Pathogenic Leptospira Research Articles

Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens

Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99 to 1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for M. ovis, Pathogenic Leptospira, C. burnetii, BTV, PPRV, Brucella, exJSRV, C. abortus, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00%; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were Mycoplasma ovis (M. ovis), Pathogenic Leptospira, Coxiella burnetii (C. burnetii), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), Brucella, exogenous jaagsiekte sheep retrovirus (exJSRV), Chlamydia abortus (C. abortus), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).

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.

The role of asymptomatic cattle for leptospirosis dynamics in a herd with imperfect vaccination

Leptospirosis is an emerging zoonotic disease with high health and economic damage. In this study, we developed a deterministic mathematical model that describes the dynamics of leptospirosis transmission within a cattle herd, incorporating asymptomatic infected and vaccinated compartments. The study examined the transmission role of asymptomatic cattle that contaminate herds without farmers’ knowledge. We proved the well-posedness of the proposed model and found the basic reproduction number using the next-generation matrix. Analytically, we demonstrated that the disease-free equilibrium point is locally and globally asymptotically stable when R0\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\mathscr {R}}_0$$\\end{document} is less than unity and is otherwise unstable. Graphically, we further established the local asymptotic stability of disease-free and endemic equilibria. Sensitivity analysis showed that the contact rate with asymptomatic infected cattle, βA\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta _A$$\\end{document}, is the most sensitive parameter in the stated model, followed by the recovery rate of asymptomatic infected cattle, σ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sigma $$\\end{document}, and the vaccination rate of susceptible cattle, τ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ au $$\\end{document}. Numerical simulations revealed that a reduction in contact rate with asymptomatic infected cattle significantly reduced pathogen Leptospira transmission in the herd. In addition, fostering the recovery rate of asymptomatic infected cattle can significantly reduce new infections in the herd. Furthermore, augmenting the vaccination rate among susceptible cattle resulted in a notable decrease in disease prevalence within the herd. Findings of this study underscore the remarkable importance of targeted interventions, such as reducing contact rates with asymptomatic infected cattle, increasing recovery rates using proper treatments, and enhancing vaccination efforts to manage leptospirosis transmission in cattle herds.

Investigation of Leptospirosis Agents in Cattle and Humans.

Leptospirosis, a global zoonotic disease, causes serious morbidity and mortality generally in low-income societies. This study aimed to investigate the prevalence of Leptospira serovars in cattle and high-risk human populations. This study investigated the presence of pathogenic Leptospira serogroups in the blood and kidney samples of cattle arriving at the Erzurum Meat and Milk Institution for slaughter between April and July, and between September and December 2022, and in the serum samples of humans. Kidney and serum samples from 289 cattle and serum samples from 100 individuals from at-risk occupational groups (58 farmers, 25 veterinarians and 17 butchers) were collected. As a control, 100 human blood samples were collected from civil servants who had no contact with animals. Microagglutination testing was used to investigate Leptospira serogroups in bovine sera, real-time polymerase chain reaction (PCR) for Leptospira DNA in kidney samples, and microagglutination testing and enzyme-linked immunosorbent assays for Leptospira antibodies in human blood serum samples. Microagglutination test in 4.5% of cattle; Leptospira DNA was positive in 0.7%. Six strains of Leptospira interrogans, two of Bratislava, one of Pomana and one of Icterohaemorrhagiae were found in cattle, as well as one strain of Leptospira kirschneri Dadas. In humans, two Icterohaemorrhagiae, one Hebdomadis and one Dadas serovar were detected in both the risk group and the control group. Using ELISA, antibody positivity was found to be 14.0% in the risk group and 11.0% in the control group. The seroprevalence of Leptospira spp. in cattle in Erzurum, Türkiye, is relatively high. In this region, the risk of encountering Leptospira in the normal population is as high as in the risk group.

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.

Revisiting oxygen toxicity: evolution and adaptation to superoxide in a SOD-deficient bacterial pathogen.

