Publications by authors named "Nobuo Koizumi"

79 Publications

Molecular and serological epidemiology of Leptospira infection in cats in Okinawa Island, Japan.

Sci Rep 2021 May 14;11(1):10365. Epub 2021 May 14.

Department of Bacteriology I, National Institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Cats have been reported to be infected with Leptospira spp. and shed the bacteria in the urine. However, the importance of cats as an infection source for humans remains unclear. In this study, Leptospira infection in cats in Okinawa Prefecture, Japan, where leptospirosis is endemic, was investigated by leptospiral antibody and DNA detection using microscopic agglutination test and nested PCR, respectively. Moreover, multilocus sequence typing (MLST) and whole genome sequencing (WGS) were conducted on the Leptospira borgpetersenii serogroup Javanica isolated from cats, black rats, a mongoose, and humans. Anti-Leptospira antibodies were detected in 16.6% (40/241) of the cats tested, and the predominant reactive serogroup was Javanica. The leptospiral flaB gene was detected in 7.1% (3/42) of cat urine samples, and their sequences were identical and identified as L. borgpetersenii. MLST and WGS revealed the genetic relatedness of L. borgpetersenii serogroup Javanica isolates. This study indicated that most seropositive cats had antibodies against the serogroup Javanica and that cats excreted L. borgpetersenii in the urine after infection. Further, genetic relatedness between cat and human isolates suggests that cats may be a maintenance host for L. borgpetersenii serogroup Javanica and a source for human infection.
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http://dx.doi.org/10.1038/s41598-021-89872-3DOI Listing
May 2021

Molecular epidemiology of Leptospira spp. among wild mammals and a dog in Amami Oshima Island, Japan.

PLoS One 2021 22;16(4):e0249987. Epub 2021 Apr 22.

Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.

Leptospirosis is a worldwide zoonosis caused by the pathogenic Leptospira spp. Canine and human leptospirosis sometimes occur on Amami Oshima Island, located in the Nansei Archipelago, southwestern Japan; however, information on the causative Leptospira spp. on this island is quite limited. This study aimed to investigate the molecular and serological characteristics of Leptospira spp. isolated from wild animals and a dog in Amami Oshima Island. We obtained seven Leptospira strains by culturing kidney tissues of wild animals, such as black rats (2), wild boars (3), and rabbit (1) as well as blood from a symptomatic dog. Using flaB sequencing and microscopic agglutination test with antisera for 18 serovars, the isolates were identified as Leptospira borgpetersenii serogroups Javanica (black rat), L. interrogans serogroup Australis (black rat and dog), and L. interrogans serogroup Hebdomadis (wild boar and rabbit). The sequence type (ST) of L. borgpetersenii serogroup Javanica was determined to be ST143 via multilocus sequence typing (MLST) using seven housekeeping genes. For L. interrogans, MLST and multiple-locus variable-tandem repeat analysis (MLVA) revealed identical ST and MLVA types in rat and canine isolates, whereas two STs and MLVA types were identified in wild boar isolates. The STs and MLVA types of rabbit and one of the wild boars were identical. Bacterial culture and flaB-nested polymerase chain reaction demonstrated a high rate of Leptospira infection in wild boars (58.3%, 7/12), whereas Leptospira spp. were detected in 4.8% of black rats (2/42). This study revealed diverse Leptospira genotype and serotype maintenance in wild mammals on Amami Oshima Island. MLST and MLVA indicated that black rats were a source of canine infection. Wild boars carry L. interrogans and are considered an important maintenance host because antibodies against serogroup Hebdomadis were detected in human and canine leptospirosis patients on this island.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249987PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061989PMC
April 2021

Rat trade and leptospirosis: Molecular epidemiology of Leptospira species in rats exported from Cambodia to Vietnam.

Transbound Emerg Dis 2021 Mar 18. Epub 2021 Mar 18.

