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Motohiro Iseki

Publications and source records attributed to Motohiro Iseki.

At least 19 recordsLinked to original sources
PubMed2006

Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences.

Cryptosporidium parvum is a well-known intestinal parasite which is associated with severe acute diarrhea in humans and animals. This parasite is composed of morphologically identical but genetically different multiple genotypes. In humans, cryptosporidiosis is mainly caused by two C. parvum genotypes, human genotype (previously known as genotype 1 and recently proposed as new species C. hominis) and cattle genotype (previously known as genotype 2). However, recent molecular studies indicate the genetic heterogeneity among the isolates of C. parvum human or cattle genotype. Therefore, identification of the isolates at the subgenotype level is more useful for control of the Cryptosporidium infection or for understanding of the population structure of C. parvum genotypes. In the present study, we identified the subgenotypes of the C. parvum human or cattle genotype isolates from humans and animals in Japan using DNA sequencing analysis of the C. parvum 60-kDa glycoprotein gene (GP60) and showed the new subgenotype in a raccoon dog isolate. This study suggested that C. parvum cattle genotype might be composed of zoonotic and host-specific multiple subgenotypes.

Animals↗
PubMed2005

Gene analysis of Cryptosporidium parvum HNJ-1 strain isolated in Japan.

We analyzed genetically Cryptosporidium parvum HNJ-1 strain, which is the Japanese reference strain isolated from human in Japan. DNA sequences of genes for thrombospondin-related adhesive protein of Cryptosporidium-1 and Cryptosporidium-2 (TRAP-C1, TRAP-C2), heat shock protein 70 (HSP70), oocyst wall protein (COWP), beta-tubulin, alpha-tubulin, polythreonine-region (Poly-T), elongation factor 1 alpha (EF-1 alpha), and 18S rRNA of this strain were determined. They showed high rate of homology to published sequences of genotype 2 strains, which were considered to be infective to both humans and animals. However, HNJ-1 had synonymous and non-synonymous substitutions in the nucleotide sequence of TRAP-C1 and beta-tubulin among HNJ-1 and published sequences of genotype 2 strains. These results implied that HNJ-1 strain was an unique subpopulation of genotype 2 strain of C. parvum.

Adult↗
PubMed2005

Infectivity of a novel type of Cryptosporidium andersoni to laboratory mice.

Previously, we reported 'a novel type' of Cryptosporidium andersoni detected from cattle in Japan, and showed that the isolate was infective to mice. In the present study, we examined the patterns of oocyst shedding in both immunocompromised and immunocompetent mice, as well as pathological lesions in the infected mice. After oral inoculation with 1 x 10(6) oocysts, all five severe combined immunodeficiency (SCID) mice began to shed endogenously produced oocysts on day 6 post-inoculation (p.i.). The number of oocysts per day (OPD) reached 1 x 10(6) on day 17 p.i., and an OPD level of 1 x 10(6) to 10(7) was maintained until 91 days p.i. when the mice were sacrificed. In the five immunocompetent mice inoculated with 1 x 10(6) oocysts, the pre-patent and patent periods were 6 and 19 days, respectively, and the maximal OPD level was 1.5 x 10(5) on average. On histological examinations of infected SCID mice, a large number of parasites were present on the surface of the gastric glands of the stomach, but not in other organs examined. In conclusion, the novel type of C. andersoni, which genetically coincides with C. andersoni reported in other countries, is infective to mice, but susceptibility was lower than that of Cryptosporidium muris infecting rodents from the perspective of infectivity to immunocompetent mice.

Animals↗
PubMed2005

Zoonotic genotype of Giardia intestinalis detected in a ferret.

Giardia intestinalis has been found in a variety of mammals, including humans, and consists of host-specific and zoonotic genotypes. There has been only 1 study of G. intestinalis infection in weasels, but the genotype of its isolate remains unclear. In this study, we report the isolation of Giardia in a ferret exhibited at a pet shop. The isolate was analyzed genetically to validate the possibility of zoonotic transmission. Giardia diagnostic fragments of the small subunit ribosomal RNA, beta-giardin, and glutamate dehydrogenase genes were amplified from the ferret isolate and sequenced to reveal the phylogenetic relationships between it and other Giardia species or genotypes of G. intestinalis reported previously. The results showed that the ferret isolate represented the genetic group A-I in assemblage A, which could be a causative agent of human giardiasis.

Animals↗
PubMed2004

Cross-reactivities with Cryptosporidium spp. by chicken monoclonal antibodies that recognize avian Eimeria spp.

