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Background: Reports of leptospirosis in horses are limited., Objectives: To describe the clinical and diagnostic findings of acute systemic leptospirosis in horses., Animals: Eleven client-owned horses presented to an equine hospital because of acute onset of disease between 2015 and 2023., Methods: Retrospective case series. Horses diagnosed with leptospirosis by 1 or more of urine PCR, serologic microscopic agglutination test (MAT), and histopathology., Results: 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., Conclusions and Clinical Importance: 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., (© 2024 The Author(s). Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Rodents are typically viewed as asymptomatic reservoirs for leptospirosis infection, as clinical disease in rodents is rarely described. This report includes three separate cases of leptospirosis in Patagonian maras ( Dolichotis patagonum ) over a 3-yr period in multiple locations within a single zoo. All three cases presented with varying clinical signs including lethargy, conjunctival hyperemia, hyperbilirubinemia, and presumed renal azotemia. Infection with Leptospira spp. was diagnosed antemortem by PCR on whole blood ( n = 1, Case 1) or urine ( n = 2, Cases 2 and 3). Leptospira antibody titers measured by serum microagglutination testing ( n = 3) were elevated or increased in all three animals over a 1-3-wk period for Leptospira serovars Bratislava and Hardjo (Case 1) and Grippotyphosa (Case 2 and 3). Two of the three animals responded to treatment with penicillin and doxycycline and supportive care, whereas one animal did not respond to treatment. Postmortem findings in this individual included conjunctivitis, chemosis, dehydration, icterus, tricavitary serosanguinous effusions, necrotizing hepatitis, diffuse pulmonary congestion, and edema. Immunohistochemical examination identified scattered Leptospira organisms within hepatocytes and renal tubular epithelial cells. A wild raccoon ( Procyon lotor ) at the institution tested positive by PCR on kidney tissue for the same Leptospira spp. serovar and was the suspected source of infection. This case series highlights the clinical importance of leptospirosis as a differential for Patagonian maras presenting with lethargy, ocular signs, acute hepatic disease, and azotemia.

In the past 20 years in Switzerland, dogs with suspect acute leptospirosis frequently showed severe glomerular changes that had not been previously reported. These features were characterized by abundant extravasated erythrocytes and fewer neutrophils accompanied by marked fibrin exudation into the urinary space that was interpreted as an exudative glomerulonephritis (GN). This retrospective study describes this significant glomerular pathological change and investigates the association with leptospirosis. Tissues from 50 dogs with exudative GN, retrieved from 2 pathology archives in Switzerland were reviewed using hematoxylin and eosin, periodic acid-Schiff, phosphotungstic acid-hematoxylin, and Warthin and Starry stains. Clinical and postmortem data were collected for each case. Immunohistochemistry (IHC) and/or polymerase chain reactions were used as confirmatory tests for leptospirosis. While all 50 cases had clinical and pathological features supporting a diagnosis of leptospirosis, 37 cases were confirmed for the disease. Using a LipL32 antibody in addition to the OMV2177 antibody raised against the lipopolysaccharide of Leptospira interrogans serovar Copenhageni increased the detection rate of Leptospira by IHC in exudative GN from 24% to 62%. Signalment, seasonality, clinical signs, blood results, and pathological changes in dogs with exudative GN were similar to those reported for dogs without GN and confirmed infection by Leptospira spp.. Exudative GN was common among Swiss dogs with leptospirosis where it caused acute severe disease. Leptospirosis should be considered as a cause of this new pathologic feature by the pathologist. The pathogenesis remains unclear, but involvement of a geographic-specific serovar with unique virulence factors is suspected and warrants further investigation., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Bacterial motility is often a crucial virulence factor for pathogenic species. A common approach to study bacterial motility is fluorescent labeling, which allows detection of individual bacterial cells in a population or in host tissues. However, the use of fluorescent labeling can be hampered by protein expression stability and/or interference with bacterial physiology. Here, we apply machine learning to microscopic image analysis for label-free motion tracking of the zoonotic bacterium Leptospira interrogans on cultured animal cells. We use various leptospiral strains isolated from a human patient or animals, as well as mutant strains. Strains associated with severe disease, and mutant strains lacking outer membrane proteins (OMPs), tend to display fast mobility and reduced adherence on cultured kidney cells. Our method does not require fluorescent labeling or genetic manipulation, and thus could be applied to study motility of many other bacterial species., (© 2023. The Author(s).)

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. having more than 300 serovars. These serovars can infect a variety of hosts, some being asymptomatic carriers and others showing varied symptoms of mild to severe infection. Since lipopolysaccharide (LPS) is the major antigen which defines serovar specificity, this different course of infection may be attributed to a differential innate response against this antigen. Previous studies have shown that Leptospira LPS is less endotoxic. However, it is unclear whether there is a difference in the ability of LPS isolated from different serovars to modulate the innate response. In this study, we purified LPS from three widely prevalent pathogenic serovars, i.e. Icterohaemorrhagiae strain RGA, Pomona, Hardjo, and from non-pathogenic L. biflexa serovar semeranga strain Potac 1 collectively termed as L-LPS and tested their ability to modulate innate response in macrophages from both resistant (mice) and susceptible (human and bovine) hosts. L-LPS induced differential response being more proinflammatory in mouse and less proinflammatory in human and bovine macrophages but overall less immunostimulatory than E. coli LPS (E-LPS). Irrespective of serovar, this response was TLR2-dependent in humans, whereas TLR4-dependent/CD14-independent in mouse using MyD88 adapter and signalling through P38 and ERK-dependent MAP kinase pathway. L-LPS-activated macrophages were able to phagocytose Leptospira and this effect was significantly higher or more pronounced when the macrophages were stimulated with L-LPS from the corresponding serovar. L-LPS activated both canonical and non-canonical inflammasome, producing IL-1β without inducing pyroptosis. Further, L-LPS induced both TNF-mediated early and NO-mediated late apoptosis. Altogether, these results indicate that L-LPS induces a differential innate response that is quite distinct from that induced by E-LPS and may be attributed to the structural differences and its atypical nature.

