Comparison of Leptospira species isolated from environmental water and soil in Japan.

Leptospira was isolated from environmental water in central Japan using selective medium comprising five antibiotics, namely sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil. Of 100 water samples 57 (57%) were culture-positive and 50 pure cultures were isolated. Of the 50 cultures isolated from water 48 were classified into a saprophytic clade on the basis of 16S ribosomal RNA gene sequences. However, it was previously reported that isolates from soil in Japan belonged to pathogenic, intermediate, and saprophytic clades, the current findings suggest less diversity of Leptospira species in environmental water than that in soil in Japan.

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.

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.

Case control study: Serological evidence that Borrelia miyamotoi disease occurs nationwide in Japan.

Since 2011, Borrelia miyamotoi disease (BMD) has been reported in five countries in the northern hemisphere. The causative agent of BMD is transmitted by Ixodes ticks, which are also vectors of Lyme disease borreliae. In this study, we examined 459 cases of clinically suspected Lyme disease (LD group), and found twelve cases that were seropositive for the glycerophosphodiester phosphodiesterase (GlpQ) antigen derived from B. miyamotoi. The retrospective surveillance revealed that the seroprevalence of anti-GlpQ in the LD group was significantly higher than in a healthy cohort. Seropositive cases were observed from spring through autumn when ticks are active, and the cases were geographically widespread, being found in Hokkaido-Tohoku, Kanto, Chubu, Kinki, and Kyushu-Okinawa regions. Seropositive cases for GlpQ were most frequent in the Chubu region (6.3%) where B. miyamotoi has been found in Ixodes ticks. Out of the twelve cases that were found in the LD group, three cases exhibited concomitant seropositivity to Lyme disease borreliae by western blot assay. This is the first report of serological surveillance for BMD in Japan, and we conclude that BMD occurs nationwide.

Borrelia yangtzensis sp. nov., a rodent-associated species in Asia, is related to Borrelia valaisiana.

Twenty-nine isolates of Lyme borreliosis (LB) group spirochaetes collected from ticks and rodents in China and Japan were included in a multilocus sequence analysis (MLSA). Using a different typing system, three of these strains had previously been identified as being divergent from other LB spirochaete species and the name 'Borrelia yangtze' sp. nov. was proposed. The data presented here confirm that the genetic distance, calculated using sequences of MLSA housekeeping genes, to other known LB group spirochaete species was < 95 % and to Borrelia valaisiana was 96.67 % (which represents the most closely related species within the group of LB spirochaetes). This and the fact that these strains are ecologically distinct from B. valaisiana (rodent-transmitted vs bird-transmitted) provide strong support for the validation of the proposed species status. We suggest the name Borrelia yangtzensis sp. nov. The type strain is Okinawa-CW62T ( = DSM 24625T = JCM 17189T).

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.

Age-Related Changes in Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1) and Receptor 2 (sVEGFR2) in Healthy Japanese Subjects.

We measured serum soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and receptor 2 (sVEGFR2) levels in healthy Japanese individuals in order to establish a reference value using a specific ELISA. Significant differences were observed in serum sVEGFR1 and sVEGFR2 levels between children and adults. To demonstrate the usefulness of the reference value for children, we measured serum sVEGFR1 and sVEGFR2 levels in children with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as a preliminary study. Serum sVEGFR2 levels in children with HUS were markedly higher than those in healthy children from the onset of D + HUS. The reference value for healthy children in the present study will allow normal and pathological conditions to be discriminated from each other in future study.

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.

Identification of Leptospira Species in Environmental Soil of Urban Settlements in Cambodia.