Defenses against oxidants are crucial for the virulence of pathogens, with superoxide scavenging enzymes (SOSEs) playing a vital role for most aerobes. However, our knowledge of superoxide adaptation primarily stems from the study of SOSE-encoding bacteria. Here, we investigated the evolution of a naturally SOSE-deficient pathogen ( Leptospira spp.), along with the alternative mechanisms it recruits to combat superoxide stress. We demonstrate that emergence of pathogenic Leptospira correlated with SOD loss, but that a long-lasting adaptation to superoxide remains possible. We reveal that cysteine and leucine biosynthesis are the most induced pathways in response to superoxide and demonstrate the importance of sulfur metabolism in superoxide adaptation in this SOSE-deficient model. We also propose cysteine oxidation as a key mediator of superoxide toxicity in the absence of SOSEs. This study challenges our conventional understanding of the oxygen toxicity theory and proposes a new model of superoxide adaptation through metabolic rewiring in bacteria.

Development of ultrasensitive genosensor targeting pathogenic Leptospira DNA detection in artificial urine

In the last decades, to access the stages of leptospirosis pathogenicity, site of infection, progression, monitor disease, and after-treatment were required especially non-invasive techniques. This becomes one of the significant goals for researchers. In this study, we developed a highly sensitive DNA-based electrochemical sensor to detect pathogenic leptospires in urine samples using a gold screen-printed electrode. The gold working electrode was electrodeposited with diazonium salt (CMA) with external carboxylic acid from the surface, and the specific Loa22 single-stranded DNA (ssDNA) probe to Leptospira interrogans was immobilized through a carbodiimide chemical reaction. The modified working electrode was characterized by contact angle measurement, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). After that, targeted DNA testing showed the EIS change in the range of 5 ag mL-1 - 16 fg mL-1 with a correlation R2 of 0.9638. The high sensitivity electrochemical sensor indicated a lower limit of detection to the attomolar level specifically 5 ag mL-1. Additionally, the developed genosensor exhibited high specificity without crossing other bacteria present in urine such as Escherichia coli, Staphylococcus aureus, and S. typhi as well as non-pathogenic leptospires L. biflexa serovar Patoc. Leptospires DNA was analyzed in both buffer solution and diluted artificial urine and detection in diluted artificial urine 1:1,000 was possible despite salt interferences. This study provides a highly sensitive lower detection platform for leptospirosis, offering potential for further development as a portable and point-of-care testing tool.

Disentangling the influence of reservoir abundance and pathogen shedding on zoonotic spillover of the Leptospira agent in urban informal settlements.

Rats are major reservoirs for pathogenic Leptospira, the bacteria causing leptospirosis, particularly in urban informal settlements. However, the impact of variation in rat abundance and pathogen shedding rates on spillover transmission to humans remains unclear. This study aimed to investigate how spatial variation in reservoir abundance and pathogen pressure affect Leptospira spillover transmission to humans in a Brazilian urban informal settlement. A longitudinal eco-epidemiological study was conducted from 2013 to 2014 to characterize the spatial distribution of rat abundance and Leptospira shedding rates in rats and determine the association with human infection risk in a cohort of 2,206 community residents. Tracking plates and live-trapping were used to measure rat abundance and quantify rat shedding status and load. In parallel, four sequential biannual serosurveys were used to identify human Leptospira infections. To evaluate the role of shedding on human risk, we built three statistical models for: (1) the relative abundance of rats, (2) the shedding rate by individual rats, and (3) human Leptospira infection, in which "total shedding", obtained by multiplying the predictions from those two models, was used as a risk factor. We found that Leptospira shedding was associated with older and sexually mature rats and varied spatially and temporally-higher at valley bottoms and with seasonal rainfall (December to March). The point estimate for "total shedding" by rat populations was positive, i.e., Leptospira infection risk increased with total shedding, but the association was not significant [odds ratio (OR) = 1.1; 95% confidence interval (CI): 0.9, 1.4]. This positive trend was mainly driven by rat abundance, rather than individual rat shedding (OR = 1.8; 95% CI: 0.6, 5.4 vs. OR = 1.0; 95% CI: 0.7, 1.4]. Infection risk was higher in areas with more vegetative land cover (OR = 2.4; 95% CI: 1.2, 4.8), and when floodwater entered the house (OR = 2.4; 95% CI: 1.6, 3.4). Our findings indicate that environmental and hydrological factors play a more significant role in Leptospira spillover than rat associated factors. Furthermore, we developed a novel approach combining several models to elucidate complex links between animal reservoir abundance, pathogen shedding and environmental factors on zoonotic spillover in humans that can be extended to other environmentally transmitted diseases.