Leptospirosis is a recurring but neglected zoonotic disease caused by pathogenic Leptospira. The explicit underlying mechanism of necroptosis and its role in Leptospira infection have not yet been elucidated. Here we reported that leptospiral pathogen-associated molecular patterns, lipopolysaccharide, and glycolipoprotein activate the necroptotic RIPK1-RIPK3-MLKL cascade through the TLR4 signaling pathway in mouse macrophages. Using the murine acute leptospirosis model, we reveal that abolition of necroptosis exhibited significantly improved outcomes in acute phases, with enhanced eradication of Leptospira from liver, mild clinical symptoms, and decreased cytokine production. RIPK3 was also found to exert a necroptosis-independent function in CXCL1 production and neutrophil recruitment, with the consequence of improved Leptospira control. These findings improve our understanding of the mechanism of Leptospira-macrophage interactions, indicating potential therapeutic values by targeting necroptosis signaling pathways., Competing Interests: Conflicts of interest. All authors declared no competing interests. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leptospirosis, caused by pathogenic spirochaetes belonging to the genus Leptospira, is a globally distributed zoonosis that can affect many species of domestic and wild animals, and humans. The red fox (Vulpes vulpes) is a native species in Croatia and, due to constant food availability and lack of interspecies competition, is becoming more abundant in urban and suburban areas. Although antibodies for Leptospira spp have been detected in red foxes, lethal disease has not been reported. We necropsied a young, male red fox that had jaundice, multifocal haemorrhages in the heart, lungs and urinary bladder, hepatomegaly, non-congestive splenomegaly and slight yellow discolouration of the renal cortex and medulla. Histology revealed multifocal haemorrhages in parenchymal organs, thrombi within lung septal capillaries and other blood vessels, interstitial lymphocytic and plasmacytic nephritis and erosions of the gastric mucosa. A microscopic agglutination test on the post-mortem cardiac blood clot revealed a high titre to Leptospira interrogans serovar Icterohaemorrhagiae, which implies contact with rats, which are natural reservoirs of this serovar in Croatia. The gross and histological findings in this fox were similar to those in dogs with leptospirosis, indicating that fatal leptospirosis can occur in foxes and suggesting that this species can act as a source of infection for other animals and humans., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The tropics are a region consisting of more than 125 countries, accounting for 40% of the world's population. The region's population is expected to increase up to 60% in the coming decades. Many tropical countries continue to experience public health problems such as high rates of infectious diseases, lack of sanitation, climate change impacts, poor regulation of herbal medicines and low access to healthcare. These conditions produce the unique problem of tropical acute kidney injury (AKI), which is associated with high morbidity and mortality. Tropical infections such as leptospirosis, dengue and malaria have varied mechanisms of AKI, including both direct kidney invasion and indirect effects, depending on the disease characteristics. Animal toxins from snakebites and arthropods along with plant toxins, such as djenkol beans, starfruit and herbal medicine, are characterized by a harmful renal effect from each toxic substance. Environmental factors such as heat stress, natural disasters and chemical compounds also lead to AKI and have a systemic effect from their own pathogenesis. The long-term kidney prognosis varies among these etiologies depending on the cause and severity of disease. However, all these conditions are potentially preventable and treatable. Prompt management and good preventive approaches are needed. This article will focus on the epidemiology, pathogenesis and management of AKI associated with tropical infections, toxins and environment impacts., (© 2022 Asian Pacific Society of Nephrology.)

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Severe leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis., Methodology/principal Findings: Male Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6-7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression., Conclusion/significance: This study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis, caused by pathogenic Leptospira, is one of the most common zoonotic diseases in the world. It is transmitted to humans through the skin and mucous membranes by contact with water or soil contaminated with urine excreted from infected animals. In human infections, gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea have been frequently observed, but there have been no reports analyzing gastrointestinal lesions in leptospirosis, and the pathological mechanism of gastrointestinal symptoms in leptospirosis remains unclear. In this study, we investigated the pathological changes and the distribution of leptospires in the intestinal wall, and the presence of leptospires in the intestinal contents and feces, of hamsters subcutaneously infected with Leptospira interrogans. Results showed that infected hamsters had macroscopic redness in the jejunum and ileum. Submucosal hemorrhage was observed histologically, and there was no infiltration of inflammatory cells such as neutrophils. There were no obvious changes in the colon, either macroscopically or histologically, and the feces were normal (solid stools). Leptospira was isolated from all the intestinal walls from the small intestine to the colon, the intestinal contents, and the feces. These findings suggest that the invasion of leptospires into the intestinal wall and the associated submucosal hemorrhage may be the cause of the gastrointestinal symptoms observed in leptospirosis. Furthermore, not only the urine of infected animals but also the feces could be a source of infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Leptospirosis is a global zoonotic disease with outcomes ranging from subclinical infection to fatal Weil's syndrome. In addition to antibiotics, some immune activators have shown protective effects against leptospirosis. However, the unclear relationship between Leptospira and cytokines has limited the development of antileptospiral immunomodulators. In this study, the particular role of interleukin-10 (IL-10) in leptospirosis was explored by using IL-10-defective (IL-10 -/- ) hamsters. After Leptospira infection, an improved survival rate, reduced leptospiral burden, and alleviation of organ lesions were found in IL-10 -/- hamsters compared with wild-type (WT) hamsters. In addition, the levels of expression of the IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) genes and the level of nitric oxide (NO) were higher in IL-10 -/- hamsters than in WT hamsters. Our results indicate that IL-10 deficiency protects hamsters from Leptospira infection.

Leptospirosis is endemic in New Caledonia. Clinical diagnosis is often difficult and its evolution can be fatal. Leptospirosis requires specific management before biological confirmation. Modified Faine criteria (Faine Score) have been suggested to diagnose leptospirosis on epidemiological (parts A and B) and biological (part C) criteria. The main objective of our study was to assess the relevance of the epidemiological-clinical modified Faine score, parts A and B (MF A + B), in patients with suspected leptospirosis in New Caledonia. A monocentric case-control study was conducted in suspect patients for whom a Leptospira polymerase chain reaction (PCR) test was done within the first 7 days of signs onset at the tertiary hospital from January 2018 to January 2019. Cases and control subjects were matched 1:2 in the gender and age categories. Bivariate, and then multivariable, analyses studied the association between the MF A + B score and a positive Leptospira PCR test, adjusted on the variables retained. In all, 35 cases and 70 control subjects matched for age and gender were analyzed. Multivariable analysis by logistic regression found a significant association between an MF A + B score taken from the categories "possible leptospirosis" (score, 20-25) and "presumed leptospirosis" (score, > 26), and the case or control subject status (P < 0.0001). Model performance was high, with an area under the curve value of 99.27%, 93.55% sensitivity, and 96.36% specificity, which classified subjects correctly in 95.35% of cases. Our study suggests using the MF A + B score to identify possible cases of leptospirosis and initiate antibiotic therapy before biological confirmation in New Caledonia. This score should be evaluated in areas where more differential diagnoses exist and where PCR is not widely available.

Despite significant progress made in elucidating the mechanism of acute human leptospirosis in different organs, there is a paucity of information in organs such as the heart, pancreas, brain, and adrenal gland. This study was designed to establish leptospire dissemination kinetics and patho-morphological changes associated with these orangs in the guinea pig infection model using cultural isolation (CI), polymerase chain reaction (PCR), Warthin Starry silver stain (WSss), immunohistochemistry (IH), and transmission electron microscopy (TEM). Twenty guinea pigs were inoculated intra-peritoneally with a low dosage of 1 × 10 7 Leptospira interrogans serovar Icterohaemorrhagiae and 10 as control using distilled water. The guinea pigs were sacrificed at post-infection day (p.i.d.) ½, 1, 3, 5, and 7 followed by the harvest of the brain, pancreas, adrenal gland, heart, lungs, liver, kidneys, and spleen for CI, PCR, HE, WSss, IH, and TEM evaluations. The study revealed early dissemination of Leptospira organism in the brain, heart, pancreas, and adrenal gland and exerted various histopathological changes that were not explicitly elucidated in previous studies. This study revealed that the virulent pathogenic isolate of Leptospira organism obtained from clinically infected dog mimicked the same clinical manifestations, gross and histopathological changes especially in organs that were not previously evaluated.