Department of Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

Rats are an important maintenance host of Leptospira spp., the causative agents of leptospirosis, a zoonotic disease with worldwide distribution. Rats are traded as food in the Mekong Delta, where Cambodia exports tons of rats to Vietnam. Handling wild rats is a potential health risk, but the information on Leptospira spp. carried by rats traded in the region remains limited. In this study, we investigated the carriage of Leptospira spp. in rats exported from Cambodia to Vietnam using bacterial culture, nested PCR and DNA sequencing. Isolates were then assessed using serological analysis and whole-genome sequencing (WGS), and Leptospira DNA detected in rat kidney tissues was also analysed by multilocus sequence typing (MLST). Seventy-two rats (2 Bandicota indica, 57 Rattus argentiventer, 11 R. losea, 1 R. norvegicus and 1 R. rattus) were subjected to bacterial culture, and three L. borgpetersenii serogroup Javanica isolates were obtained from R. argentiventer (5.3%). WGS revealed that although Cambodian isolates were genetically related to L. borgpetersenii serogroup Javanica strains widely distributed in East and South-East Asian countries, they formed a different cluster from other strains. In addition to the three L. borgpetersenii sequences, the flaB sequence of L. interrogans was detected in 18 R. argentiventer and 7 R. losea kidney tissue samples (38.9%) using nested PCR followed by DNA sequencing. The L. interrogans flaB-positive samples were further analysed by MLST, revealing that seven housekeeping genes (glmU, pntA, sucA, tpiA, pfkB, mreA and caiB) contained novel sequences with distinct lineages from other sequence types. This study revealed a high prevalence of Leptospira spp. among rats exported from Cambodia to Vietnam, indicating a potential risk to people engaging in rat trade and demonstrating that a fastidious L. interrogans strain circulates among Cambodian rats.
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http://dx.doi.org/10.1111/tbed.14077DOI Listing
March 2021

Antimicrobial resistance in Escherichia coli isolated from brown rats and house shrews in markets, Bogor, Indonesia.

J Vet Med Sci 2021 Apr 21;83(3):531-534. Epub 2021 Jan 21.

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

The prevalence of antimicrobial resistance (AMR) in small mammals dwelling in the city was used as an indicator of AMR bacteria in the environment. We captured 87 small mammals (79 brown rats and 8 house shrews) in four markets, Bogor, Indonesia in October 2019, and we obtained 20 AMR Escherichia coli (E. coil) from 18 brown rats and two house shrews. Of these, eight isolates were determined to be multi-drug resistant (MDR) E. coli, suggesting the potential contamination of AMR E. coli in the markets in Bogor, Indonesia, and that mammals, including humans, are at risk of infection with AMR E. coli from environment.
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http://dx.doi.org/10.1292/jvms.20-0558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025418PMC
April 2021

Crawling Motility on the Host Tissue Surfaces Is Associated With the Pathogenicity of the Zoonotic Spirochete .

Front Microbiol 2020 5;11:1886. Epub 2020 Aug 5.

Department of Applied Physics, Graduate School of Engineering, Tohoku University, Sendai, Japan.

Bacterial motility is crucial for many pathogenic species in the process of invasion and/or dissemination. The spirochete bacteria spp. cause symptoms, such as hemorrhage, jaundice, and nephritis, in diverse mammals including humans. Although loss-of-motility attenuate the spirochete's virulence, the mechanism of the motility-dependent pathogenicity is unknown. Here, focusing on that spp. swim in liquid and crawl on solid surfaces, we investigated the spirochetal dynamics on the host tissues by infecting cultured kidney cells from various species with pathogenic and non-pathogenic leptospires. We found that, in the case of the pathogenic leptospires, a larger fraction of bacteria attached to the host cells and persistently traveled long distances using the crawling mechanism. Our results associate the kinetics and kinematic features of the spirochetal pathogens with their virulence.
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http://dx.doi.org/10.3389/fmicb.2020.01886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419657PMC
August 2020

Multilocus sequence typing reveals diverse known and novel genotypes of Leptospira spp. circulating in Sri Lanka.

PLoS Negl Trop Dis 2020 08 25;14(8):e0008573. Epub 2020 Aug 25.

Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

Background: Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking.

Methodology/principal Findings: We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat.

Conclusions/significance: This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.
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http://dx.doi.org/10.1371/journal.pntd.0008573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473516PMC
August 2020

Genetic relatedness of Leptospira interrogans serogroup Autumnalis isolated from humans, dogs, and mice in Japan.

