Characterization of Leptospira species isolated from soil collected in Japan.

Leptospira were isolated from soil obtained from Hokkaido, the northernmost island, to Okinawa, the southernmost island, of Japan using sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5- fluorouracil. Fifty of 132 soil samples (37.9%) were culture-positive. On the basis of 16S-rDNA sequences, 12 of the isolated Leptospira were classified into a pathogenic species clade that is closely associated with L. alstonii and L. kmetyi. Nine isolates were classified as intermediate species and were found to be similar to L. licerasiae. Twenty-seven isolates were classified as non-pathogenic species, of which 23 were found to be related to L. wolbachii. Non-pathogenic Leptospira are commonly distributed in environmental soil.

[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.

The usefulness of semi-solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka, Japan.

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi-solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira-positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi-solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi-solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.

High virulence in hamsters of four dominant Leptospira serovars isolated from rats in the Philippines.

Leptospirosis is caused by pathogenic species of Leptospira. The aim of this study was to determine and characterize the pathogenicity of four dominant Leptospira isolates prevailing among rats in the Philippines. The isolates were Leptospira interrogans serovar Manilae strain K64, L. interrogans serovar Losbanos strain K37, L. interrogans serovar Ratnapura strain K5 and Leptospira borgpetersenii serovar Javanica strain K6. Pathogenicities were studied using hamsters, which reproduce severe human leptospirosis. The minimum lethal doses were 10(0) ( = 1) leptospires for K64, K37 and K5, and 10(1) leptospires for K6. Weight loss amongst the Leptospira-infected hamsters was observed from 1 day before death (K64-, K37- and K5-infected hamsters) to as much as 1 week before death for K6-infected hamsters. Similar and varied gross and microscopic lesions were observed amongst infected hamsters, even for strains belonging to the same species (i.e. L. interrogans). The most significant and common histopathological findings were congestion of the glomerulus, disarrangement of hepatic cords and erythrophagocytosis. Other findings were foamy splenic macrophages for K6, severe petechial pulmonary haemorrhage for K64, and hematuria and severe pulmonary congestion for K37. Immunostaining and culture revealed the presence of leptospires in different organs of the infected hamsters. Based on these results, Leptospira isolates from rats in the Philippines were shown to be highly virulent, causing pulmonary haemorrhage, severe hepato-renal damage and death in hamsters even at lower doses. The present findings on experimental leptospirosis support clinical data showing that patients with severe manifestations of leptospirosis, such as pulmonary haemorrhage, are increasing in the Philippines. These findings may serve as a basis to strengthen the early diagnosis and treatment of human leptospirosis.

PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during Super Typhoon Haiyan (Yolanda).

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof's medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection.

Natural defense by saliva and mucosa against oral infection by Leptospira.

Leptospirosis caused by drinking water has not been as frequently reported as percutaneous infection. Resistance to oral infection by pathogenic Leptospira was examined in an experimental hamster infection model. The results suggested some natural defenses against oral infection by Leptospira. First, we found that characteristic linear agglutination of Leptospira rapidly occurs when mixed with human saliva. That human saliva attenuated the infectivity of the treated leptospires by its agglutination activity suggested saliva to be the first line of defense against oral infection by leptospires. Second, only 10(1) Leptospira organisms caused death after submucosal injection into oral mucosa in hamsters, but oral infection with drinking water containing 10(5) organisms/mL did not cause death. This result showed that the mucosa plays the role of a physical barrier. Third, hamsters intragastrically infected by leptospires, with doses lethal to hamsters in oral infection, showed no signs of illness, which suggested that gastric acid plays an important role in preventing oral infection. Based on these results, saliva, mucosa, and gastric acid make up a natural defense, which confers high resistance to hosts against oral infection by leptospires.

Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan.

