RESUMEN

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.

Asunto(s)

Brotes de Enfermedades , Leptospira , Leptospirosis , Filogenia , Roedores , Animales , Leptospirosis/epidemiología , Leptospirosis/veterinaria , Leptospirosis/microbiología , Leptospirosis/diagnóstico , Humanos , Venezuela/epidemiología , Bovinos , Leptospira/genética , Leptospira/aislamiento & purificación , Leptospira/clasificación , Femenino , Roedores/microbiología , Adulto , Masculino , Persona de Mediana Edad , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/epidemiología , Adolescente , Leptospira interrogans/genética , Leptospira interrogans/aislamiento & purificación , Leptospira interrogans/clasificación , Adulto Joven , Estudios Prospectivos , Niño , Anciano , Enfermedades Endémicas , Zoonosis/epidemiología , Zoonosis/microbiología , Preescolar

RESUMEN

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1-130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1. SIGNIFICANCE: The comparative analysis established an array of specific proteins in pathogenic strain that will narrow down the identification of immune protective proteins that will help fight leptospirosis.

Asunto(s)

Leptospira interrogans , Leptospira , Leptospirosis , Humanos , Proteoma/metabolismo , Factores de Virulencia/metabolismo

RESUMEN

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

RESUMEN

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1.

RESUMEN

Leptospirosis is a neglected disease of man and animals that affects nearly half a million people annually and causes considerable economic losses. Current human vaccines are inactivated whole-cell preparations (bacterins) of Leptospira spp. that provide strong homologous protection yet fail to induce a cross-protective immune response. Yearly boosters are required, and serious side-effects are frequently reported so the vaccine is licensed for use in humans in only a handful of countries. Novel universal vaccines require identification of conserved surface-exposed epitopes of leptospiral antigens. Outer membrane ß-barrel proteins (ßb-OMPs) meet these requirements and have been successfully used as vaccines for other diseases. We report the evaluation of 22 constructs containing protein fragments from 33 leptospiral ßb-OMPs, previously identified by reverse and structural vaccinology and cell-surface immunoprecipitation. Three-dimensional structures for each leptospiral ßb-OMP were predicted by I-TASSER. The surface-exposed epitopes were predicted using NetMHCII 2.2 and BepiPred 2.0. Recombinant constructs containing regions from one or more ßb-OMPs were cloned and expressed in Escherichia coli. IMAC-purified recombinant proteins were adsorbed to an aluminium hydroxide adjuvant to produce the vaccine formulations. Hamsters (4-6 weeks old) were vaccinated with 2 doses containing 50 - 125 µg of recombinant protein, with a 14-day interval between doses. Immunoprotection was evaluated in the hamster model of leptospirosis against a homologous challenge (10 - 20× ED50) with L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. Of the vaccine formulations, 20/22 were immunogenic and induced significant humoral immune responses (IgG) prior to challenge. Four constructs induced significant protection (100%, P < 0.001) and sterilizing immunity in two independent experiments, however, this was not reproducible in subsequent evaluations (0 - 33.3% protection, P > 0.05). The lack of reproducibility seen in these challenge experiments and in other reports in the literature, together with the lack of immune correlates and commercially available reagents to characterize the immune response, suggest that the hamster may not be the ideal model for evaluation of leptospirosis vaccines and highlight the need for evaluation of alternative models, such as the mouse.

Asunto(s)

Leptospira , Leptospirosis , Cricetinae , Humanos , Ratones , Animales , Hidróxido de Aluminio , Reproducibilidad de los Resultados , Leptospirosis/prevención & control , Vacunas Bacterianas , Antígenos Bacterianos/genética , Proteínas Recombinantes , Escherichia coli , Inmunoglobulina G , Epítopos