BMC Res Notes 2020 Aug 3;13(1):369. Epub 2020 Aug 3.

Department of Bacteriology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640, Japan.

Objective: Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of Leptospira spp., and peridomiciliary rodents are the most important reservoir animals for human infection. Dogs are known to be the reservoir animal of L. interrogans serovar Canicola, but the importance of dogs in zoonotic transmission of other Leptospira serotypes/genotypes remains unclear. This study reports the isolation of L. interrogans serogroup Autumnalis from two human patients in Japan and describes the genetic comparison between canine and mouse isolates using multiple-locus variable-number tandem repeat analysis (MLVA).

Results: MLVA revealed that 8 out of the 11 loci compared were identical between the two human isolates. The human isolates clustered with the dog but not the mouse isolates. Moreover, the profile of one of the human isolates was identical to that of one of the dog isolates.
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http://dx.doi.org/10.1186/s13104-020-05211-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398373PMC
August 2020

Multiple Pulmonary Nodules in Leptospirosis.

Intern Med 2020 Nov 21;59(22):2941-2944. Epub 2020 Jul 21.

Disease Control and Prevention Center, National Center for Global Health and Medicine, Japan.

A 21-year-old man presented with the chief complaints of fever and sore throat after visiting Cambodia and Thailand. Computed tomography revealed multiple pulmonary nodules. After performing antibiotic therapy, the pulmonary nodules without bacteremia disappeared completely. Paired microscopic agglutination tests revealed seroconversion against Leptospira serogroup Autumnalis. Thus, he was diagnosed with multiple pulmonary nodules caused by leptospirosis. Leptospirosis is a common zoonosis that occurs in tropical and subtropical areas. Its various clinical features include unspecified fever and Weil's disease. Although diffuse alveolar hemorrhaging is known to occur in severe leptospirosis, multiple pulmonary nodules resembling septic emboli or vasculitis are a rare complication.
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http://dx.doi.org/10.2169/internalmedicine.4305-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725640PMC
November 2020

Laboratory Diagnosis of Leptospirosis.

Authors:
Nobuo Koizumi

Methods Mol Biol 2020 ;2134:277-287

Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.

The diagnosis of leptospirosis depends on specific laboratory tests because nonspecific and diverse clinical manifestations make clinical diagnosis difficult and it is easily confused with other infectious diseases in the tropics. Suitable laboratory diagnostic tests vary depending on the stage of the disease, requiring the combination of diagnostic tests using appropriate specimens at each disease stage.
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http://dx.doi.org/10.1007/978-1-0716-0459-5_25DOI Listing
March 2021

Comparative genomic analysis of Leptospira borgpetersenii serogroup Javanica isolated from Rattus species in Southern Japan, Philippines, and Taiwan.

Infect Genet Evol 2020 11 30;85:104447. Epub 2020 Jun 30.

Department of Bacteriology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.

Leptospira spp. are the causative agents of leptospirosis, a zoonosis that occurs worldwide, and are maintained by various mammals. Recent molecular typing studies have indicated the existence of a specific Leptospira genotype-host association; however, few studies have shown the colonization of a single genotype in multiple animal genera. To gain further insights into the Leptospira genotype-host association, we conducted whole genome sequencing of 35 strains of L. borgpetersenii serogroup Javanica isolated from various Rattus species in Southern Japan, Philippines, and Taiwan. The SNP-based phylogenetic tree showed that the strains were grouped into five clusters based on their geographic location, indicating geographical structuring of genetic diversity in L. borgpetersenii serogroup Javanica strains. Furthermore, phylogenetic analysis indicated that the L. borgpetersenii serogroup Javanica can infect various animal species, thus making these strains generalist pathogens. This study reports the whole genome comparison of L. borgpetersenii serogroup Javanica strains isolated from Rattus species in East Asian states. The data herein suggest that the serogroup Javanica strains can infect the dominant Rattus species in each region and can be genetically diversified in a geographic manner.
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http://dx.doi.org/10.1016/j.meegid.2020.104447DOI Listing
November 2020

Novel genotypes of serogroup Sejroe isolated from human patients in Okinawa Prefecture, Japan.