High-incidence regions of leptospirosis caused by Leptospira spp. coincide with chronic kidney disease. This study investigated whether asymptomatic leptospirosis is an emerging culprit that predisposes to progressive chronic kidney disease when superimposed on secondary nephrotoxic injury. Kidney histology/function and whole transcriptomic profiles were evaluated for Leptospira -infected C57/BL6 mice with adenine-induced kidney injury. The extent of tubulointerstitial kidney lesions and expression of inflammation/fibrosis genes in infected mice with low-dose (0.1%) adenine, particularly in high-dose (0.2%) adenine-fed superimposed on Leptospira -infected mice, were significantly increased compared with mice following infection or adenine diet alone, and the findings are consistent with renal transcriptome analysis. Pathway enrichment findings showed that integrin-β- and fibronectin-encoding genes had distinct expression within the integrin-linked kinase-signaling pathway, which were upregulated in 0.2% adenine-fed Leptospira -infected mice but not in 0.2% adenine-fed mice, indicating that background subclinical Leptospiral infection indeed enhanced subsequent secondary nephrotoxic kidney injury and potential pathogenic molecules associated with secondary nephrotoxic leptospirosis. Comparative analysis of gene expression patterns with unilateral ureteric obstruction-induced mouse renal fibrosis and patients with chronic kidney disease showed that differentially expressed orthologous genes such as hemoglobin-α 2 , PDZ-binding kinase, and DNA topoisomerase II-α were identified in infected mice fed with low-dose and high-dose adenine, respectively, revealing differentially expressed signatures identical to those found in the datasets and may serve as markers of aggravated kidney progression. This study indicates that background subclinical leptospirosis, when subjected to various degrees of subsequent secondary nephrotoxic injury, may predispose to exacerbated fibrosis, mimicking the pathophysiological process of progressive chronic kidney disease. NEW & NOTEWORTHY Leptospira -infected mice followed by secondary nephrotoxic injury exacerbated immune/inflammatory responses and renal fibrosis. Comparison with the murine model revealed candidates involved in the progression of renal fibrosis in chronic kidney disease (CKD). Comparative transcriptome study suggests that secondary nephrotoxic injury in Leptospira -infected mice recapitulates the gene expression signatures found in CKD patients. This study indicates that secondary nephrotoxic injury may exacerbate CKD in chronic Leptospira infection implicating in the progression of CKD of unknown etiology.

In the summer of 2017, an estimated 745,000 Rohingya fled to Bangladesh in what has been described as one of the largest and fastest growing refugee crises in the world. Among numerous health concerns, an outbreak of acute jaundice syndrome (AJS) was detected by the disease surveillance system in early 2018 among the refugee population. This paper describes the investigation into the increase in AJS cases, the process and results of the investigation, which were strongly suggestive of a large outbreak due to hepatitis A virus (HAV). An enhanced serological investigation was conducted between 28 February to 26 March 2018 to determine the etiologies and risk factors associated with the outbreak. A total of 275 samples were collected from 18 health facilities reporting AJS cases. Blood samples were collected from all patients fulfilling the study specific case definition and inclusion criteria, and tested for antibody responses using enzyme-linked immunosorbent assay (ELISA). Out of the 275 samples, 206 were positive for one of the agents tested. The laboratory results confirmed multiple etiologies including 154 (56%) samples tested positive for hepatitis A, 1 (0.4%) positive for hepatitis E, 36 (13%) positive for hepatitis B, 25 (9%) positive for hepatitis C, and 14 (5%) positive for leptospirosis. Among all specimens tested 24 (9%) showed evidence of co-infections with multiple etiologies. Hepatitis A and E are commonly found in refugee camps and have similar clinical presentations. In the absence of robust testing capacity when the epidemic was identified through syndromic reporting, a particular concern was that of a hepatitis E outbreak, for which immunity tends to be limited, and which may be particularly severe among pregnant women. This report highlights the challenges of identifying causative agents in such settings and the resources required to do so. Results from the month-long enhanced investigation did not point out widespread hepatitis E virus (HEV) transmission, but instead strongly suggested a large-scale hepatitis A outbreak of milder consequences, and highlighted a number of other concomitant causes of AJS (acute hepatitis B, hepatitis C, Leptospirosis), albeit most likely at sporadic level. Results strengthen the need for further water and sanitation interventions and are a stark reminder of the risk of other epidemics transmitted through similar routes in such settings, particularly dysentery and cholera. It also highlights the need to ensure clinical management capacity for potentially chronic conditions in this vulnerable population., Competing Interests: The authors have declared that no competing interests exist.

The spirochete bacterium Leptospira interrogans serovar Pomona is enzootic to California sea lions (CSL; Zalophus californianus ) and causes periodic epizootics. Leptospirosis in CSL is associated with a high fatality rate in rehabilitation. Evidence-based tools for estimating prognosis and guiding early euthanasia of animals with a low probability of survival are critical to reducing the severity and duration of animal suffering. Classification and regression tree (CART) analysis of clinical data was used to predict survival outcomes of CSL with leptospirosis in rehabilitation. Classification tree outputs are binary decision trees that can be readily interpreted and applied by a clinician. Models were trained using data from cases treated from 2017 to 2018 at The Marine Mammal Center in Sausalito, CA, and tested against data from cases treated from 2010 to 2012. Two separate classification tree analyses were performed, one including and one excluding data from euthanized animals. When data from natural deaths and euthanasias were included in model-building, the best classification tree predicted outcomes correctly for 84.7% of cases based on four variables: appetite over the first 3 days in care, and blood urea nitrogen (BUN), creatinine, and sodium at admission. When only natural deaths were included, the best model predicted outcomes correctly for 87.6% of cases based on BUN and creatinine at admission. This study illustrates that CART analysis can be successfully applied to wildlife in rehabilitation to establish evidence-based euthanasia criteria with the goal of minimizing animal suffering. In the context of a large epizootic that challenges the limits of a facility's capacity for care, the models can assist in maximizing allocation of resources to those animals with the highest predicted probability of survival. This technique may be a useful tool for other diseases seen in wildlife rehabilitation.

Leptospira interrogans is a pathogenic spirochete responsible for leptospirosis, a neglected, zoonotic reemerging disease. Humans are sensitive hosts and may develop severe disease. Some animal species, such as rats and mice can become asymptomatic renal carriers. More than 350 leptospiral serovars have been identified, classified on the basis of the antibody response directed against the lipopolysaccharide (LPS). Similarly to whole inactivated bacteria used as human vaccines, this response is believed to confer only short-term, serogroup-specific protection. The immune response of hosts against leptospires has not been thoroughly studied, which complicates the testing of vaccine candidates. In this work, we studied the immunoglobulin (Ig) profiles in mice infected with L. interrogans over time to determine whether this humoral response confers long-term protection after homologous challenge six months post-infection. Groups of mice were injected intraperitoneally with 2×107 leptospires of one of three pathogenic serovars (Manilae, Copenhageni or Icterohaemorrhagiae), attenuated mutants or heat-killed bacteria. Leptospira-specific immunoglobulin (IgA, IgM, IgG and 4 subclasses) produced in the first weeks up to 6 months post-infection were measured by ELISA. Strikingly, we found sustained high levels of IgM in mice infected with the pathogenic Manilae and Copenhageni strains, both colonizing the kidney. In contrast, the Icterohaemorrhagiae strain did not lead to kidney colonization, even at high dose, and triggered a classical IgM response that peaked at day 8 post-infection and disappeared. The virulent Manilae and Copenhageni serovars elicited high levels and similar profiles of IgG subclasses in contrast to Icterohaemorrhagiae strains that stimulated weaker antibody responses. Inactivated heat-killed Manilae strains elicited very low responses. However, all mice pre-injected with leptospires challenged with high doses of homologous bacteria did not develop acute leptospirosis, and all antibody responses were boosted after challenge. Furthermore, we showed that 2 months post-challenge, mice pre-infected with the attenuated M895 Manilae LPS mutant or heat-killed bacterin were completely protected against renal colonization. In conclusion, we observed a sustained IgM response potentially associated with chronic leptospiral renal infection. We also demonstrated in mice different profiles of protective and cross-reactive antibodies after L. interrogans infection, depending on the serovar and virulence of strains., Competing Interests: The authors have declared that no competing interests exist.