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.

Diagnostic Pathways of Leptospira spp. in Dogs with Kidney Injury.

Pathogenic Leptospira spp. causes leptospirosis in animals and humans globally, leading to systemic infections that can impact vital organs in affected animals. The purpose of this study was to evaluate kidney injury and to perform a retrospective analysis of leptospirosis infection in follow-up dog samples. The retrospective study collected epidemiological information obtained through paraclinical exams, immunohistochemistry (IHC), and molecular biology (qPCR) of cases from the Faculty of Veterinary Medicine from Timisoara between September 2016 and May 2023. No correlations were found between Leptospira infection and breed (p = 0.714), gender or castration status (p = 0.890), and anatomic pathology exam results (p = 0.608). Significant associations were found in cases with high levels of azotemia (p = 0.000) and immunological status (vaccinated vs. unvaccinated, p = 0.000), with the leptospirosis risk in unvaccinated animals calculated at OR = 85.500 (95%CI, 6.82-1071.26, p = 0.000). Retrospectively, leptospirosis was diagnosed in 27/65 cases (42%) using the IHC method, while the qPCR assay detected 29/65 cases (45%). This study demonstrates that qPCR is a robust and specific method for postmortem diagnosis of Leptospira spp. infection in dogs, offering higher specificity and reliability compared to traditional IHC methods, which showed 94.74% specificity in our study.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Socio-environmental factors associated with small mammal assemblage and Leptospira prevalence in Suburban Areas of Terengganu, Malaysia

Objective: To examine the socio-environmental factors associated with the assemblage of small mammals and the prevalence of Leptospira pathogen in poor suburban communities of Terengganu, Malaysia. Methods: We trapped small mammals from 119 trapping points scattered around three suburban communities of Terengganu using sausage-baited live traps. On the average, we set up five traps for three nights at each sampling point during the trapping period. Kidneys of captured animals were harvested and processed for Leptospira investigation. Additionally, environmental survey was conducted at each trapping point to obtain information about possible variables supporting small mammal assemblage. We used a generalized linear model to evaluate the effect of different socio-environmental variables on small mammals’ assemblage. Results: A total of 89 small mammals, specifically, Rattus norvegicus (n=39), Rattus rattus (n=27), Rattus exulans (n=10), Suncus murinus (n=11), and Tupaia glis (n=2) were captured from 1 385 trap nights. Fourteen individuals (15.7%) of the captured animals tested positive for Leptospira bacteria using PCR detection. Results of our generalized linear model showed only residences bordering vacant lots as the variable positively associated with small mammal occurrence in the three study sites. Conclusions: Small mammal community, especially the often neglected species, could harbour and potentially contribute towards pathogenic Leptospira maintenance in the study sites. To adequately control small mammals’ population and subsequent human zoonoses transmission, it is critical to advocate and promote appropriate infrastructure and suburban services, together with good hygiene practices that can reduce the animals’ access to food and harborage.

Leptospira interrogans encodes a canonical BamA and three novel noNterm Omp85 outer membrane protein paralogs

The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three ‘noNterm’ Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

Acute leptospirosis in horses: A retrospective study of 11 cases (2015-2023).