RESUMEN

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

RESUMEN

The Darwin's fox (Lycalopex fulvipes) is one of the most endangered carnivores worldwide, with the risk of disease spillover from domestic dogs being a major conservation threat. However, lack of epidemiologic information about generalist, non-dog-transmission-dependent protozoal and bacterial pathogens may be a barrier for disease prevention and management. To determine the exposure of some of these agents in Darwin's fox populations, 54 serum samples were collected from 47 Darwin's foxes in Southern Chile during 2013-18 and assessed for the presence of antibodies against Brucella abortus, Brucella canis, Coxiella burnetii, pathogenic Leptospira (serovars Grippotyphosa, Pomona, Canicola, Hardjo, and Copehageni), Toxoplasma gondii, and Neospora caninum. The highest seroprevalence was detected for T. gondii (78%), followed by pathogenic Leptospira (14%). All the studied Leptospira serovars were confirmed in at least one animal. Two foxes seroconverted to Leptospira and one to T. gondii during the study period. No seroconversions were observed for the other pathogens. No risk factors, either intrinsic (sex, age) or extrinsic (season, year, and degree of landscape anthropization), were associated with the probability of being exposed to T. gondii. Our results indicate that T. gondii exposure is widespread in the Darwin's fox population, including in areas with minimal anthropization, and that T. gondii and pathogenic Leptospira might be neglected threats to the species. Further studies identifying the causes of morbidity and mortality in Darwin's fox are needed to determine if these or other pathogens are having individual or population-wide effects in this species.

Asunto(s)

Enfermedades de los Perros , Leptospira , Leptospirosis , Neospora , Toxoplasma , Animales , Anticuerpos Antibacterianos , Enfermedades de los Perros/epidemiología , Perros , Leptospirosis/epidemiología , Leptospirosis/veterinaria , Estudios Seroepidemiológicos

RESUMEN

In this study, we genotyped samples from environmental reservoirs (surface water and soil), colonized rat specimens, and cases of human severe leptospirosis from an endemic urban slum in Brazil, to determine the molecular epidemiology of pathogenic Leptospira and identify pathways of leptospirosis infection. We identified a well-established population of Leptospira interrogans serovar Copenhageni common to human leptospirosis cases, and animal and environmental reservoirs. This finding provides genetic evidence for a potential environmental spillover pathway for rat-borne leptospirosis through the environment in this urban community and highlights the importance of environmental and social interventions to reduce spillover infections.

Asunto(s)

Ambiente , Leptospira/aislamiento & purificación , Leptospirosis/epidemiología , Microbiología del Suelo , Microbiología del Agua , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Animales , Brasil/epidemiología , Humanos , Leptospira/genética , Leptospira interrogans/genética , Leptospirosis/diagnóstico , Epidemiología Molecular , Filogenia , Ratas , Análisis de Secuencia de ADN

RESUMEN

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

RESUMEN

Background: Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). Methodology/Principal findings: L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent DE of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. Conclusions/Significance: Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.

RESUMEN

Leptospirosis and Lyme borreliosis are zoonotic emerging diseases of global importance and wide distribution. The aim of this study was to detect by molecular testing to Leptospira interrogans and Borrelia burgdorferi sensu lato in wild rodents from Nuevo Leon, Quintana Roo, and Campeche, Mexico. This study is the first in report to Chaetodipus nelsoni, Dipodomys merriami, and Peromyscus eremicus infected with L. interrogans in Mexico. Besides, Chaetodipus hispidus, Heteromys gaumeri, Heteromys irroratus, Neotoma micropus, Peromyscus leucopus, Peromyscus maniculatus, and Sigmodon hispidus infected with B. burgdorferi s.l. in Mexico. Also, is the first report in identify coinfection of L. interrogans and B. burgdorferi s.l. in wild rodents such as H. irroratus and S. hispidus in Nuevo Leon, and H. gaumeri in Quintana Roo, Mexico. These wild rodent species infected represent a risk factor for the exposed population in these sylvatic and rural areas of Mexico.