Background: Leptospirosis is one of the most widespread zoonosis in the world and Brazil has the highest number of cases in Latin America. Transmission occurs mainly through exposure to water and soil contaminated by the urine of infected animals. The goals of this study are to describe the geographic distribution, demographic characteristics and exposure factors of urban and rural cases of leptospirosis, and identify spatial clusters in urban and rural areas of Brazil., Methods/results: A retrospective epidemiological study was carried out using 16 years (2000-2015) of surveillance data from the Brazilian Ministry of Health. Cases were described by age, sex and race, and exposure factors were characterized in urban and rural areas. A spatial autocorrelation analysis was conducted using local Moran's I to identify urban and rural clusters of disease. On average 3,810 leptospirosis cases were reported annually with higher numbers in urban areas. National urban and rural incidence rates were the same (1.9 cases/100,000 population), however, regional differences were observed. Urban incidence rates were higher in the North and Northeast regions, while rural incidence rates were higher in the Southeast and South. The main exposure factor reported in urban and rural areas was exposure to places with signs of rodents, followed by flood in urban areas and agriculture and animal farming in rural areas. Clusters of leptospirosis were identified in densely populated urban areas of the North, Southeast and South regions, while rural clusters were concentrated in of the Southern region with large agriculture and animal farming practices., Conclusions: This study highlights that leptospirosis is an important public health problem in both urban and rural areas of Brazil. The results provide decision-makers with detailed information about where disease incidence is high and can be used in the development of prevention and control strategies for priority areas and risk groups., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is an important zoonotic disease with worldwide distribution and nonspecific clinical manifestation. We report a case of fatal leptospirosis in a previously healthy woman with a causative agent. A young adult Indian woman was brought in dead to the forensic department. Ten days before, she developed fever, dizziness with headache, myalgia, diarrhea, and vomiting. Routine inquest and autopsy were performed on the deceased, revealing hemorrhagic lungs with extensive intra-alveolar hemorrhages, pale liver with dissociation and separation of hepatocyte plates, and edematous brain with histiocyte and lymphocyte infiltration in the parenchyma and meninges. Heart tissue depicts myocarditis and pericarditis inflammatory changes. Cerebrospinal fluid (CSF) was turbid in appearance with mildly elevated leukocytes, predominantly lymphocytes. Real-time PCR targeting lipL32 gene of pathogenic Leptospira was detected in the blood, CSF, brain, kidney, heart, and liver. The genetic profile of the causative agent was ST149 (multi-locus sequence typing Scheme 3). This study illustrates the usefulness of Leptospira PCR assay in postmortem diagnosis and addresses the need for further surveillance to identify the epidemiological link of the disease.

In bacteria, DNA methylation can be facilitated by 'orphan' DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

With over one million cases worldwide annually and a high fatality in symptomatic forms, human leptospirosis is a growing public health concern for the most vulnerable populations, especially in the context of global warming and unplanned urbanization. Although the Asia-Pacific region is particularly affected, accurate epidemiological data are often lacking. We conducted an eleven-year retrospective laboratory-based epidemiological survey of human leptospirosis in New Caledonia. From 2006 to 2016, 904 cases were laboratory-confirmed, including 29 fatalities, corresponding to an average annual incidence of 30.6/100,000 and a case fatality rate of 3.2%. Over the period, there was a major shift from indirect serological diagnosis by MAT to direct diagnosis by real-time PCR, a more specific and sensitive test when performed early in the course of the disease. The systematic implementation of genotyping informed on the variety of the infective strains involved, with a predominance of serogroups Icterohaemorrhagiae and Pyrogenes. The epidemiological pattern showed a marked seasonality with an annual peak in March-April. Interestingly, the seasonal peak in children of school age was significantly earlier and corresponded to school holidays, suggesting that attending school from February on could protect children from environment-borne leptospirosis., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is an important global zoonosis caused by pathogenic Leptospira. It is estimated that more than 1 million people are infected by Leptospira each year, and the death toll is about 60,000. Some studies showed that delayed immune response was associated with severe leptospirosis, and TLR4 was very important in the control of leptospirosis. In this study, we aimed to explore the effect of the classical activator (LPS) of TLR4 on leptospirosis in susceptible and resistant hosts. The results showed that LPS pretreatment increased the survival rate of hamsters to 80%. And LPS pre-treatment also significantly reduced the leptospiral load and alleviated the pathological injury in organs of hamsters and mice. The result detected by ELISA in mice showed that the levels of TNF-α and IL-1β were increased in the LPS-treated group compared to the control group before infection. However, two days after infection, the level of cytokines in LPS group was down-regulated compared with that in control group. In addition, in vitro results showed that LPS pre-treatment enhanced the phagocytosis and bactericidal ability of macrophages on Leptospira. Collectively, our results indicated that the pre-activated immune response induced by LPS enhanced the ability of host against leptospirosis., Competing Interests: The authors have declared that no competing interests exist.

Biomarkers to predict the severity of leptospirosis are still lacking. This study aimed to identify and validate microRNAs in patients with severe leptospirosis, that could potentially be used as biomarkers for predicting an unfavorable outcome. Serum samples were collected from participants with definite diagnosis of leptospirosis. The participants were divided into two groups, non-severe and severe leptospirosis, as defined by the Specific Organ Sequential Organ Failure (SOFA) Score of more than two in any organ. Microtranscriptome analysis was performed using the NanoString miRNA Expression Assay. The expression level of candidate miRNAs was then validated by quantitative RT-PCR. Based on the NanoString, the microtranscriptome profile of the severe group was significantly different from that of the non-severe group. Upregulation of miR155-5p, miR362-3p, miR502-5p, miR601, miR1323, and miR630 in the severe group were identified, and further investigated. A total of 119 participants were enrolled in the validation cohort. Serum miR155-5p and miR630 levels were significantly higher in the severe group compared to the non-severe group. The combined use of miR155-5p or miR-630 with serum bicarbonate levels had an AUC of 0.79 (95%CI; 0.69-0.89, p<0.001) in identifying the severity of the disease. This data provides the first evidence that the microtranscriptome profiles of patients with severe leptospirosis were different from the non-severe group. Serum miR155-5p and miR630 levels might be novel biomarkers for identifying severe leptospirosis., Competing Interests: The authors have declared that no competing interests exist.