There have been few reports on the epidemiological analysis of environmental Leptospira isolates. This is probably because the isolation of leptospires from the environment was usually unsuccessful due to the overgrowth of contaminants and the slow growth of Leptospira. In this study, we collected a total of 88 samples of soil and water from three sites: Metro Manila and Nueva Ecija, Philippines (an area where Leptospira is now endemic), and Fukuoka, Japan (an area where Leptospira was once endemic). We succeeded in isolating Leptospira from 37 samples by using the novel combination of five antimicrobial agents reported in 2011. The frequencies of positive isolation of Leptospira in the Philippines and Japan were 40 and 46%, respectively. For Leptospira-positive samples, five colonies from each sample were isolated and analyzed by pulsed-field gel electrophoresis (PFGE). The isolates from each area showed their respective characteristics in phylogenetic trees based on the PFGE patterns. Some isolates were closely related to each other across borders. Based on 16S rRNA gene-based phylogenetic analysis, four isolates in Fukuoka were identified as a pathogenic species, L. alstonii; however, its virulence had been lost. One isolate from Nueva Ecija was identified as the intermediate pathogenic species Leptospira licerasiae. Most of the isolates from the environment belonged to nonpathogenic Leptospira species. We also investigated the strain variation among the isolates in a puddle over 5 months. We demonstrated, using PFGE analysis, that Leptospira survived in the wet soil on dry days and appeared in the surface water on rainy days. These results showed that the soil could be a reservoir of leptospires in the environment.

Proposal for effective treatment of Shiga toxin-producing Escherichia coli infection in mice.

Previously, we reported that minocycline, kanamycin and norfloxacin improved the survival rate in the E32511 model that we developed (FEMS Immunol Med Microbiol 26, 101-108, 1999), but fosfomycin did not. In this study, we investigated the effectiveness of azithromycin (AZM) against Stx2d-producing EHEC O91:H21 strain B2F1 or Stx2c-producing Escherichia coli strain E32511 treated with mitomycin C in vivo. Recently, we reported the effectiveness of AZM in our model and AZM strongly inhibited the release of Stx2c from E32511 in vitro (PLOS ONE e58959, 2013). However, it was very difficult to completely eliminate E32511 in the mouse feces by treatment with AZM alone. In this report, only AZM or Daio effectively promoted survival of mice infected with B2F1 compared to untreated mice. Furthermore, Daio inhibited the colonization of GFP-expressing B2F1 in the mouse intestine. Similarly, a combination of AZM and Daio in the E32511-infected mice reduced E32511 in the mouse feces and significantly improved survival.

Leptospira idonii sp. nov., isolated from environmental water.

Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthof's medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).

Genetic Diversity and Dispersal of DENGUE Virus among Three Main Island Groups of the Philippines during 2015-2017.

Dengue has been one of the major public health concerns in the Philippines for more than a century. The annual dengue case burden has been increasing in recent years, exceeding 200,000 in 2015 and 2019. However, there is limited information on the molecular epidemiology of dengue in the Philippines. We, therefore, conducted a study to understand the genetic composition and dispersal of DENV in the Philippines from 2015 to 2017 under UNITEDengue. Our analyses included 377 envelope (E) gene sequences of all 4 serotypes obtained from infections in 3 main island groups (Luzon, Visayas, and Mindanao) of the Philippines. The findings showed that the overall diversity of DENV was generally low. DENV-1 was relatively more diverse than the other serotypes. Virus dispersal was evident among the three main island groups, but each island group demonstrated a distinct genotype composition. These observations suggested that the intensity of virus dispersal was not substantive enough to maintain a uniform heterogeneity among island groups so that each island group behaved as an independent epidemiological unit. The analyses suggested Luzon as one of the major sources of DENV emergence and CAR, Calabarzon, and CARAGA as important hubs of virus dispersal in the Philippines. Our findings highlight the importance of virus surveillance and molecular epidemiological analyses to gain deep insights into virus diversity, lineage dominance, and dispersal patterns that could assist in understanding the epidemiology and transmission risk of dengue in endemic regions.

Leptospira Is an Environmental Bacterium That Grows in Waterlogged Soil.