Background: Leptospirosis represents a major public health threat in Cambodia. To help establish preventive strategies for leptospirosis, we isolated and identified Leptospira from soil collected in Phnom Penh and its neighboring province during the dry and rainy seasons. Materials and Methods: Soil samples were collected from 15 sites in the Phnom Penh metropolitan area and Kandal Province in 2019 and 2020. A total of 120 soil samples were collected and analyzed after culturing. Cultured isolates were identified at the species level using 16S rRNA and gyrB gene sequencing analysis. Results: The positive culture rate for Leptospira was significantly higher for samples collected during the rainy season (31 out of 60 samples, 51.7%) than during the dry season (19 out of 60 samples; p < 0.05, chi-squared test). In terms of sampling area, 10 (35.7%) and 13 (46.9%) soil samples from Phnom Penh and 9 (28.1%) and 18 (56.3%) soil samples from Kandal Province were culture-positive when collected during the dry and rainy seasons, respectively. Three isolates from the Kandal samples collected during the dry season were categorized as pathogenic clade type P1 according to 16S rRNA and gyrB sequence analyses. In addition, samples collected during the rainy season from Phnom Penh and Kandal Province (three each) were categorized as clade type P1. The samples with isolates in clade type P1 were collected from riverbanks during both the dry and rainy seasons and from a hospital garden during the rainy season. Conclusions: This is the first report on the isolation of clade P1 Leptospira species in environmental soil samples from Cambodia. Environmental soil appears to be a reservoir for Leptospira in the capital city and surrounding areas of Cambodia. This study contributes to the understanding of Leptospira species distribution in Asian countries.

Brazilian propolis inhibits the differentiation of Th17 cells by inhibition of interleukin-6-induced phosphorylation of signal transducer and activator of transcription 3.

Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants. It has been reported to show immunomodulatory activity. Previously, we reported the protective effect of Brazilian propolis ethanolic extract against the pathogenesis of collagen-induced arthritis (CIA), an experimental animal model of rheumatoid arthritis (RA). Moreover, we found that the protective effect against CIA was involved in suppression of the production of interleukin-17 (IL-17) in CIA mice. In the present study, we demonstrated for the first time that propolis inhibited IL-6 plus transforming growth factor-ß induced Th17 differentiation in vitro. Propolis also suppressed the IL-6-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a cytokine-activated essential transcription factor in Th17 development, concomitantly with the enhanced suppressor of cytokine signaling 3 expression involved in the downregulation of STAT3 phosphorylation. These data suggest that the suppressive effect of propolis on Th17 differentiation might be useful for controlling unbalanced cytokine networks in autoimmune diseases.

Suppression of interleukin 17 production by Brazilian propolis in mice with collagen-induced arthritis.

Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants. Propolis has been reported to have immunomodulatory activity. We hypothesized that propolis would be able to reduce the disease severity of rheumatoid arthritis. We evaluated the effect of Brazilian propolis ethanolic extract on the pathogenesis of collagen-induced arthritis (CIA) in mice. Mice fed propolis exhibited significant lower clinical arthritis scores than those fed the control diet. To investigate the mechanism of the effect of propolis on CIA mice, we examined interleukin-17 (IL-17) production in CIA mice fed propolis using an enzyme-linked immunospot assay and flow cytometric analysis. The numbers of IL-17-producing cells in the CIA mice fed propolis were significantly decreased. To determine direct influence of propolis on cytokine production, splenocytes were stimulated with phorbol myristate acetate in the presence of propolis extract in vitro. Concentration-dependent declines in IL-17 expression were observed by ELISA and real-time PCR methods. We further found that propolis significantly inhibited the differentiation of Th17 cells from murine splenocytes in a concentration-dependent manner. Taken together, our results may provide a new light on the potential mechanism of the immunosuppressive and anti-inflammatory effects of propolis.

Age-Related Changes of Serum Soluble Interleukin 9 Receptor (sIL-9Rα) in Healthy Subjects.

Most cytokine receptors including interleukin (IL)-9 have soluble counterparts in body fluids. We planned to investigate the pathophysiological significance of the serum soluble IL-9 receptor (sIL-9R) level. We determined the serum sIL-9Rα chain (sIL-9Rα) levels in 96 healthy Japanese individuals to establish a control value by means of specific human sIL-9Rα ELISA, followed by a preliminary application in a patient with diarrhea positive hemolytic uremic syndrome. Age was negatively correlated with the sIL-9Rα level (Spearman r = -0.241, n = 96, p = 0.0180). The serum sIL-9Rα level showed a progressive decline to the normal adult level by the age of 30. The serum sIL-9Rα level of the patient with HUS was markedly higher than those of the age-matched control from the onset of the disease. Because of the remarkable age-dependent variability of sIL-9Rα in healthy subjects, disease-related changes, as well as therapy-dependent alterations, should be considered with caution. Thus, it is recommended that when the serum sIL-9Rα levels of patients are evaluated, the values should be compared with those of age-matched controls. The established control value will be used to discriminate between normal and the pathological conditions in our future studies.