Shedding of DNA of pathogenic Leptospira spp. has been documented in naturally infected cats in several countries, but urinary shedding of infectious Leptospira spp. has only recently been proven. The climate in Southern Chile is temperate rainy with high annual precipitations which represents ideal preconditions for survival of Leptospira spp., especially during spring and summer. The aims of this study were to investigate shedding of pathogenic Leptospira spp. in outdoor cats in Southern Chile, to perform molecular characterization of isolates growing in culture, and to assess potential risk factors associated with shedding. Urine samples of 231 outdoor cats from rural and urban areas in southern Chile were collected. Urine samples were investigated for pathogenic Leptospira spp. by 4 techniques: qPCR targeting the lipL32 gene, immunomagnetic separation (IMS)-coupled qPCR (IMS-qPCR), direct culture and IMS-coupled culture. Positive urine cultures were additionally confirmed by PCR. Multilocus sequence typing (MLST) was used to molecularly characterize isolates obtained from positive cultures. Overall, 36 urine samples (15.6%, 95% confidence interval (CI) 11.4-20.9) showed positive results. Eighteen (7.8%, 95% CI 4.9-12.1), 30 (13%, 95% CI 9.2-18), 3 (1.3%, 0.3-3.9) and 4 cats (1.7%; 95% CI 0.5-4.5) were positive in qPCR, IMS-qPCR, conventional culture, and IMS-coupled culture, respectively. MLST results of 7 culture-positive cats revealed sequences that could be assigned to sequence type 17 (6 cats) and sequence type 27 (1 cat) corresponding to L. interrogans (Pathogenic Leptospira Subgroup 1). Shedding of pathogenic Leptospira spp. by cats might be an underestimated source of infection for other species including humans. The present study is the first one reporting growth of leptospires from feline urine in culture in naturally infected cats in South-America and characterisation of culture-derived isolates. So far, very few cases of successful attempts to culture leptospires from naturally infected cats are described worldwide., Competing Interests: H.L.B.M. Klaasen is an employee of a company (MSD Animal Health) developing and marketing animal vaccines and pharmaceuticals. Otherwise the authors declare that they have no conflict of interest with respect to the research, authorship and/or publication of this article. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood., Methods: Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by quantitative reverse-transcription polymerase chain reaction, Western blot assay, and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters., Results: Leptospira interrogans but not saprophytic Leptospira biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed extracellular matrix proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 μmol/L Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys, and urine, and 10/13-fold decreased 50% lethal dose and milder histopathologic injury in hamsters., Conclusions: Lep-MP1 and 3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

In the past decade, two leptospirosis outbreaks occurred among strawberry harvesters in Germany, with 13, and 45 reported cases respectively. In both outbreaks, common voles (Microtus arvalis) infected with Leptospira kischneri serovar Grippotyphosa were identified as the most likely outbreak source. In an univariate analysis, eating unwashed strawberries was identified as one of the risk factors associated with Leptospira infection. The aim of this study was to evaluate the survival time of L. kirschneri serovar Grippotyphosa on strawberries under varying conditions. Strawberries were spiked with 5x109 of both a laboratory reference strain (strain Moskva V) and an outbreak field strain (94-6/2007) of L. kirschneri serovar Grippotyphosa sequence type 110. Survival times were investigated in a fully crossed design with three incubation times (2h, 4h, 6h and 8h) and three temperatures (15°C, 21°C and 25°C) with three replicated for each condition. A wash protocol was developed and recovered Leptospira were determined by qPCR, dark field microscopy and culturing. Viable L. kirschneri of both the reference strain and the field strain were identified in all samples at 25°C and an incubation time of 2h, but only 1/9 (11%) and 4/9 (44%) of the samples incubated at 15°C were positive, respectively. Both reference and field strain were viable only in 2/9 (22%) at 25° after 6h. After an 8h incubation, viable Leptospira could not be identified on the surface of the strawberries or within the fruit for any of the tested conditions. Based on these results, the exposure risk of consumers to viable Leptospira spp. through the consumption of strawberries bought at the retail level is most likely very low. However, there is a potential risk of Leptospira infection by consumption of strawberries on pick-your-own farms., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is a globally distributed disease associated with reproductive failures in livestock; however, its pathogenesis has not been fully elucidated. Results from the present study indicate there is a presence of Leptospira sp. in organs and fluids of fetuses from ewes slaughtered in the semiarid region of Brazil. Twenty-nine fetuses from 23 ewes determined to be Leptospira sp.-positive using PCR were sampled (14 and 15 in dry and rainy seasons, respectively). Fetal samples of blood, central nervous system (CNS), lung, liver, spleen, stomach contents, peritoneal fluid, kidney, bladder, urine and reproductive system were collected. Diagnostic methods included the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Of the 29 fetuses, 24 (82.8 %) had at least one Leptospira sp.-positive organ or fluid, as determined using PCR, and of a total of 209 samples, 62 (29.7 %) contained leptospiral DNA. Of the 99 samples collected during the dry season, 42 (42.4 %) were positive, and of 110 samples collected during the rainy season, 20 (18.2 %) were positive (P = 0.0001). There was deoxyribonucleic acid (DNA) sequencing of three samples of kidney, CNS and liver, and in all of these, there was 99.3 % similarity with Leptospira interrogans. Leptospires were present in cultures of pooled samples from fetuses with deformities. Results indicate there is vertical (maternal-to-fetus) transmission which would represent an alternative transmission route for the spread of Leptospira sp. in ewes, suggesting molecular detection is essential in the investigation of leptospirosis in fetuses to identify animals that have been infected with this bacterium., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Leptospires are highly invasive spirochetes equipped with efficient strategies for dissemination in the host. The Leptospira genus currently comprises 64 species divided into two major clades: the saprophytes composed of nonpathogenic, free-living organisms, and the pathogens encompassing all the species that cause mild or severe infections in humans and animals. While saprophytes are highly susceptible to the lytic action of the complement system, pathogenic (virulent) strains have evolved virulence strategies that allow efficient colonization of a variety of hosts and target organs, including mechanisms to circumvent hosts' innate and acquired immune responses. Pathogenic Leptospira avoid complement-mediated killing by recruiting host complement regulatory proteins and by targeting complement proteins using own and host-expressed proteases. This review outlines the role of complement in eradicating saprophytic Leptospira and the stratagems adopted by pathogenic Leptospira to maneuver the host complement system for their benefit., (© 2020 Federation of European Biochemical Societies.)

Human leptospirosis is considered as one of the most widespread and potentially fatal zoonotic diseases that causes high mortality and morbidity in the endemic regions of tropical and subtropical countries. The infection can arise from direct or indirect exposure of human through contaminated environment that contains leptospires or animal reservoirs that carry leptospires. The clinical manifestations during human leptospirosis ranges from asymptomatic, mild infections to severe and life-threatening complications involving multi-organ failures with kidneys, lungs and liver severely affected. Despite much efforts have been put in to unravel the pathogenesis during human leptospirosis, it remains obscure to which extent the host factors or the pathogen itself contribute towards the pathogenesis. Host innate immunity, especially, polymorphonuclear neutrophils and complement system are involved in the first line of defense during human leptospirosis. However, pathogenic Leptospira has acquired diverse evasion strategies to evade from host immunity and establish infection in infected hosts. Hence, in this review, we focus on organs pathology during human leptospiral infection and host evasion strategies employed by Leptospira. A profound understanding on leptospiral immunity and how Leptospira subvert the immune system may provide new insights on the development of therapeutic regimens against this species in future.