J Med Microbiol 2020 Apr;69(4):587-590

Department of Bacteriology I, National Institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of species. It is a public health issue in the tropics, including Okinawa, the southernmost prefecture of Japan. This study reports the first isolation of serogroup Sejroe from two human patients in Japan, and describes its molecular characterization using multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). MLST on the two isolates, 168036 and 178129, showed that in 178129 is a novel allele, and that both isolates constitute novel sequence types (STs); ST286 for 168036 and ST287 for 178129. A minimum spanning tree based on seven alleles of indicates that both isolates are genetically close, but are distinct from known serogroup Sejroe strains. MLVA using 11 loci demonstrated that seven of the 11 loci were identical between the two isolates, whereas the identity between the isolates and the seven reference strains of serogroup Sejroe was zero to three loci. These results indicate that the isolates investigated in this study have novel genotypes, and are genetically closest to each other among the known serogroup Sejroe strains.
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http://dx.doi.org/10.1099/jmm.0.001169DOI Listing
April 2020

Antibiotic-resistant Escherichia coli isolated from urban rodents in Hanoi, Vietnam.

J Vet Med Sci 2020 May 30;82(5):653-660. Epub 2020 Mar 30.

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

Antimicrobial resistance (AMR) is a global public health concern for both clinical and veterinary medicine. Rodent feces are one of the major infectious sources of zoonotic pathogens including AMR bacteria. So far, there are limited studies reported focused on Escherichia coli isolated in rodent feces from rural and suburban areas in Vietnam. In this study, we investigated the prevalence of antimicrobial resistance in E. coli isolated from feces samples of 144 urban rodents caught in Hanoi, Vietnam. A total of 59 AMR E. coli was isolated from urban rodents of which 42 were multidrug-resistant (MDR) isolates (resistance to at least three classes of antimicrobial agents), four were extended-spectrum β-lactamase (ESBL) producing isolates and five were colistin-resistant isolates. The highest prevalence of the resistance was against ampicillin (79.7%: 47/59), followed by tetracycline (78.0%: 46/59), nalidixic acid (67.8%: 40/59), sulfamethoxazole-trimethoprim (59.3%: 35/59), chloramphenicol (45.8%: 27/59), ciprofloxacin (44.1%: 26/59), cefotaxime (30.5%: 18/59), cefodizime (23.7%: 14/59), amoxicillin-clavulanate (22.0%: 13/59), and gentamicin (22.0%: 13/59). With regard to the virulence genes associated with diarrheagenic E. coli (DEC), only aaiC gene found in one AMR isolate. In general, the use of antimicrobials does not aim to treat rodents except for companion animals. However, our findings show the carriage of AMR and MDR E. coli in urban rodents and highlight the potential risk of rodents in Hanoi acting as a reservoir of transferable MDR E. coli, including ESBL-producing, colistin-resistant E. coli, and virulence-associated with DEC.
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http://dx.doi.org/10.1292/jvms.19-0697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273608PMC
May 2020

Detection of pathogenic leptospires in the urine of domesticated elephants in Sri Lanka.

Acta Trop 2019 Jul 29;195:78-82. Epub 2019 Apr 29.

Department of Microbiology, Faculty of Medicine, University of Peradeniya, Sri Lanka. Electronic address:

Leptospirosis is a globally common zoonotic infectious disease in humans and animals. This disease is caused by pathogenic spirochetes belonging to the genus Leptospira. The pathogen is able to survive in mammalian kidneys after infection and is excreted in urine intermittently. Pathogenic leptospires infect humans either by direct contact with infected animal urine or through contaminated soil or water. In Sri Lanka, some studies have demonstrated the involvement of animals, such as livestock species and peridomestic rats, in the transmission of leptospirosis to humans. However, none of the previous studies focused on domesticated elephants, which are in close contact with humans during cultural and religious events and bathe in rivers together with humans. If domesticated elephants act as carriers of pathogenic leptospires, it could be a major public health issue in the country. In this study, 13 healthy domesticated elephants were subjected to leptospiral DNA detection from urine samples collected on three consecutive days. Four elephants (31%) were confirmed to shed pathogenic leptospires in their urine. DNA sequencing followed by phylogenetic distance measurements revealed that all positive elephants were infected with L. interrogans. This study reveals the possibility that elephants act as a source of infection of leptospires to humans and recommends the screening of all domesticated elephants that are in close contact with humans for the shedding of pathogenic leptospires.
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http://dx.doi.org/10.1016/j.actatropica.2019.04.029DOI Listing
July 2019

Molecular epidemiology of Leptospira interrogans in Rattus norvegicus in Hanoi, Vietnam.

