Machine learning-based motion tracking reveals an inverse correlation between adhesivity and surface motility of the leptospirosis spirochete.

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.

Differential modulation of innate immune response by lipopolysaccharide of Leptospira.

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-1ß 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.

Lipopolysaccharide and Glycolipoprotein Coordinately Triggered Necroptosis Contributes to the Pathogenesis of Leptospira Infection in Mice.

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.

Acute lethal leptospirosis in a red fox (Vulpesvulpes).

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.

Acute kidney injury in the tropics.

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.

Identifying Leptospira interrogans putative virulence factors with a yeast protein expression screen.

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.

Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358.

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-1ß, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.

Dissemination of Leptospira into the intestinal tract resulting in fecal excretion in a hamster model of subcutaneous infection with Leptospirainterrogans.

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.

Clinical Evaluation of the Modified Faine Criteria in Patients Admitted with Suspected Leptospirosis to the Territorial Hospital, New Caledonia, 2018 to 2019.

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.

IL-10 Deficiency Protects Hamsters from Leptospira Infection.

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-1ß, 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.

Caracterização funcional de proteínas hipotéticas de Leptospira interrogans como adesinas e potenciais antígenos vacinais e para diagnóstico / Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.

Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

An outbreak of acute jaundice syndrome (AJS) among the Rohingya refugees in Cox's Bazar, Bangladesh: Findings from enhanced epidemiological surveillance.

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.

CLASSIFICATION AND REGRESSION TREE ANALYSIS FOR PREDICTING PROGNOSIS IN WILDLIFE REHABILITATION: A CASE STUDY OF LEPTOSPIROSIS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

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.

Transcriptomic signatures of exacerbated progression in leptospirosis subclinical chronic kidney disease with secondary nephrotoxic injury.

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-ß- 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-α2, 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 & NOTEWORTHYLeptospira-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.

Epidemiology of human leptospirosis in urban and rural areas of Brazil, 2000-2015.

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.

Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization.

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.

Dissemination kinetics and pathology of canine Leptospira icterohaemorrhagiae isolate in a guinea pig infection model.

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 × 107 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.

Case Report: Fatal Human Leptospirosis Caused by Leptospira interrogans Genotype ST149.

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.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.

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.