Leptospirosis is caused by pathogenic Leptospira transmitted through contact with contaminated environments. Most mammalian species are infectable by Leptospira but only few act as efficient reservoir being capable of establishing long term kidney colonization and shedding Leptospira in urine. In Madagascar, a large diversity of pathogenic Leptospira display a tight specificity towards their endemic volant or terrestrial mammalian hosts. The basis of this specificity is unknown: it may indicate some genetically determined compatibility between host cells and bacteria or only reflect ecological constraints preventing contacts between specific hosts. In this study, Rattus norvegicus was experimentally infected with either Leptospira interrogans, Leptospira borgpetersenii or Leptospira mayottensis isolated from rats, bats or tenrecs, respectively. Leptospira borgpetersenii and L. mayottensis do not support renal colonization as featured by no shedding of live bacteria in urine and low level and sporadic detection of Leptospira DNA in kidneys. In contrast 2 out of the 7 R. norvegicus challenged with L. interrogans developed renal colonization and intense Leptospira shedding in urine throughout the 3 months of experimental infection. These data suggest that host-Leptospira specificity in this biodiversity hotspot is driven at least in part by genetic determinants likely resulting from long-term co-diversification processes.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. The commercially available vaccines are bacterins that offer limited protection, short-term effect, and serovar-specific immunity. The development of novel immunization strategies is crucial to control the infection and decrease the chances of new outbreaks. In this study, purified monoclonal antibodies (mAbs) anti-LipL32 (1D9 and mAb3) were evaluated by their capacity to bind and neutralize the pathogen improving host survival. For that, an in vitro growth inhibition assay, and in vivo passive immunization were performed in animal model. Syrian hamsters were passively immunized by three different strategies. Hamsters immunized with mAb3 6 h prior to the lethal challenge showed a significantly higher survival rate of 61.1%, and a significant reduction in tissue damage in the lungs. Cumulatively, our results showed that anti-LipL32 mAbs inhibited the growth of L. interrogans in vitro, and that passive immunization offered significant protection in animal model when administered prior to infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bats can harbor zoonotic pathogens, but their status as reservoir hosts for Leptospira bacteria is unclear. During 2015-2017, kidneys from 47 of 173 bats captured in Grenada, West Indies, tested PCR-positive for Leptospira. Sequence analysis of the Leptospira rpoB gene from 31 of the positive samples showed 87-91% similarity to known Leptospira species. Pairwise and phylogenetic analysis of sequences indicate that bats from Grenada harbor as many as eight undescribed Leptospira genotypes that are most similar to known pathogenic Leptospira, including known zoonotic serovars. Warthin-Starry staining revealed leptospiral organisms colonizing the renal tubules in 70% of the PCR-positive bats examined. Mild inflammatory lesions in liver and kidney observed in some bats were not significantly correlated with renal Leptospira PCR-positivity. Our findings suggest that Grenada bats are asymptomatically infected with novel and diverse Leptospira genotypes phylogenetically related to known pathogenic strains, supporting the hypothesis that bats may be reservoirs for zoonotic Leptospira., Competing Interests: The authors have declared that no competing interests exist.

Bovine leptospirosis is often associated with host-adapted leptospires infections, such as strains belonging to the Sejroe serogroup. Although bovine leptospirosis by adapted strains may result in abortions, fetal death, premature births and the birth of weak and/or low-weight calves, this infection is more closely associated with subtler syndromes, such as subfertility and early embryonic death. In this way, this silent disease can go unnoticed and undiagnosed, compromising reproductive efficiency with a consequent decrease in the productivity of herds over long periods. For many years, genital tract infection has been considered a secondary effect of renal infection with the assumption being that leptospires are found in the genital tract due to bacteremia caused by renal colonization. In contrast to this hypothesis, there is some evidence in the literature that suggests that genital leptospirosis should be considered a specific syndrome dissociated from renal/systemic disease. Therefore, this paper aims to gather and critically analyze information about genital leptospirosis in cattle, considering the disease a distinct syndrome, herein denominated bovine genital leptospirosis (BGL), that requires a unique approach to diagnosis and treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Severe leptospirosis can have a case-fatality rate of over 50%, even with intensive care unit (ICU) support. Multiple strategies-including protective ventilation and early renal replacement therapy (RRT)-have been recommended to improve outcomes. However, management guidelines vary widely around the world and there is no consensus on the optimal approach., Methodology/principal Findings: All cases of leptospirosis admitted to the ICU of Cairns Hospital in tropical Australia between 1998 and 2018 were retrospectively reviewed. The patients' demographics, presentation, management and clinical course were examined. The 55 patients' median (interquartile range (IQR)) age was 47 (32-62) years and their median (IQR) APACHE III score was 67 (48-105). All 55 received appropriate antibiotic therapy, 45 (82%) within the first 6 hours. Acute kidney injury was present in 48/55 (87%), 18/55 (33%) required RRT, although this was usually not administered until traditional criteria for initiation were met. Moderate to severe acute respiratory distress syndrome developed in 37/55 (67%), 32/55 (58%) had pulmonary haemorrhage, and mechanical ventilation was required in 27/55 (49%). Vasopressor support was necessary in 34/55 (62%). Corticosteroids were prescribed in 20/55 (36%). The median (IQR) fluid balance in the initial three days of ICU care was +1493 (175-3567) ml. Only 2/55 (4%) died, both were elderly men with multiple comorbidities., Conclusion: In patients with severe leptospirosis in tropical Australia, prompt ICU support that includes early antibiotics, protective ventilation strategies, conservative fluid resuscitation, traditional thresholds for RRT initiation and corticosteroid therapy is associated with a very low case-fatality rate. Prospective studies are required to establish the relative contributions of each of these interventions to optimal patient outcomes., Competing Interests: The authors have declared that no competing interests exist.

Clinical manifestations of leptospirosis range from mild, common cold-like illness, to a life-threatening condition. The host immune response has been hypothesized to play a major role in leptospirosis outcome. Increased levels of inflammatory mediators, such as cytokines, may promote tissue damage that lead to increased disease severity. The question is whether cytokines levels may predict the outcome of leptospirosis and guide patient management. This study aimed to assess the association between Th1-, Th2-, and Th17-related cytokines with the clinical outcome of patients with leptospirosis. Different cytokine levels were measured in fifty-two plasma samples of hospitalized patients diagnosed with leptospirosis in Malaysia (January 2016-December 2017). Patients were divided into two separate categories: survived (n = 40) and fatal outcome (n = 12). Nineteen plasma samples from healthy individuals were obtained as controls. Cytokine quantification was performed using Simple Plex™ assays from ProteinSimple (San Jose, CA, USA). Measurements were done in triplicate and statistical analysis was performed using GraphPad software and SPSS v20. IL-6 (p = 0.033), IL-17A (p = 0.022), and IL-22 (p = 0.046) were significantly elevated in fatal cases. IL-17A concentration (OR 1.115; 95% CI 1.010-1.231) appeared to be an independent predictor of fatality of leptospirosis. Significantly higher levels of TNF-α (p ≤ 0.0001), IL-6 (p ≤ 0.0001), IL-10 (p ≤ 0.0001), IL-12 (p ≤ 0.0001), IL17A (p ≤ 0.0001), and IL-18 (p ≤ 0.0001) were observed among leptospirosis patients in comparison with healthy controls. Our study shows that certain cytokine levels may serve as possible prognostic biomarkers in leptospirosis patients.