Acta Trop 2019 Jun 6;194:204-208. Epub 2019 Apr 6.

Department of Bacteriology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Leptospirosis is a zoonotic disease that is caused by pathogenic spirochaetes of Leptospira spp. and it has become a public health concern in urban localities in the tropics. Rats are important reservoir animals for the transmission of leptospirosis in urban areas. Leptospirosis is considered endemic in Vietnam. However, information on the causative Leptospira genotypes and serotypes in the country is limited. We investigated the carrier status of Leptospira spp. in rats captured in Hanoi by culturing and DNA detection. Isolates were characterized using a serological method and multiple-locus variable-number tandem repeat analysis (MLVA). We captured 144 rats (1 Rattus argentiventer, 135 R. norvegicus, and 8 R. rattus) and obtained 17 L. interrogans, determined by rrs sequencing, from R. norvegicus (12.6%). Sixteen of the isolates were serogroup Bataviae. Five of the 16 isolates exhibited an MLVA type identical to that of the serovar Bataviae reference strain Van Tienen, while there were nine repeats for the other 11 isolates at VNTR31 compared with the reference strain. The remaining isolate grew poorly, and we were unable to determine its serogroup. However, it had an MLVA type matching those of serogroup Pomona strains isolated from R. norvegicus in Japan. Three different flaB sequences were detected in 23 out of 81 R. norvegicus kidney tissue samples (28.4%) using nested PCR followed by DNA sequencing. Two of the sequences were identical with those of serogroups Bataviae and Pomona, and no strain with another sequence was detected in the present study. The present study reveals a high prevalence rate of L. interrogans among R. norvegicus in Hanoi, Vietnam, indicating a potential risk of rat-borne leptospirosis in the area. The present study also demonstrates that a fastidious L. interrogans strain circulates among rats and that molecular detection is crucial in facilitating the accurate determination of reservoir animals.
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http://dx.doi.org/10.1016/j.actatropica.2019.02.008DOI Listing
June 2019

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.

Microbiol Immunol 2019 Mar 29;63(3-4):89-99. Epub 2019 Mar 29.

Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Aichi.

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 , = JCM 32515  = CIP111620 ), Leptospira ellinghausenii sp. nov., (type strain E18 , = JCM 32516  = CIP111618 ) and Leptospira ryugenii sp. nov., (type strain YH101 , = JCM 32518  = CIP111617 ) are proposed.
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http://dx.doi.org/10.1111/1348-0421.12671DOI Listing
March 2019

Leptospiral 3-hydroxyacyl-CoA dehydrogenase as an early urinary biomarker of leptospirosis.

Heliyon 2018 Apr 30;4(4):e00616. Epub 2018 Apr 30.

Department of Bacteriology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.

Leptospirosis, caused by spirochetes of the genus , is a globally widespread, neglected and emerging zoonotic disease. The currently used diagnostic tests are time-consuming, require technical expertise or require the use of sophisticated equipment. Clinicians have pointed out the urgent need to develop a rapid test for the diagnosis of acute leptospirosis with a non-invasive and easy sampling method. In this study, we have focused on a leptospiral enzyme, 3-hydroxyacyl-CoA dehydrogenase (3-HADH), as a urinary biomarker of acute leptospirosis. A specific antiserum for pathogenic spp. was produced, targeting a peptide corresponding to amino acids 410 to 424 of 3-HADH. The antiserum was used to investigate whether 3-HADH is excreted in the urine by Western blotting. Among 70 suspected leptospirosis patients, 40 were laboratory confirmed by microscopic agglutination test (MAT) using paired sera samples and/or polymerase chain reaction (PCR). In the acute phase of the laboratory-confirmed leptospirosis cases, sensitivity for 3-HADH, blood PCR and urine PCR were 52.5%, 57.5% and 12%, respectively. 3-HADH was detected from 2 days post-onset of illness (p.o) and could be detected at least until 9 days p.o. The combination of PCR and 3-HADH detection increased sensitivity of diagnosis to 100% in samples collected between 1 and 3 days p.o., and to 82% in samples collected between 4 and 9 days p.o. Our results suggested that the detection of 3-HADH can support a clinical diagnosis of leptospirosis, especially when serological methods are negative during the acute phase.
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http://dx.doi.org/10.1016/j.heliyon.2018.e00616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968168PMC
April 2018