Reports of leptospirosis in horses are limited. To describe the clinical and diagnostic findings of acute systemic leptospirosis in horses. Eleven client-owned horses presented to an equine hospital because of acute onset of disease between 2015 and 2023. Retrospective case series. Horses diagnosed with leptospirosis by 1 or more of urine PCR, serologic microscopic agglutination test (MAT), and histopathology. Common clinical signs included lethargy (10), anorexia (10), fever (9), tachypnea (9), abnormal lung sounds (9), and epistaxis (6). Acute kidney injury was present in all cases. Evidence of pulmonary hemorrhage and liver disease was found in 8 (73%) and 6 (55%) horses, respectively. In 6 (55%) horses, kidneys, lungs, and liver were affected. Urine quantitative polymerase chain reaction for detection of pathogenic Leptospira spp. was positive in 6 (55%) cases. On serology Leptospira interrogans serovar Australis, Autumnalis, and Bratislava accounted for 86% of all titers ≥1 : 800. Overall case fatality rate was 4/11 (36%). Main findings on necropsy were tubular necrosis, interstitial nephritis, hemorrhage in the alveoli, pulmonary edema, periportal hepatitis and necrosis, cholestasis, and cholangitis. Leptospirosis should be considered as a differential diagnosis in horses with evidence of acute systemic inflammation and acute renal injury, epistaxis, or hepatic disease. For increased likelihood of identifying positive cases, both MAT serology and urine PCR should be performed.

Molecular epidemiology of Leptospira spp. serogroup Sejroe associated with chronic bovine leptospirosis

Pathogenic bacteria Leptospira spp. are commonly associated with bovine leptospirosis, characterized chiefly by chronic and subclinical reproductive disorders. Strains from the Sejroe serogroup play a significant role in these chronic genital infections known as Bovine Genital Leptospirosis (BGL), which notably impact cattle health. This study aims to deepen our understanding of BGL by investigating the genetic diversity, geographical distribution, and specific anatomical sites of infection of the causative agents. Initially, uterine fragments and cervicovaginal mucus were collected from 47 cows and subjected to PCR targeting the lipL32 gene. Positive samples in lipL32-PCR (9 samples) underwent genotyping based on the secY gene. Subsequently, sequences were aligned with GenBank entries (108 sequences) and analyzed in silico. All nine sequences from this study were identified as L. interrogans with an identity >99 % to serogroup Sejroe reference strains (Norma and L53). In the broader analysis, the most prevalent species observed was L. borgpetersenii, followed by L. interrogans and L. santarosai. The haplotype network of L. interrogans revealed that haplogroups B and C exclusively included L. interrogans strains of genital origin, while haplogroup A encompassed strains from renal sources as well. These findings underscore the significance of the L. borgpetersenii genotype Hardjobovis and L. interrogans genotype Hardjoprajitno as the predominant circulating strains and highlight the existence of distinct haplogroups of pathogenic leptospires originating from genital sources. We advocate for the use of secY as an effective genetic marker for Leptospira spp. and stress the necessity for additional research prioritizing the genital tract. The outcomes of this study contribute to the development of improved control measures for chronic cattle diseases and provide valuable guidance for future investigations.

Monitoring of pathogenic Leptospira infection in wolves (Canis lupus) from Spain and Italy

Leptospirosis is a bacterial disease of worldwide distribution with relevant implications for animal and human health. Different large wild carnivore species can act as reservoirs of this zoonotic pathogen. This study aimed to evaluate the circulation of Leptospira spp. in free-ranging wolves (Canis lupus) from southern Europe. A total of 281 kidney samples of wolves from Spain and Italy were collected between 2017 and 2023. The presence of Leptospira DNA was analysed by real-time PCR and phylogenetic analyses were carried out using a Bayesian approach. The overall prevalence was 3.2 % (9/281; 95 %CI: 1.1–5.3). Leptospira DNA was detected in nine of the 180 wolves from Spain (5.0 %; 95 %CI: 1.8–8.2), but not in the Italian wolf population (0 %; 0/101). Molecular analyses revealed high homology between the sequences obtained in the present study and isolates of Leptospira interrogans and Leptospira borgpetersenii from different rodent and domestic ungulate species. Our results provide evidence of a low and spatially heterogeneous circulation of this pathogen in wolf populations of southern Europe. The detection of zoonotic Leptospira species in this survey supports the need to consider wolf populations in monitoring programs for leptospirosis with a One Health approach.