In a previous study, we have developed several chicken monoclonal antibodies (mAbs) against Eimeria acervulina (EA) in order to identify potential ligand molecules of Eimeria. One of these mAbs, 6D-12-G10, was found to recognize a conoid antigen of EA sporozoites and significantly inhibited the sporozoite invasions of host T lymphocytes in vitro. Furthermore, some of these chicken mAbs showed cross-reactivities with several different avian Eimeria spp. and the mAb 6D-12-G10 also demonstrated cross-reactivities with the tachyzoites of Neospora caninum and Toxoplasma gondii. Cryptosporidium spp. are coccidian parasites closely related to Eimeria spp., and especially C. parvum is an important cause of diarrhea in human and mammals. In the present study, to assess that the epitopes recognized by these chicken mAbs could exist on Cryptosporidium parasites, we examined the cross-reactivity of these mAbs with Cryptosporidium spp. using an indirect immunofluorescent assay (IFA) and Western blotting analyses. In IFA by chicken mAbs, the mAb 6D-12-G10 only showed a immunofluorescence staining at the apical end of sporozoites of C. parvum and C. muris, and merozoites of C. parvum. Western blotting analyses revealed that the mAb 6D-12-G10 reacted with the 48-kDa molecular weight band of C. parvum and C. muris oocyst antigens, 5D-11 reacted the 155 kDa of C. muris. Furthermore, these epitopes appeared to be periodate insensitive. These results indicate that the target antigen recognized by these chicken mAbs might have a shared epitope, which is present on the apical complex of apicomplexan parasites.

Animals↗
PubMed2004

First record of Cryptosporidium infection in a raccoon dog (Nyctereutes procyonoides viverrinus).

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis.

Animals↗
PubMed2004

Identification of Cryptosporidium isolates from cockatiels by direct sequencing of the PCR-amplified small subunit ribosomal RNA gene.

Cryptosporidium is a significant pathogen in humans and animals. Cases of infection by C. meleagridis or C. baileyi with zoonotic potential have also been reported in domestic birds; and recent studies indicate the presence of new host-adapted species or genotype in birds. Therefore, accurately identifying isolates is important for understanding the epizootiology of Cryptosporidium infection in birds and for the control of human cryptosporidiosis. Cryptosporidium has been detected in cockatiels, but the species or genotype of isolates remains unclear because identification was performed using conventional microscopy. We report herein the species or genotype of isolates from two cockatiels distinguished by a PCR-based diagnostic method. The isolates were found to be C. meleagridis and C. baileyi, respectively. This study documents the first discovery of C. meleagridis and C. baileyi in cockatiels and suggests that pet birds may play an important role in the epidemiology of cryptosporidiosis.

Animals↗
PubMed2004

Molecular characterization of a Cryptosporidium isolate from a banded mongoose Mungos mungo.

Cryptosporidium spp. has been found in more than 150 species of mammals, but there has been no report in mongooses. In this study, we report the isolation of Cryptosporidium sp. in a banded mongoose Mungos mungo, which was brought from Tanzania to Japan; the isolate was analyzed genetically to validate the occurrence of a new, host-adapted genotype. Cryptosporidium diagnostic fragments of 18S ribosomal RNA and 70-kDa heat shock protein genes were amplified from this isolate and compared with the other Cryptosporidium species and genotypes reported previously. Analyses showed that the mongoose isolate represents a new genotype, closely related to that of bears.

Animals↗
PubMed2003

Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries.

Since the genotype of human Blastocystis hominis isolates is highly polymorphic, PCR-based genotype classification using known sequenced-tagged site (STS) primers would allow the identification or classification of different genotypes. Five populations of human B. hominis isolates obtained from Japan, Pakistan, Bangladesh, Germany, and Thailand were subjected to genotype analysis by using seven kinds of STS primers. Ninety-nine out of 102 isolates were identified as one of the known genotypes, while one isolate from Thailand showed two distinct genotypes and two isolates from Japan were negative with all the STS primers. The most dominant genotype among four populations, except for all four isolates from Thailand, was subtype 3 and it varied from 41.7% to 92.3%. The second most common genotype among four populations was either subtype 1 (7.7-25.0%) or subtype 4 (10.0-22.9%). Subtype 2, subtype 5, and/or subtype 7 were only rarely detected among the isolates from Japan and Germany, while subtype 6 was not detected. The phylogenetic position of the two isolates which were negative with all STS primers, was inferred from the small subunit rRNA (SSU rRNA) genes with the known sequence data of 20 Blastocystis isolates. Since the two isolates were positioned in an additional clade in the phylogenetic tree, this suggested they were a new genotype. These results demonstrated that PCR-based genotype classification is a powerful tool with which to analyse genotypes of Blastocystis isolates obtained from clinical samples. In addition, two groups of the isolates from 15 symptomatic and 11 asymptomatic patients in Bangladesh were compared with the PCR-based subtype classification. Since both groups were only classified into two distinct genotypes of subtype 1 or subtype 3 and no statistically significant difference was observed between the two groups, in this study it could not be shown that the specific genotype correlated with the pathogenic potential of B. hominis.