The mechanism of two-phase motility in the spirochete : Swimming and crawling.

Sci Adv 2018 05 30;4(5):eaar7975. Epub 2018 May 30.

Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579, Japan.

Many species of bacteria are motile, but their migration mechanisms are considerably diverse. Whatever mechanism is used, being motile allows bacteria to search for more optimal environments for growth, and motility is a crucial virulence factor for pathogenic species. The spirochete , having two flagella in the periplasmic space, swims in liquid but has also been previously shown to crawl over solid surfaces. The present motility assays show that the spirochete movements both in liquid and on surfaces involve a rotation of the helical cell body. Direct observations of cell-surface movement with amino-specific fluorescent dye and antibody-coated microbeads suggest that the spirochete attaches to the surface via mobile, adhesive outer membrane components, and the cell body rotation propels the cell relative to the anchoring points. Our results provide models of how the spirochete switches its motility mode from swimming to crawling.
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http://dx.doi.org/10.1126/sciadv.aar7975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976277PMC
May 2018

Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa.

PLoS One 2018 10;13(4):e0194923. Epub 2018 Apr 10.

Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ΔfcpA strains, the L. biflexa ΔfcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ΔfcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ΔfcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194923PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892894PMC
July 2018

Building prognostic models for adverse outcomes in a prospective cohort of hospitalised patients with acute leptospirosis infection in the Philippines.

Trans R Soc Trop Med Hyg 2017 12;111(12):531-539

School of Tropical Medicine and Global Health, Nagasaki University, Japan.

Leptospirosis is endemic to the Philippines. Ten per cent of cases will develop severe or fatal disease. Predicting progression to severity is difficult. Risk factors have been suggested, but few attempts have been made to create predictive models to guide clinical decisions. We present two models to predict the risk of mortality and progression to severe disease. Data was used from a prospective cohort study conducted between 2011 and 2013 in San Lazaro Hospital, Manila. Predictive factors were identified from a literature review. A strategy utilizing backwards stepwise-elimination and multivariate fractional polynomials identified key predictive factors. A total of 203 patients met the inclusion criteria. The overall mortality rate was 6.84%. Multivariable logistic regression revealed that neutrophil counts [OR 1.38, 95% CI 1.15-1.67] and platelet counts [OR 0.99, 95% CI 0.97-0.99] were predictive for risk of mortality. Multivariable logistic regression revealed that male sex (OR 3.29, 95% CI 1.22-12.57) and number of days between symptom onset and antibiotic use (OR 1.28, 95% CI 1.08-1.53) were predictive for risk of progression to severe disease. The multivariable prognostic models for the risks of mortality and progression to severe disease developed could be useful in guiding clinical management by the early identification of patients at risk of adverse outcomes.
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http://dx.doi.org/10.1093/trstmh/try015DOI Listing
December 2017

Leptospirosis Cases in the Tokyo Metropolitan Area, Japan.

Jpn J Infect Dis 2017 Nov 11;70(6):669-671. Epub 2017 Sep 11.

Disease Control and Prevention Center, National Center for Global Health and Medicine.

