and patient outcomes. Leptospira interrogans is considered as the leading causative agent of leptospirosis worldwide and it is the most studied species. However, the genomic features and phylogeography of other Leptospira pathogenic species remain to be determined. Here we investigated the genome diversity of the main pathogenic Leptospira species based on a collection of 914 genomes from strains isolated around the world. Genome analyses revealed species-specific genome size and GC content, and an open pangenome in the pathogenic species, except for L. mayottensis. Taking advantage of a new set of genomes of L. santarosai strains isolated from patients in Costa Rica, we took a closer look at this species. L. santarosai strains are largely distributed in America, including the Caribbean islands, with over 96% of the available genomes originating from this continent. Phylogenetic analysis showed high genetic diversity within L. santarosai, and the clonal groups identified by cgMLST were strongly associated with geographical areas. Serotype identification based on serogrouping and/or analysis of the O-antigen biosynthesis gene loci further confirmed the great diversity of strains within the species. In conclusion, we report a comprehensive genome analysis of pathogenic Leptospira species with a focus on L. santarosai. Our study sheds new light onto the genomic diversity, evolutionary history, and epidemiology of leptospirosis in America and globally. Our findings also expand our knowledge of the genes driving O-antigen diversity. In addition, our work provides a framework for understanding the virulence and spread of L. santarosai and for improving its surveillance in both humans and animals.

Monitoring the spread of leptospirosis agent as one of the reasons of low-quality milk

On the global scale of the zoonoses problem, leptospirosis is among the five diseases that pose the greatest threat to humankind today. Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira species. In general, leptospirosis has been registered in more than 150 species of mammals. There are about 300 serovars of Leptospira spp. Serovar Hardjo is one of the most common causes of leptospirosis among cattle globally. In cows, the infection can be completely asymptomatic or cause abortions, stillbirths, infertility, and mastitis. The study's relevance is determined by the negative impact on the economy – productivity loss, and high cost of medical-preventive activities. Leptospirosis also affects humans. In this regard, the present study aimed to determine the prevalence of antibodies to Leptospira interrogans serovar Hardjo in tank milk samples from cows selected from farms in different regions of Ukraine. The method of indirect enzyme-multiplied immunoassay was used for this problem to be investigated. We have investigated 114 tank samples from 66 Ukrainian farms, 63.2% positive, and 36.8% negative. It was established that antibodies to the causative agent of leptospirosis were recorded in different regions of Ukraine. It has been established that the largest number of positive samples was from Kyiv and Cherkasy regions. Our study results complement the study results of other authors and indicate the circulation of this causative agent among the cows in Ukraine, as well as being of practical value for diagnosing and controlling leptospirosis among the cattle.