Asunto(s)

Borrelia burgdorferi/aislamiento & purificación , Leptospira interrogans/aislamiento & purificación , Leptospirosis/veterinaria , Enfermedad de Lyme/veterinaria , Enfermedades de los Roedores/microbiología , Animales , Animales Salvajes , Leptospirosis/epidemiología , Leptospirosis/microbiología , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/microbiología , México/epidemiología , Enfermedades de los Roedores/epidemiología , Roedores

RESUMEN

This study aimed to evaluate the occurrence of canine leptospirosis and the possible risk factors associated with the disease in the municipality of Patrocínio, MG, Brazil. A cross-sectional observational study was carried out from July through August 2017. The municipality was divided into four regions (north, south, east and west) and a predefined number of neighborhoods (25) were randomly sampled in each region. Samples of blood serum were collected from 241 domiciled male and female dogs of different breeds and ages. To investigate the risk factors for canine leptospirosis, the owners of the animals were asked to fill out an epidemiological questionnaire. The following factors were evaluated: breed, sex, age, presence of rodents, type of diet, access to the street, vaccination, presence of flooded areas, and educational level of the owners. Blood serum samples were evaluated by the Microscopic Agglutination Test (MAT), using a collection of 24 live antigens. Of the 241 dogs evaluated, 32 (13.2%) were reactive. The most frequent serovars were: Copenhageni (37.5%) and Canicola (21.8%), followed by Icterohaemorrhagiae and Grippotyphosa (12.5%), Pomona, Tarassovi and Butembo (9.3%) and Hardjo (6.2%). The presence of canine leptospirosis was associated with purebred dogs (OR=0.3059 [95% CI: 0.1430 ­ 0.6547]) and vaccination (OR=2.581 [95% CI: 1.198 ­ 5.563]). It was concluded that some dogs in the municipality of Patrocínio, MG have anti-Leptospiraspp. antibodies and that the serovars most frequently identified were Copenhageni (37.5%) and Canicola (21.8%). Pure breeds and vaccination were factors associated with the prevalence of infection.

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O objetivo deste estudo foi avaliar a ocorrência da leptospirose canina e os possíveis fatores de riscos associados à doença no município de Patrocínio ­ MG. Foi realizado um estudo observacional transversal, durante os meses de Julho à Agosto de 2017. O município foi divido em quatro regiões (norte, sul, leste e oeste) e um número predefinido de bairros (25) foi amostrado aleatoriamente em cada região. Foram colhidas 241 amostras de soro sanguíneo de cães domiciliados de ambos os sexos e de diferentes raças e idades. Para investigação dos fatores de risco da leptospirose canina foi aplicado um questionário epidemiológico aos tutores dos animais, foram avaliados os fatores: raça, sexo, idade, presença de roedores, tipo de dieta, acesso à rua, vacinação, presença de áreas alagadas, terrenos baldios e grau de escolaridade dos tutores. As amostras de soro sanguíneo foram avaliadas pelo exame de Soroaglutinação Microscópica (SAM), com uma coleção de vinte e quatro antígenos vivos. Dos 241 cães avaliados, 32 (13,2%) apresentaram-se reagentes. Os sorovares de maior frequência foram: Copenhageni (37,5%) e Canicola (21,8%), seguido por Icterohaemorrhagiae e Grippotyphosa (12,5%), Pomona, Tarassovi e Butembo (9,3%) e Hardjo (6,2%). A presença de leptospirose canina foi associada em cães com raça definida (OR = 0,3059 [IC 95%: 0,1430 ­ 0,6547]) e vacinação (OR = 2,581 [IC 95%: 1.198 ­ 5.563]). Concluiu-se que existem cães que apresentam anticorpos anti-Leptospira spp., no município de Patrocínio-MG e que os sorovares Copenhageni (37,5%) e Canicola (21,8%) foram os de maior ocorrência. Apresentar raça definida e a vacinação foram fatores associados à prevalência da infecção.

Asunto(s)

Perros , Leptospira interrogans

RESUMEN

Leptospira is a genus of spirochete bacteria highly motile that includes pathogenic species responsible to cause leptospirosis disease. Chemotaxis and motility are required for Leptospira infectivity, pathogenesis, and invasion of bacteria into the host. In prokaryotes, the most common chemoreceptors are methyl-accepting chemotaxis proteins that have a role play to detect the chemical signals and move to a favorable environment for its survival. Here, we report the first crystal structure of CACHE domain of the methyl-accepting chemotaxis protein (McpA) of L. interrogans. The structural analysis showed that McpA adopts similar α/β architecture of several other bacteria chemoreceptors. We also found a typical dimerization interface that appears to be functionally crucial for signal transmission and chemotaxis. In addition to McpA structural analyses, we have identified homologous proteins and conservative functional regions using bioinformatics techniques. These results improve our understanding the relationship between chemoreceptor structures and functions of Leptospira species.