Isolation and characterization of Leptospira licerasiae in Austrian swine — a first-time case report in Europe

BackgroundLeptospiraceae comprise a diverse family of spirochetal bacteria, of which many are involved in infectious diseases of animals and humans. Local leptospiral diversity in domestic animals is often poorly understood. Here we describe the incidental detection of Leptospira (L.) licerasiae in an Austrian pig.Case presentationDuring an experiment to characterize the pathogenesis of L. interrogans serovar Icterohaemorrhagiae in pigs, cultivation of a urine sample from a non-challenged contact pig resulted in growth of a spirochetal bacterium that tested negative for pathogenic Leptospira (LipL32 gene). PCR, Sanger sequencing and standard serotyping further confirmed that the recovered isolate was clearly different from the challenge strain L. interrogans serovar Icterohaemorrhagiae used in the animal experiment. Whole genome sequencing revealed that the isolate belongs to the species L. licerasiae, a tropical member of the Leptospiraceae, with no prior record of detection in Europe.ConclusionsThis is the first report describing the occurrence of L. licerasiae in Europe. Since L. licerasiae is considered to have intermediate pathogenicity, it will be important to follow the geographical distribution of this species and its pathogenic and zoonotic potential in more detail.

Pathogenic Leptospira Detection in Environmental Contaminant Water Sources by Highly Performance Antibody Absorption Polystyrene Agglutinating Particles

AbstractLeptospirosis is a re‐emerging bacterial zoonotic disease that affects both humans and animals, with a significantly higher incidence in tropical and sub‐tropical regions. Disease control, epidemiology, and surveillance rely on a One Health approach, as accurate detection can be applied to humans, animals, and the environment. This study represents the first attempt to develop a method for detecting the pathogenic Leptospira santarosai serovar Shermani based on the latex agglutination reaction. The serological activity of the antibody is examined to achieve a high titer of antibody before adsorption onto polystyrene particles. Using a pH medium of 6.8–7.8, total antibody adsorption of up to 3 mg m−2 is achieved. Particle agglutination is observed after incubating the antibody‐adsorbed PS with leptospiral culture for 4 min, revealing a detection limit of 1.7 × 102 leptospires mL−1. Interestingly, the detection limit increased by 1000 times when observing agglutination using spectrophotometer. The test exhibits high specificity with Shermani and shows negligible cross‐agglutination with non‐pathogenic Leptospira and water‐borne bacteria. Agglutination testing in collected water samples from natural sources demonstrates a good correlation with culture technique. This simple and rapid leptospires agglutination detection method can be applied as a screening test in environmental, human, and animal specimens.

Metagenomic Detection of Bacterial Zoonotic Pathogens among Febrile Patients, Tanzania, 2007-20091.

Bacterial zoonoses are established causes of severe febrile illness in East Africa. Within a fever etiology study, we applied a high-throughput 16S rRNA metagenomic assay validated for detecting bacterial zoonotic pathogens. We enrolled febrile patients admitted to 2 referral hospitals in Moshi, Tanzania, during September 2007-April 2009. Among 788 participants, median age was 20 (interquartile range 2-38) years. We performed PCR amplification of V1-V2 variable region 16S rRNA on cell pellet DNA, then metagenomic deep-sequencing and pathogenic taxonomic identification. We detected bacterial zoonotic pathogens in 10 (1.3%) samples: 3 with Rickettsia typhi, 1 R. conorii, 2 Bartonella quintana, 2 pathogenic Leptospira spp., and 1 Coxiella burnetii. One other sample had reads matching a Neoerhlichia spp. previously identified in a patient from South Africa. Our findings indicate that targeted 16S metagenomics can identify bacterial zoonotic pathogens causing severe febrile illness in humans, including potential novel agents.

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.