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.

A machine learning model of microscopic agglutination test for diagnosis of leptospirosis.

Leptospirosis is a zoonosis caused by the pathogenic bacterium Leptospira. The Microscopic Agglutination Test (MAT) is widely used as the gold standard for diagnosis of leptospirosis. In this method, diluted patient serum is mixed with serotype-determined Leptospires, and the presence or absence of aggregation is determined under a dark-field microscope to calculate the antibody titer. Problems of the current MAT method are 1) a requirement of examining many specimens per sample, and 2) a need of distinguishing contaminants from true aggregates to accurately identify positivity. Therefore, increasing efficiency and accuracy are the key to refine MAT. It is possible to achieve efficiency and standardize accuracy at the same time by automating the decision-making process. In this study, we built an automatic identification algorithm of MAT using a machine learning method to determine agglutination within microscopic images. The machine learned the features from 316 positive and 230 negative MAT images created with sera of Leptospira-infected (positive) and non-infected (negative) hamsters, respectively. In addition to the acquired original images, wavelet-transformed images were also considered as features. We utilized a support vector machine (SVM) as a proposed decision method. We validated the trained SVMs with 210 positive and 154 negative images. When the features were obtained from original or wavelet-transformed images, all negative images were misjudged as positive, and the classification performance was very low with sensitivity of 1 and specificity of 0. In contrast, when the histograms of wavelet coefficients were used as features, the performance was greatly improved with sensitivity of 0.99 and specificity of 0.99. We confirmed that the current algorithm judges the positive or negative of agglutinations in MAT images and gives the further possibility of automatizing MAT procedure.

Correlation between renal distribution of leptospires during the acute phase and chronic renal dysfunction in a hamster model of infection with Leptospira interrogans.

BACKGROUND: Leptospirosis has been described as a biphasic disease consisting of hematogenous dissemination to major organs in the acute phase and asymptomatic renal colonization in the chronic phase. Several observational studies have suggested an association between leptospirosis and chronic kidney disease (CKD). We investigated the dynamics of leptospires and histopathological changes in the kidney to understand the relationship between them, and also investigated the extent of renal dysfunction in the acute and chronic phases of leptospirosis using a hamster model. FINDINGS: Hamsters (n = 68) were subcutaneously infected with 1 × 104 cells of the Leptospira interrogans serovar Manilae strain UP-MMC-SM. A total of 53 infected hamsters developed fatal acute leptospirosis, and the remaining 15 hamsters recovered from the acute phase, 13 of which showed Leptospira colonization in the kidneys in the chronic phase. Five asymptomatic hamsters also had renal colonization in the chronic phase. Immunofluorescence staining showed that leptospires were locally distributed in the renal interstitium in the early acute phase and then spread continuously into the surrounding interstitium. The kidneys of the surviving hamsters in the chronic phase showed patchy lesions of atrophic tubules, a finding of chronic tubulointerstitial nephritis, which were substantially consistent with the distribution of leptospires in the renal interstitium. The degree of atrophic tubules in kidney sections correlated statistically with the serum creatinine level in the chronic phase (rs = 0.78, p = 0.01). CONCLUSION: Subcutaneous infection with pathogenic leptospires could cause acute death or chronic leptospirosis in hamsters after surviving the acute phase. We suggest that the renal distribution of leptospires during the acute phase probably affected the extent of tubular atrophy, leading to CKD.

Transbronchial Invasion and Proliferation of Leptospira interrogans in Lung without Inflammatory Cell Infiltration in a Hamster Model.

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 (LD50) of the Leptospira interrogans strain UP-MMC-SM (L495) for hamsters in transtracheal infection was 3.2 × 102 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.

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov. and Leptospira ryugenii sp. nov. isolated from soil and water in Japan.

In a previous study, 50 of 132 soil samples collected throughout Japan were found to be Leptospira-positive. In the present study, three strains identified in the collected specimens, three, E8, E18 and YH101, were found to be divergent from previously described Leptospira species according to 16S ribosomal RNA gene sequence analysis. These three strains have a helical shape similar to that of typical Leptospira and were not re-isolated from experimental mice inoculated with the cultured strains. Upon 16S ribosomal RNA gene sequence analysis, E8 was found to belong to the intermediate Leptospira species clade and E18 and YH101 to belong to the saprophytic Leptospira species clade. Based on analyses of genome-to-genome distances and average nucleotide identity in silico using whole genome sequences and DNA-DNA hybridization in vitro, these isolates were found to be distinct from previously described Leptospira species. Therefore, these three isolates represent novel species of the genus Leptospira for which the names Leptospira johnsonii sp. nov., (type strain E8 T , = JCM 32515 T = CIP111620 T ), Leptospira ellinghausenii sp. nov., (type strain E18 T , = JCM 32516 T = CIP111618 T ) and Leptospira ryugenii sp. nov., (type strain YH101 T , = JCM 32518 T = CIP111617 T ) are proposed.

[The Current Status of Diagnostic Tools for Leptospirosis].

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. The severity of leptospirosis vary from mild, flu-like disease to a more severe form, Weil's disease causing jaundice, hemorrhage, renal failure, and even death. Every year, 300,000‒500,000 cases of severe leptospirosis are reported around the world, with the case fatality rate being 10‒30%. The usual diagnostic tools for leptospirosis are 1) direct observation of leptospires in blood and urine under dark-field microscope, 2) isolation of leptospires from blood, cerebrospinal fluid (CSF), or urine samples by culture, 3) microscopic agglutination test (MAT) to detect anti-Leptospira antibodies in serum, and 4) PCR to detect Leptospira DNA. At presents, the gold standards for diagnosis are culture isolation and MAT. However, it is actually not easy to isolate leptospires from clinical samples. On the other hand, it takes several days before the results of MAT become positive after the onset of illness. Moreover, MAT requires skilled handling, and also needs the maintenance of live Leptospira cells representing all serogroups. Hence other simple or rapid diagnostic tests are needed at the bedside. The micro capsule agglutination test (MCAT) to detect antibody and immunochromatographic assay to detect urinary antigen are currently in the research and development phases. In this paper, the characteristics of each diagnostic test for leptospirosis are described.