RESUMEN

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Asunto(s)

Animales , Femenino , Proteínas Bacterianas/administración & dosificación , Factores de Transcripción/administración & dosificación , Vacunas Bacterianas/administración & dosificación , Vacunas de ADN/administración & dosificación , Leptospirosis/prevención & control , Antígenos Bacterianos/administración & dosificación , Proteínas Bacterianas/inmunología , Factores de Transcripción/inmunología , Vacunas Bacterianas/inmunología , Cricetinae , Técnica del Anticuerpo Fluorescente Indirecta , Vacunas de ADN/inmunología , Modelos Animales de Enfermedad , Nanopartículas , Leptospira interrogans/inmunología , Leptospirosis/inmunología , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología

RESUMEN

BACKGROUND: Ferredoxins are small iron-sulfur proteins that participate as electron donors in various metabolic pathways. They are recognized substrates of ferredoxin-NADP+ reductases (FNR) in redox metabolisms in mitochondria, plastids, and bacteria. We previously found a plastidic-type FNR in Leptospira interrogans (LepFNR), a parasitic bacterium of animals and humans. Nevertheless, we did not identify plant-type ferredoxins or flavodoxins, the common partners of this kind of FNR. METHODS: Sequence alignment, phylogenetical analyses and structural modeling were performed for the identification of a 2[4Fe4S] ferredoxin (LepFd2) as a putative redox partner of LepFNR in L. interrogans. The gene encoding LepFd2 was cloned and the protein overexpressed and purified. The functional properties of LepFd2 and LepFNR-LepFd2 complex were analyzed by kinetic and mutagenesis studies. RESULTS: We succeeded in expressing and purifying LepFd2 with its FeS cluster properly bound. We found that LepFd2 exchanges electrons with LepFNR. Moreover, a unique structural subdomain of LepFNR (loop P75-Y91), was shown to be involved in the recognition and binding of LepFd2. This structural subdomain is not found in other FNR homologs. CONCLUSIONS: We report for the first time a redox pair in L. interrogans in which a plastidic FNR exchanges electron with a bacterial 2[4Fe4S] ferredoxin. We characterized this reaction and proposed a model for the productive LepFNR-LepFd2 complex. GENERAL SIGNIFICANCE: Our findings suggest that the interaction of LepFNR with the iron-sulfur protein would be different from the one previously described for the homolog enzymes. This knowledge would be useful for the design of specific LepFNR inhibitors.

Asunto(s)

Ferredoxina-NADP Reductasa/metabolismo , Ferredoxinas/metabolismo , Leptospira interrogans/enzimología , Secuencia de Aminoácidos , Ferredoxina-NADP Reductasa/química , Ferredoxinas/química , Modelos Moleculares , Oxidación-Reducción , Filogenia , Conformación Proteica , Alineación de Secuencia

RESUMEN

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. Neutrophils are key cells against bacterial pathogens but can also contribute to tissue damage. Because the information regarding the role of human neutrophils in leptospirosis is scant, we comparatively analysed the human neutrophil's response to saprophytic Leptospira biflexa serovar Patoc (Patoc) and the pathogenic Leptospira interrogans serovar Copenhageni (LIC). Both species triggered neutrophil responses involved in migration, including the upregulation of CD11b expression, adhesion to collagen, and the release of IL-8. In addition, both species increased levels of pro-inflammatory IL-1ß and IL-6 associated with the inflammasome and NFκB pathway activation and delayed neutrophil apoptosis. LIC was observed on the neutrophil surface and not phagocytized. In contrast, Patoc generated intracellular ROS associated with its uptake. Neutrophils express the TYRO3, AXL, and MER receptor protein tyrosine kinases (TAM), but only LIC selectively increased the level of AXL. TLR2 but not TLR4-blocking antibodies abrogated the IL-8 secretion triggered by both Leptospira species. In summary, we demonstrate that Leptospira species trigger a robust neutrophil activation and pro-inflammatory response. These findings may be useful to find new diagnostic markers and therapeutic strategies against leptospirosis.