Leptospirosis is a zoonotic disease caused by infection with pathogenic leptospires. Consistent with recent studies by other groups, leptospires were isolated from 89 out of 110 (80.9%) soil or water samples from varied locations in the Philippines in our surveillance study, indicating that leptospires might have a life cycle that does not involve animal hosts. However, despite previous work, it has not been confirmed whether leptospires multiply in the soil environment under various experimental conditions. Given the fact that the case number of leptospirosis is increased after flood, we hypothesized that waterlogged soil, which mimics the postflooding environment, could be a suitable condition for growing leptospires. To verify this hypothesis, pathogenic and saprophytic leptospires were seeded in the bottles containing 2.5 times as much water as soil, and bacterial counts in the bottles were measured over time. Pathogenic and saprophytic leptospires were found to increase their number in waterlogged soil but not in water or soil alone. In addition, leptospires were reisolated from soil in closed tubes for as long as 379 days. These results indicate that leptospires are in a resting state in the soil and are able to proliferate with increased water content in the environment. This notion is strongly supported by observations that the case number of leptospirosis is significantly higher in rainy seasons and increased after flood. Therefore, we reached the following conclusion: environmental soil is a potential reservoir of leptospires. IMPORTANCE Since research on Leptospira has focused on pathogenic leptospires, which are supposed to multiply only in animal hosts, the life cycle of saprophytic leptospires has long been a mystery. This study demonstrates that both pathogenic and saprophytic leptospires multiply in the waterlogged soil, which mimics the postflooding environment. The present results potentially explain why leptospirosis frequently occurs after floods. Therefore, environmental soil is a potential reservoir of leptospires and leptospirosis is considered an environment-borne as well as a zoonotic disease. This is a significant report to reveal that leptospires multiply under environmental conditions, and this finding leads us to reconsider the ecology of leptospires.

A novel combination of selective agents for isolation of Leptospira species.

A novel combination of antimicrobial agents (sulfamethoxazole, 40 µg/mL; trimethoprim, 20 µg/mL; amphotericin B, 5 µg/mL; fosfomycin, 400 µg/mL; and 5-fluorouracil, 100 µg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10(1) cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.

Electron microscopic study of the effects of antimicrobial agents on the cellular architecture of Leptospira.

Morphological changes of Leptospira induced by ampicillin, streptomycin, or ciprofloxacin were studied by transmission electron microscopy after staining with uranyl acetate. Irregularities in coiling, loss of hooks, spherical swellings, membrane vesicle formation, disruption of outer membrane, exposure and detachment of flagella, penetration of dye, and lysis of cells were observed. These changes appeared in a time-dependent manner and appeared to be associated with the sites of action of antimicrobial agents. This is the first report delineating the action of aminoglycoside and new quinolone on the morphology of Leptospira cells.

In vitro sensitivity and resistance of 46 Leptospira strains isolated from rats in the Philippines to 14 antimicrobial agents.

The in vitro susceptibilities of 46 Leptospira isolates from rats to 14 antimicrobial agents were tested. All of the strains were found to be sensitive to ampicillin, cefotaxime, ciprofloxacin, norfloxacin, doxycycline, erythromycin, and streptomycin. In contrast, the tested isolates showed resistance to amphotericin B, 5-fluorouracil, fosfomycin, trimethoprim, sulfamethoxazole, neomycin, and vancomycin. These findings will help in selecting effective and ineffective antimicrobials for treatment of leptospirosis and for the development of new selective media, respectively.

Successful steroid pulse therapy for brain lesion caused by Shiga toxin 2 in rabbits.

Betamethasone sodium phosphate (BSP) is usually used as a steroid therapy for human brain edema. High doses of BSP (36mg/kg) twice a day for two days statistically reduced the mortality rate and improved the survival period of Stx2 (1.4mug/kg; 1.6LD(50))-toxemic rabbits. We made evaluations on three kinds of magnetic resonance images (MRI) including T1-weighted, T2-weighted, and enhanced MRI using gadopentetate dimeglumine (Gd-DTPA) to detect brain lesion induced by an intravenous injection of Stx2 in rabbits. Enhanced T1-weighted MRI was the most sensitive tool to find leakage of Gd-DTPA suggesting impairment of the blood brain barrier caused by Stx2. Enhanced MRI revealed that BSP treatment inhibited the leakage of Gd-DTPA, as directly evidenced by the protective effect of BSP against brain edema induced by intravenous injection of Stx2. Interleukin 1beta was not induced after Stx2 treatment in brain primary mixed cell culture.

Leptospirosis Pathophysiology: Into the Storm of Cytokines.