In Japan, 20-50 cases of leptospirosis are reported annually, typically involving farmers or returnees from tropical areas. Here, we report on 5 indigenous leptospirosis cases that occurred in the Tokyo urban area. All patients were men (mean age, 66.4 ± 4.6 [SD] years) and presented with clinical features that included high fever, acute kidney injury, and jaundice. Three patients presented with pulmonary infiltration and ground-glass opacity on admission. Two of the 5 patients were intubated and underwent continuous hemodiafiltration. The mean length of hospitalization was 38.0 ± 17.0 days, and all patients were treated successfully with antibiotics. Notably, all patients had a history of exposure to rats prior to becoming symptomatic, suggesting that exposure to rats is a risk factor for urban leptospirosis. Although rare, even in metropolitan areas, leptospirosis should be considered by physicians when patients present with severe sepsis, acute kidney injury, and a history of exposure to rats.
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http://dx.doi.org/10.7883/yoken.JJID.2017.108DOI Listing
November 2017

Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection.

Microb Pathog 2017 Oct 12;111:33-40. Epub 2017 Aug 12.

Department of Bacteriology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640, Japan. Electronic address:

Leptospirosis causes severe clinical signs more frequently in men than in women, but the mechanism underlying the gender differences in leptospirosis remains unclear. In this study, petechial hemorrhage was observed in male but not in female hamster lung tissues infected with Leptospira interrogans serovar Hebdomadis at 120 h pi, demonstrating that male hamsters were more susceptible to the development of a severe disease upon Leptospira infection. No leptospiral DNA was detected in the lung tissues at 120 h pi when pulmonary hemorrhage was observed, indicating that pulmonary hemorrhage is attributable to the immune reactions of the host rather than from the direct effect of leptospires. The upregulation of nitric oxide synthase genes in the hamsters without pulmonary hemorrhage, inos and enos in female hamsters at 96 h pi and enos in male animals without hemorrhage at 120 h pi, may suggest that nitric oxide has a suppressive effect on leptospirosis-associated pulmonary hemorrhage.
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http://dx.doi.org/10.1016/j.micpath.2017.08.016DOI Listing
October 2017

An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

PLoS Negl Trop Dis 2017 Jul 20;11(7):e0005728. Epub 2017 Jul 20.

Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India.

Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.
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http://dx.doi.org/10.1371/journal.pntd.0005728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544247PMC
July 2017

Imported Leptospira licerasiae Infection in Traveler Returning to Japan from Brazil.

Emerg Infect Dis 2017 03;23(3):548-549

We describe a case of intermediate leptospirosis resulting from Leptospira licerasiae infection in a traveler returning to Japan from Brazil. Intermediate leptospirosis should be included in the differential diagnosis for travelers with fever returning from South America. This case highlights the need for strategies that detect pathogenic and intermediate Leptospira species.
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http://dx.doi.org/10.3201/eid2303.161262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382744PMC
March 2017

Molecular epidemiology of pathogenic Leptospira spp. among large ruminants in the Philippines.

J Vet Med Sci 2016 Dec 23;78(11):1649-1655. Epub 2016 Jul 23.

Department of Bioresources, Research Center for Zoonosis Control, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan.

The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013-2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.
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http://dx.doi.org/10.1292/jvms.16-0289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138416PMC
December 2016

Serological investigation of Leptospira infection and its circulation in one intensive-type water buffalo farm in the Philippines.

Jpn J Vet Res 2016 Feb;64(1):15-24

Water buffalo is an indispensable livestock in the Philippines. Leptospirosis is a serious zoonosis that can be fatal to humans and cause reproductive problems in livestock. Leptospirosis has been reported in some countries where water buffaloes are commercially raised, highlighting the Leptospira prevalence in this farming system, but information on leptospirosis in water buffalo farms in the Philippines is limited. In this study, we collected blood samples from rats (n = 21), and water buffaloes (n = 170) from different groups and locations in one intensive-type buffalo farm in the Philippines. Serum was analyzed by microscopic agglutination test (MAT). Anti-Leptospira antibodies reacting with serogroups Canicola, Icterohaemorrhagiae and Pomona were found in sera of 30% tested rats, and 48% of water buffalo sera tested positive for at least one Leptospira strain, in which serogroups Mini, Hebdomadis, Tarassovi and Pyrogenes were predominantly agglutinated. The number of seropositive young water buffaloes (< 1 year-old) was lower than that of older seropositive ones. Furthermore, sera from younger water buffaloes were reactive with single serotypes with low MAT titers, but older animals were reactive with multiple Leptospira strains with variable MAT titers. In addition, antibodies against serogroups Icterohaemorrhagiae and Pomona were detected in both animals. Finally, Leptospira infection was found associated with age and animal grouping, highlighting the impact of management in the persistence of leptospirosis at intensive-type buffalo farm settings in the Philippines. Further investigation and appropriate control strategies are required to prevent leptospirosis from causing risks to public health and economic losses to the water buffalo farming industry.
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February 2016