Asunto(s)

Leptospira/inmunología , Leptospirosis/inmunología , Leptospirosis/patología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Antígeno CD11b/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leptospira interrogans/inmunología , Leptospirosis/microbiología

RESUMEN

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Asunto(s)

Filogenia , Microbiología del Agua , Leptospira interrogans/aislamiento & purificación , Leptospira interrogans/genética , Virulencia , Datos de Secuencia Molecular , Genoma Bacteriano

RESUMEN

Leptospirosis is a globally distributed zoonosis that can be transmitted through direct or indirect contact with the urine or tissues of infected animals. In Argentina, leptospirosis is endemic in the province of Santa Fe and epidemic outbreaks occur during floods. However, very little is known about the role that wild rodents play in the spread of the disease in Argentina. The objective of this study was to identify the host species of pathogenic Leptospira among rodents in a riverine settlement in the province of Santa Fe.We conducted a trapping session in October 2015. Kidneys of the captured animals were analyzed by real-time PCR for the LipL32 gene of pathogenic Leptospira. Animals that were positive were subjected to microscopic agglutination test (MAT) and molecular typing by amplification of the 16S rRNA gene and two multilocus sequence typing (MLST) schemes.A total of 37 rodents of the species Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus, and Scapteromys aquaticus were captured. Real-time PCR found one male Scapteromys aquaticus that was positive. The serum of this individual and of the rest of the S. aquaticus captured (n = 18) were analyzed by MAT and were non-reactive for the 10 serovars tested. Amplification of the 16S rRNA gene identified the infective species as Leptospira interrogans, while amplification could not be obtained for the two MLST schemes.The findings of this study contribute new information concerning the presence of pathogenic Leptospira in wild rodents, which is relevant in this region because the species is widely distributed in swampy and flood-prone environments of South America.

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A leptospirose é uma doença zoonótica de distribuição mundial transmitida pelo contato direto ou indireto com a urina ou os tecidos de animais infectados. Na Argentina, a leptospirose é endêmica na Província de Santa Fé com surtos epidêmicos ocorrendo com as enchentes. Sabe-se pouco sobre o papel dos roedores silvestres na propagação da doença no país. O objetivo deste estudo foi identificar as espécies hospedeiras de leptospiras patogênicas em roedores encontrados em um núcleo de povoamento ribeirinho na Província de Santa Fé.A amostragem dos roedores foi feita no mês de outubro de 2015. Os tecidos dos rins dos animais capturados foram analisados com a técnica de reação em cadeia da polimerase em tempo real (PCR-RT) quanto à presença do gene LipL32 de leptospiras patógenas. Para os animais com resultados positivos, foi realizado o teste de microaglutinação (MAT) e tipagem molecular baseada na amplificação do gene 16S rRNA e dois esquemas de tipagem por sequenciamento de locos múltiplos (MLST).Ao todo, foram capturados 37 roedores das espécies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus e Scapteromys aquaticus. O ensaio de PCR-RT foi positivo em um roedor macho da espécie Scapteromys aquaticus. Os soros deste animal e dos outros S. aquaticus capturados (n = 18) foram analisados com o MAT e os resultados foram não reagentes para os 10 sorovares testados. A amplificação do gene 16S rRNA permitiu identificar a espécie infetante como sendo Leptospira interrogans e não houve amplificação nos dois esquemas de MLST.O achado deste estudo fornece um novo dado quanto à presença de leptospiras patogênicas em roedores silvestres, importante para esta área por se tratar de uma espécie de ampla distribuição em terras pantanosas e inundáveis da América do Sul.

RESUMEN

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader.[...]

Asunto(s)

Adhesión Bacteriana , Biopelículas , Leptospira interrogans/aislamiento & purificación , Matriz Extracelular

RESUMEN

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader.[...](AU)

Asunto(s)

Leptospira interrogans/aislamiento & purificación , Biopelículas , Matriz Extracelular , Adhesión Bacteriana