Leptospirosis is a neglected tropical zoonosis caused by pathogenic spirochetes of the genus Leptospira. Infected reservoir animals, typically mice and rats, are asymptomatic, carry the pathogen in their renal tubules, and shed pathogenic spirochetes in their urine, contaminating the environment. Humans are accidental hosts of pathogenic Leptospira. Most human infections are mild or asymptomatic. However, 10% of human leptospirosis cases develop into severe forms, including high leptospiremia, multi-organ injuries, and a dramatically increased mortality rate, which can relate to a sepsis-like phenotype. During infection, the triggering of the inflammatory response, especially through the production of cytokines, is essential for the early elimination of pathogens. However, uncontrolled cytokine production can result in a cytokine storm process, followed by a state of immunoparalysis, which can lead to sepsis and associated organ failures. In this review, the involvement of cytokine storm and subsequent immunoparalysis in the development of severe leptospirosis in susceptible hosts will be discussed. The potential contribution of major pro-inflammatory cytokines in the development of tissue lesions and systemic inflammatory response, as well as the role of anti-inflammatory cytokines in contributing to the onset of a deleterious immunosuppressive cascade will also be examined. Data from studies comparing susceptible and resistant mouse models will be included. Lastly, a concise discussion on the use of cytokines for therapeutic purposes or as biomarkers of leptospirosis severity will be provided.

Molecular analysis of Streptococcus pyogenes strains isolated from patients with recurrent pharyngitis after oral amoxicillin treatment.

PURPOSE: The most common illness caused by Streptococcus pyogenes (Group A streptococcus; GAS) is acute pharyngitis. It has been reported that a small percentage of patients experience recurrent GAS pharyngitis after 10 days of treatment with oral amoxicillin. The aim of this study was to clarify whether recurrent GAS pharyngitis is reactivation caused by the primary strain remaining at the infection site, or if the reinfection is caused by newly acquired strains. METHODOLOGY: A total of 135 GAS clinical strains were isolated from the tonsils of 116 pediatric patients with acute GAS pharyngitis between November, 2012 and April, 2014 in Saga, Japan. These strains were analysed by pulsed-field gel electrophoresis (PFGE)-typing methods. RESULTS: The isolates were grouped into 16 PFGE-types. The epidemic PFGE types that caused pharyngitis were found to change dynamically during 18 months. Eleven strains caused recurrent pharyngitis within 40 days after the last treatment, all of them showing the same PFGE-type as the primary strains. Eight of the strains caused recurrence more than 40 days after the treatment. Among them, six showed different PFGE-types from the primary strains. CONCLUSION: When recurrent pharyngitis emerges more than 40 days after the last treatment, penicillin can be prescribed again because reinfection is suspected. However, when recurrent pharyngitis takes place within 40 days after completing the treatment, alternative drugs should be considered for retreatment because the pharyngitis is likely to be due to reactivation.

Current status of leptospirosis in Japan and Philippines.

Leptospirosis is an acute febrile illness with a wide variety of clinical manifestations and is encountered throughout the world, prominently in tropical areas with high rainfall. In this paper, we review the current status of leptospirosis in two Asian countries, Japan and Philippines, which have quite different situations in terms of economy, environment, infrastructures and prevailing infectious diseases. In Japan, until 1960, more than 200 deaths due to leptospirosis had been reported yearly. After 1960, the number of reported cases had rapidly decreased. Now, leptospirosis cases notified every year were less than 20. After the early 1960s, modernization of agriculture was introduced, at the same time, inactivated vaccine against Leptospira was applied for humans and there was good maintenance of infrastructures such as water works and sewage systems. In Philippines, on the other hand, leptospirosis patients tend to be frequently found in flood-prone areas of urban setting such as Metro Manila, which was found to be the endemic foci of leptospirosis. Morbidity in a rural area (Cabatuan, Iloilo) was 147 cases per 100,000 populations. From 1998 to 2001, about 70% of 1200 suspected leptospirosis patients in Philippines were serologically positive. The average age of patients was 32 years old where 87% of the cases were males and 70% were outdoor workers. Case fatality rate was found to be 12-14%. The estimated major serovars in Metro Manila and neighboring provinces were Manilae, Losbanos, Tarassovi, Poi and an unknown serovar. Outbreaks of leptospirosis in the Philippines are thought to be associated with heavy rainfall, rapid urbanization (dramatic increase in populations), deforestation, increasing number of flood-prone areas, poor infrastructures and many others.

Adipose tissue is the first colonization site of Leptospira interrogans in subcutaneously infected hamsters.

Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, "Weil's disease," may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil's disease, by using an in vivo imaging system to observe the whole bodies of animals infected with Leptospira interrogans and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 104 bioluminescent leptospires, were analyzed by in vivo imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster's body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil's disease.