Pathogenic Leptospira Research Articles

STUDY OF HUMAN LEPTOSPIROSIS IN A TERTIARY CARE HOSPITAL, VISAKHAPATNAM

Objective: Prevalence of leptospirosis in humans is found in an alarming number in recent times. This endemic zoonotic disease is widespread and potentially fatal. It is usually seen in areas with poor socioeconomic conditions, water stagnation, garbage accumulation, which becomes breeding sites for the rodents. Infection may turn epidemic during monsoon. Reservoir of infection is rat scientifically named as Rattus rattus and infection results from direct or indirect exposure to host animals that carry the pathogen in their renal tubules and shed pathogenic leptospires in their urine. The main objective of this study was to estimate the seropositivity of leptospirosis in patients presenting with undifferentiated acute febrile illness for 5 to 7 days during one year period from February 2023 to February 2024 in a tertiary care hospital, Visakhapatnam, India. Methods: A total of 279 blood samples received from suspected cases of leptospirosis were subjected to IgM ELISA. Results: Out of total 279 cases, 20 were positive for leptospirosis (OD ratio ≥1.00). All the positive cases presented with a fever more than 99.5°F, chills and headache with elevated serum procalcitonin levels. Peak incidence of cases was recorded during July and August 2023. Conclusion: Seroprevalence of human leptospirosis in this study was found to be 7.2%.

Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, Leptospira interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis.

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, Leptospira interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Insights into Leptospira spp. Infection in Pet Cats in Croatia: Clinical, Serological and Molecular Findings with Emphasis on the Potential Important Role of Serogroup Pomona.

Leptospirosis, a globally re-emerging zoonosis caused by pathogenic Leptospira spp., poses a significant threat to public health. Leptospirosis in cats is often neglected due to its high underdiagnosis. Therefore, the role of cats in disease transmission and bacterial maintenance in the environment remains unclear. For this study, 54 serum samples, 54 urine samples and 27 EDTA-anticoagulated blood samples from pet cats presenting to the Veterinary Teaching Hospital due to health problems were used. The serum samples were tested for antibodies against 12 pathogenic serovars of Leptospira spp. using the microscopic agglutination test (MAT). EDTA-anticoagulated blood and urine samples were tested for the lipL32 gene of pathogenic Leptospira spp. by conventional (PCR) and real-time (qPCR) polymerase chain reaction. Agglutinating antibodies were detected in 18.52% (10/54) of the sera with a titre range of 1:50 to 1:12800. The most common serogroup was Pomona, followed by Sejroe, Icterohaemorrhagiae, Australis and Javanica. Leptospira spp. DNA was found in 1.85% (1/54) of the urine samples, while all EDTA-anticoagulated blood samples were negative. A statistically significant difference in seropositivity regarding lifestyle was found between outdoor/indoor and indoor-only cats, while the presence of another cat in the household significantly increased the likelihood of seropositivity. Cats with immunocompromising conditions showed a significantly increased risk of seropositivity, especially those undergoing immunosuppressive treatment. In addition, respiratory signs and changes in lung structure associated with the presence of leptospiral antibodies, and these cats were more likely to be infected with the Pomona serogroup. Moreover, cats with anaemia, leucocytosis, and thrombocytopenia were significantly more likely to have antibodies against Leptospira spp., while seropositive cats had significantly lower urine-specific gravity compared to seronegative cats. The results underline the importance of raising awareness of feline leptospirosis in veterinary care and recognising pet cats as potential carriers of leptospires. Further research is needed to clarify the specific role of the Pomona serogroup as a potentially highly evolutionary drifting serogroup in terms of pathogenicity and to clarify the zoonotic potential of infected cats, which is crucial for the implementation of effective public health measures and veterinary interventions.

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.

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.