Light dependent synthesis of a nucleotide second messenger controls the motility of a spirochete bacterium.

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and in functions such as virulence and biofilm formation, mainly via gene regulation. Here, we show that the swimming motility of the soil bacterium Leptospira kobayashii is rapidly modulated by light stimulation. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis identified the novel sensory gene, and its expression in Escherichia coli through codon optimization elucidated the light-dependent synthesis of cyclic adenosine monophosphate (cAMP). GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. These findings suggest a new role for cAMP in rapidly controlling the flagella-dependent motility of Leptospira and highlight the global distribution of the newly discovered photoactivated cyclase among diverse microbial species.

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.

Multilocus sequence typing implicates rodents as the main reservoir host of human-pathogenic Borrelia garinii in Japan.

Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.

Determination of soluble tumor necrosis factor-α receptor type I (TNFRI) and II (TNFRII) in the urine of healthy Japanese subjects.

We determined the urinary soluble tumor necrosis factor-α receptor type I (sTNFRI) and type II (sTNFRII) levels in healthy Japanese individuals to establish a reference value by means of specific ELISA. It was found that there were no significant differences between the urine sTNFRI and sTNFRII levels of children and adults. To demonstrate the usefulness of the reference value for children, we measured the urine sTNFRI and sTNFRII levels of children with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as a preliminary study. The urine sTNFRI and sTNFRII levels of the patients with HUS were markedly higher than those of healthy children from the onset of D + HUS. Our reliable reference value for healthy children will allow us to discriminate between normal and pathological conditions in future studies.

Complete Genome Sequence of Leptospira kobayashii Strain E30, Isolated from Soil in Japan.

The spirochete bacterium Leptospira kobayashii is a recently designated species of the genus Leptospira. Here, we report the complete genome sequence of L. kobayashii strain E30, consisting of two circular chromosomes and two plasmids.

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.

Potential infectivity of Anaplasma phagocytophilum strains in Ixodes ricinus ticks from Serbia.

Anaplasma phagocytophilum strains from different geographical regions are characterised by diverse potential infectivity for humans and domesticated animals. We investigated the potential pathogenicity of A. phagocytophilum strains in Ixodes ricinus ticks from 11 geographically different localities in Serbia. Sequences obtained in this study showed a high variability of p44 paralogues. Some of them, however, formed groups with similarities greater than 86% ('similarity groups'). Previous studies showed that 'similarity groups' were nearly always country specific. Our results correlated with this observation, and we also observed significant clustering of paralogues according to vector and reservoir origin of A. phagocytophilum strains. According to the high genetic similarity of sequences isolated from ticks collected in four localities, namely Avala, Batrovci, Hajducka cesma and Ljubovija, with paralogues with proven pathogenicity isolated from human granulocytic anaplasmosis (HGA) patients and A. phagocytophilum infected sheep, we could assume that strains with potential infectivity for humans and domestic animals were present in Serbia.

Detection of Asian-Type Borrelia miyamotoi from Ixodes ricinus Inhabiting Tver Province (Russia): A Sympatric Region for I. ricinus and Ixodes persulcatus.

Borrelia miyamotoi, a hard tick-borne relapsing fever agent, was sampled in Ixodes ricinus and Ixodes persulcatus ticks from the Tver province in Russia (a sympatric region of both tick species) and examined by TaqMan-PCR targeting the 16S rRNA gene. Borrelia was detected in 4 out of 168 ticks: 2 out of 58 I. ricinus ticks (infection rate 2.9%) and 2 out of 110 I. persulcatus ticks (1.8%). The agent was identified as B. miyamotoi on the basis of the 16S and 23S rDNA intergenic spacer and glycerophosphodiester phosphodiesterase gene sequencing analyses. Interestingly, the genes sequences detected from one I. ricinus tick were identical to those of Asian-type B. miyamotoi from I. persulcatus. This tick was identified as I. ricinus by sequencing analysis of internal transcribed spacer 2 and mitochondrial cytochrome oxidase subunit 1. The results suggest that the I. ricinus ticks were infected with Asian-type B. miyamotoi in a sympatric region for I. ricinus and I. persulcatus.