Leptospirosis caused by pathogenic Leptospira is one of the most common zoonoses in the world. It is believed that humans become infected with it mainly through their skin and mucous membranes by contact with water or soil that is contaminated with urine excreted from infected animals. Recently, outbreaks have frequently occurred in the tropics, especially after flooding, but how leptospires cause mass infection remains poorly understood. In this study, we injected leptospires into the tracheas of hamsters under direct view and prove for the first time that leptospires can infect through the respiratory tract. We determined that a 50% lethal dose (LD 50 ) of the Leptospira interrogans strain UP-MMC-SM (L495) for hamsters in transtracheal infection was 3.2 × 10 2 cells. The results of culture, macroscopic findings, and histopathological analysis suggested that intratracheally injected leptospires invaded the lung tissue, proliferated in the collagen-rich stroma adjacent to the bronchus and blood vessels, and then spread throughout the body via the bloodstream. In the lung, leptospires continuously infiltrated the alveolar wall without inflammatory cell infiltration, spread throughout the lung, and finally caused pulmonary hemorrhage. Our results revealed that the respiratory tract might be a portal of entry for leptospires. We speculate that some cases of leptospirosis might be caused by transbronchial infection from inhaling infectious aerosols containing leptospires during floods. Leptospira was also confirmed to be a unique pathogen that invades through the bronchus, proliferates in the collagen-rich lung stroma, and spreads through the alveolar interstitium throughout the lung without causing pneumonia., (Copyright © 2019 American Society for Microbiology.)

Leptospirosis, caused by pathogenic Leptospira species, has emerged as an important neglected zoonotic disease. Few studies have reported the preventable effects of immunoregulators, except for antibiotics, against leptospirosis. Generally, immunostimulatory agents are considered effective for enhancing innate immune responses. Many studies have found that beta-glucan (β-glucan) could be a potent and valuable immunostimulant for improving immune responses and controlling diseases. In this study, we investigated the preventable role of β-glucan against Leptospira infection in hamsters. First, β-glucan was administered 24 h prior to, during and after infection. The results showed that β-glucan increased the survival rate to 100%, alleviated tissue injury, and decreased leptospire loads in target organs. Additionally, we found using quantitative real-time PCR that application of β-glucan significantly enhanced the expression of Toll-like receptor (TLR) 2, interleukin (IL)-1β and iNOS at 2 dpi (days post infection) and reduced the increase of TLR2, IL-1β and iNOS induced by Leptospira at 5 dpi. Furthermore, to induce memory immunity, β-glucan was administered 5 days prior to infection. β-Glucan also significantly increased the survival rates and ameliorated pathological damage to organs. Moreover, we demonstrated that β-glucan-trained macrophages exhibited elevated expression of proinflammatory cytokines (IL-1β and IL-6) in vitro, indicating that β-glucan induces an enhanced inflammatory response against Leptospira infection. These results indicate that administration of β-glucan and other immunostimulants could be potential valuable options for the control of Leptospira infection., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is considered a neglected disease with an estimated more than one million cases every year. Since rodents are at the same time the main reservoir and generally asymptomatic to Leptospira infection, understanding why some animal species are resistant and others are susceptible to this infection would shed some light in how to control this important zoonosis. The innate immune response against Leptospira is mainly dependent on phagocytosis and activation of the Complement System. In this context, cytokines may drive the early control of infection and the adaptive response. Since the Complement System is important to eliminate leptospires in vivo , we investigated if Complement C5 in A/J mice would modulate the cytokine production during infection by Leptospira interrogans serovar Kennewicki type Pomona Fromm (LPF). Thus, our aim was to investigate the systemic levels of pro- and anti-inflammatory cytokines during Leptospira infection in the blood, liver, lung, and kidney on the third and sixth days of infection in A/J C5 +/+ and A/J C5 -/- mice. Blood levels of TNF- α , IL-6, IFN- γ , and MCP-1 reached a peak on the third day. Although both mouse strains developed splenomegaly, similar histopathological alterations in the liver and the lung, levels of pro- and anti-inflammatory cytokines were different. A/J C5 +/+ mice had higher levels of liver IL-10, IL-1 β , IL-12p40, and IL-12p70 and kidney IL-1 β , IL-12p40, and IL-12p70 on the sixth day of infection when compared to A/J C5 -/- mice. Our results showed that in A/J genetic background, the Complement component C5 modulates a cytokine profile in the liver and kidney of infected mice, which may play a role in the control of disease progression., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Lorena Bavia et al.)

Leptospirosis is a life-threatening zoonotic disease and it has been hypothesized that the innate immune system fails to control the infection through ill-characterized mechanisms. The aim of this observational study was to better evaluate the activation processes of monocytes at the early stage of the disease. Blood samples were taken from healthy donors (n = 37) and patients hospitalized for either non-severe (n = 25) or severe (n = 32) leptospirosis. Monocyte cell counts and phenotypes were assessed by flow cytometry. We analysed the expression of several cell activation markers: CD14, CD16, HLA-DR, CD69, TLR2, TLR4, CD11b and CD11c. Although monocyte values at admittance were not significantly different from controls, patients experienced significant monocytosis at 1.33 × 10 9 /L (p < 0.0001 compared to controls: 0.56 × 10 9 /L) during their hospital stay. This monocytosis observed during hospital stay was correlated to several surrogate markers of organ injury. Non-classical (CD14-CD16+) and intermediate (CD14+CD16+) monocyte subsets increased compared to controls (p < 0.05). Accordingly, classical monocyte subset (CD14+CD16-) showed decreased percentages (p < 0.0001). Levels of several cell surface activation molecules were decreased: HLA-DR involved in MHC class II antigen presentation, integrins CD11b and CD11c implicated in phagocytosis and cell recruitment (p < 0.0001). None of these parameters had a prognostic value. Results from this study showed that during acute human leptospirosis, patients experienced monocytosis with a switch toward an inflammation-related phenotype contrasted by low expression levels of markers implicated in monocyte function.

The study aimed to evaluate the histopathological characteristics of renal lesions in chronically infected sheep and with low titers of anti-Leptospira antibodies from a slaughterhouse. In the serological analysis, 24.74% (48/194) presented seroreactivity with a titer equal to or greater than 100. Among these seroreactive sheep, titers of 100 were predominant (58.33%, 28/48), with the highest titer being 1,600 (2.08%, 1/48). Serogroup Sejroe (sv. Hardjo) was the most frequent at 35.42% (17/48). Leptospiral DNA was verified in 4.12% (8/194) of the kidney samples tested, and no urine sample was positive. All the samples corresponded to the pathogenic species L. interrogans. The eight amplicons with 202-nucleotides were identical with two mismatches (presented 100% of identity) using the PCR targeting to secY gene. Histological sections of PCR-positive kidneys were submitted to direct detection by the anti-LipL32 immunohistochemistry (IHC) technique. The Leptospira spp. antigen was evident in 62.5% (5/8) of the kidneys. Positive staining was observed in the cytoplasm of tubular cells and in the form of brownish aggregates that adhered to tubular epithelial cells and projected into the lumen. Inflammatory lymphoplasmacytic infiltrate, ranging from mild to moderate, with multifocal distribution, was the predominant finding in seroreactive animals (33.33%, 16/48). The demonstration of the leptospiral antigen lining the renal tubules through IHC of naturally infected sheep confirmed by PCR characterizes renal colonization in a species with the presence of histological changes compatible with leptospirosis., Competing Interests: The authors have declared that no competing interests exist.