Combined antibody and DNA detection for early diagnosis of leptospirosis after a disaster.

Diagn Microbiol Infect Dis 2016 Apr 8;84(4):287-91. Epub 2016 Jan 8.

Division of Disaster-Related Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, Sendai, Japan; Division of Emerging Infectious Diseases, Department of Internal Medicine, Graduate School of Medicine, Tohoku University, Sendai, Japan. Electronic address:

Early diagnosis based on laboratory confirmation is essential for managing leptospirosis. This study investigated the effectiveness of a novel method of detecting leptospirosis that combines measurement of anti-Leptospira antibodies by the microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT) and leptospiral DNA by loop-mediated isothermal amplification (LAMP) and real-time PCR in plasma and 2 types of urine pellets. Of 113 suspected cases, 68.1%, 76.1%, and 60.2% were positive by MAT, ELISA, and ICT, respectively. Real-time PCR using DNA purified from urine pellets collected by low-speed centrifugation yielded positive signals for patients in late acute as well as early phase who were positive by LAMP using plasma DNA or urine pellets. Among antibody-negative patients, 9.5% were positive by DNA detection. These findings indicate that the leptospirosis detection rate is increased by combining antibody and DNA detection, providing a new tool for timely diagnosis of infection.
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http://dx.doi.org/10.1016/j.diagmicrobio.2016.01.001DOI Listing
April 2016

Imported Flood-Related Leptospirosis From Palau: Awareness of Risk Factors Leads to Early Treatment.

J Travel Med 2015 Nov-Dec;22(6):422-4

Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan.

We describe two Japanese travelers with leptospirosis who visited Palau. Both travelers swam in Ngardmau Falls, which was flooded for two days after typhoon Phanfone. The diagnoses were confirmed by microscopic agglutination test or polymerase chain reaction. This is the first report of leptospirosis in travelers who returned from Palau. It should be noted that choosing the appropriate test to biologically confirm leptospirosis was highly time-dependent. Awareness of the risk factors for leptospirosis, mainly that of the exposure to contaminated fresh water after a flooding, would lead to an early and appropriate treatment before the confirmed diagnosis.
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http://dx.doi.org/10.1111/jtm.12241DOI Listing
August 2016

Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents.

J Vet Med Sci 2016 Feb 27;78(2):221-30. Epub 2015 Sep 27.

Department of Microbiology, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan.

The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87-188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.
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http://dx.doi.org/10.1292/jvms.15-0325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785110PMC
February 2016

Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia.

Infect Genet Evol 2015 Dec 18;36:434-440. Epub 2015 Aug 18.

Department of Bacteriology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors.
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http://dx.doi.org/10.1016/j.meegid.2015.08.013DOI Listing
December 2015

Complete Genome Sequences of Low-Passage Virulent and High-Passage Avirulent Variants of Pathogenic Leptospira interrogans Serovar Manilae Strain UP-MMC-NIID, Originally Isolated from a Patient with Severe Leptospirosis, Determined Using PacBio Single-Molecule Real-Time Technology.

Genome Announc 2015 Aug 13;3(4). Epub 2015 Aug 13.

Okinawa Institute of Advanced Sciences, Uruma, Okinawa, Japan.

Here, we report the complete genome sequences of low-passage virulent and high-passage avirulent variants of pathogenic Leptospira interrogans serovar Manilae strain UP-MMC-NIID, a major causative agent of leptospirosis. While there were no major differences between the genome sequences, the levels of base modifications were higher in the avirulent variant.
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http://dx.doi.org/10.1128/genomeA.00882-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536678PMC
August 2015