This study evaluated the seropositivity of Brucella abortus and Leptospira interrogans in ewes with reproductive disturbances in southern Brazil and verified the creatine kinase (CK) activity and oxidation status via assessment of superoxide dismutase, glutathione peroxidase and glutathione transferase in serum of seropositive animals for L. interrogans serovar Icterohaemorrhagiae. For Leptospira infection 381 animals with clinical history of reproductive disturbance from Planalto Serrano de Santa Catarina (Brazil) were analyzed, showing an occurrence for L. interrogans of 20.2% from which 81.8% were seropositive for L. interrogans Icterohaemorrhagiae. Serovars Wolfii, Grippothyphosa, Bratislava, Canicola and Butembo were also identified. In the case of B. abortus, positive cases were identified by buffered acidified antigen, finding 14 positive samples, but none of them were positive after a second test (2-mercaptoethanol), showing the absence of relationship between infection with B. abortus and abortion in the tested individuals. Serum reactive oxygen species (ROS) levels and CK activity were found higher in animals positive for Leptospira infection, presenting higher titrations (1:320) than non-infected individuals. Serum glutathione peroxidase activity was higher in positive animals with titrations 1:160 and 1:320, while serum glutathione S-transferase was higher in positive individuals only for titrations 1:320. Serum superoxide dismutase showed lower activity in infected animals with titrations of 1:320. Our results show the region of Planalto Serrano de Santa Catarina with a high occurrence levels of sheep infected by L. interrogans serovar Icterohaemorrhagiae, from which animals with high titrations (1:320) present oxidative stress elicited by excessive ROS production, triggering the stimulation of antioxidant systems to counter this excess. In summary, ovine with higher titrations (1:320) present oxidative damage that can contribute to disease pathophysiology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background : The aim of this study was to investigate factors associated with poor outcomes among elderly hospitalized patients with leptospirosis. Methods : This is a retrospective cohort study with leptospirosis patients admitted to three tertiary hospitals in Fortaleza, Brazil, from January 1985 to July 2017. Patients were divided into two groups: elderly (age ≥60 years) and young (age <60 years). A comparison of demographical, clinical and laboratory data, treatment and outcomes was executed in order to investigate differences between groups. Results : A total of 507 hospitalized patients were included, with mean age 38 ± 15 years. Elderly group presented lower incidence of myalgia, vomiting, and dyspnea, as well as, higher medium systolic blood pressure. Elderly also manifested higher frequency of AKI (85.9 vs. 74.7%, p = 0.05), hemodialysis requirement (54.7 vs. 37.0%, p = 0.007) and death (32.8 vs. 12.2%, p < 0.001). In multivariate analysis, age ≥60 years was a predictor of hemodialysis requirement (p = 0.008, OR = 2.049, 95% CI = 1.207-3.477) and death (p < 0.001, OR = 3.520, 95% CI = 1.940-6.386). Conclusion : Leptospirosis in the elderly is associated with less hemodynamic impairment and higher frequency of AKI. Advanced age was also a predictor of poor outcomes, such as hemodialysis requirement and death, mostly due to kidney involvement.

The article describes the results of a retrospective analysis of medical records of 395 patients with a clinical diagnosis of leptospirosis treated at the Lviv Oblast Infectious Disease Clinical Hospital (Ukraine) between 2002 and 2016. The main risk factors for leptospirosis were contact with rodents or their excrements (26.84%) and bathing in ponds, small lakes, and reservoirs (10.63%). Among 276 patients in whom the anti-leptospira antibodies were detected by the microscopic agglutination test (MAT), the most common serotypes were Leptospira icterohaemorrhagiae (33.33%) and Leptospira grippotyphosa (25.0%). The mortality rate was significantly higher in patients where leptospirosis diagnosis was established based on clinical symptoms without confirmation by MAT (15.13% vs. 5.43%, p  < 0.01).

Objectives: To compare microscopic and immunologic features in the spleens of patients who died of pulmonary hemorrhage and shock caused by leptospirosis (11 cases) or Gram-positive/-negative bacterial septic shock (10 cases) to those from control spleens (12 cases from splenectomy). Methodology: Histological features in the red pulp and white pulp were analyzed using archived samples by a semi quantitative score. Immunohistochemistry was used for the recognition of immune cell markers, cytokines, caspase-3 and Leptospira antigens. Results: The control group differed significantly from the leptospirosis and septic shock patients which demonstrate strong similarities: diffuse congestion in the red pulp with a moderate to intense infiltration of plasma cells and polymorphonuclear cells; follicles with marked atrophy; high density of CD20 + cells; low density of NK, TCD4 + and active caspase-3 positive cells and strong expression of IL-10; leptospirosis patients had higher S100 and TNF-α positive cells in the spleen than the other groups. Conclusion: The results suggest that an immunosuppressive state develops at the terminal stage of severe leptospirosis with pulmonary hemorrhage and shock similar to that of patients with septic shock, with diffuse endothelial activation in the spleen, splenitis, and signs of disturbance in the innate and adaptive immunity in the spleen. The presence of leptospiral antigens in 73% of the spleens of the leptospirosis patients suggests the etiological agent contributes directly to the pathogenesis of the lesions. Our results support therapeutic approaches involving antibiotic and immunomodulatory treatments for leptospirosis patients and suggest that leptospirosis patients, which are usually young men with no co-morbidities, form a good group for studying sepsis and septic shock.

It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC-737 showed strong co-aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix-like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin., (© 2019 The Societies and John Wiley & Sons Australia, Ltd.)

Background: The case-fatality rate of severe leptospirosis can exceed 50%. While prompt supportive care can improve survival, predicting those at risk of developing severe disease is challenging, particularly in settings with limited diagnostic support., Methodology/principal Findings: We retrospectively identified all adults with laboratory-confirmed leptospirosis in Far North Queensland, Australia, between January 1998 and May 2016. Clinical, laboratory and radiological findings at presentation were correlated with the patients' subsequent clinical course. Medical records were available in 402 patients; 50 (12%) had severe disease. The presence of oliguria (urine output ≤500 mL/24 hours, odds ratio (OR): 16.4, 95% confidence interval (CI): 6.9-38.8, p<0.001), abnormal auscultatory findings on respiratory examination (OR 11.2 (95% CI: 4.7-26.5, p<0.001) and hypotension (systolic blood pressure ≤100 mmHg, OR 4.3 (95% CI 1.7-10.7, p = 0.002) at presentation independently predicted severe disease. A three-point score (the SPiRO score) was devised using these three clinical variables, with one point awarded for each. A score could be calculated in 392 (98%) patients; the likelihood of severe disease rose incrementally: 8/287 (3%), 14/70 (20%), 18/26 (69%) and 9/9 (100%) for a score of 0, 1, 2 and 3 respectively (p = 0.0001). A SPiRO score <1 had a negative predictive value for severe disease of 97% (95% CI: 95-99%)., Conclusions/significance: A simple, three-point clinical score can help clinicians rapidly identify patients at risk of developing severe leptospirosis, prompting early transfer to referral centres for advanced supportive care. This inexpensive, bedside assessment requires minimal training and may have significant utility in the resource-limited settings which bear the greatest burden of disease., Competing Interests: The authors have declared that no competing interests exist.

Clinical presentation of leptospirosis ranges from asymptomatic infection to fulminant, life-threatening disease. Pulmonary involvement in terms of severe pulmonary haemorrhage syndrome (SPHS) has recently become a more frequently reported facet of leptospirosis and correlates with high mortality rates. It has not yet been described in returning German travellers. We present a case of a healthy young man developing massive pulmonary haemorrhage and severe ARDS requiring mechanical ventilation and high-dose catecholamines after travelling to Indonesia. Leptospirosis was verified by blood PCR as well as serology and treated with high-dose, intravenous penicillin. Outcome was favourable, the patient recovered completely. Leptospirosis and SPHS should be taken into account as an emerging infectious disease in patients with fever and lung involvement.