Animals↗
PubMed2003

Identification of genotypes of Cryptosporidium parvum isolates from ferrets in Japan.

Recent molecular studies show that Cryptosporidium parvum is composed of at least eight genotypes including zoonotic genotypes. Therefore, it is important to screen the genotypes of the isolates harbored in animals for the control of human cryptosporidiosis. The ferret is a popular pet, but also a reservoir of Cryptosporidium. Since the infectivity of zoonotic genotypes in ferrets remains unclear, there is a possibility these hosts harbor zoonotic genotypes. In the present study, we examined the genotypes of C. parvum isolates from ferrets in Japan using polymerase chain reaction direct sequencing. The sequences of the isolates examined clustered with the ferret-adapted genotype (ferret genotype). Our study suggests that ferrets harbor the ferret genotype which is conserved across geographical areas. Further study is required to determine whether ferrets are a significant reservoir for human cryptosporidiosis.

Animals↗
PubMed2003

[Identification of species of Cyclospora isolates from patients by the PCR-based diagnostic methods].

Cyclospora cayetanensis is an intestinal coccidian parasite and known as a human pathogen causing watery diarrhea. Recently, Cyclospora organisms, morphologically indistinguishable from C. cayetanensis, were detected from the several species of primates, and three new species named, C. cercopitheci, C. colobi, and C. papionis, have been proposed to the isolates on the basis of the genetic differences. The infectivity of these species to humans is strictly unknown, and there is a possibility of infection with not only C. cayetanensis but also the species from primates among patients. Therefore, it is necessary for the accurate diagnosis of Cyclospora infection to distinguish among the Cyclospora species. In the present study, we identified species of Cyclospora isolates from patients by the PCR-based diagnostic method. Since the sequence of Cyclospora 18S ribosomal RNA gene generated with the primary pair CYC1 FE and CYC4RB is found to be variable among Cyclospora species, we applied the PCR-direct sequencing using above primers to identify species of the isolates. Consequently, the diagnostic fragment was amplified by the PCR in all isolates, and the sequence of the PCR product obtained from each isolate was completely identical to that of C. cayetanensis. Therefore, we identified the isolates from patients as C. cayetanensis. On the basis of the results obtained in the present study, it is supposed that the PCR-direct sequencing using the primer pair CYC1FE and CYC4RB is a useful tool for the distinction among Cyclospora species.

Animals↗
PubMed2003

Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene.

Giardia has been detected in domestic dogs in Japan, but the genotype of isolates has remained unclear because identification has relied on conventional microscopy. Here we tried to identify the genotypes of four isolates from dogs in Japan by direct sequencing of the PCR amplified Giardia glutamate dehydrogenase (GDH) gene. The primer pair GDHF3 and GDHB5, targeting the GDH gene, was designed to prime a region of the GDH gene sequence conserved in the strains found to have the dog-specific genotype. The specific PCR product (approximately 220 bp), amplified with this primer pair, was only observed when Giardia DNA was used as the template. The sequences of the diagnostic fragments were identical among the isolates from dogs, and were differed by 15 bp or 1 bp from the strains, which were found to be the dog-specific genotypes, Assemblage C or D respectively. To verify the identity of the amplified DNA, a phylogenetic analysis was performed. Consequently, the sequence of the isolates from dogs clearly clustered with the strain found to be Assemblage D with neighbor-joining analyses. Therefore, all the isolates from dogs examined were identified as the dog-specific genotype, Assemblage D. In the present study, we revealed the genotype of Giardia isolates in Japan, and showed that direct sequencing of the PCR product amplified with the primer pair GDHF3 and GDHB5 was a useful tool for distinguishing between the zoonotic and dog-specific genotypes.

Animals↗
PubMed2003

Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.

Animals↗
PubMed2003

[Cryptosporidiosis].

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AIDS-Related Opportunistic Infections↗
PubMed2002

Cryptosporidium infection in dogs in Osaka, Japan.

Cryptosporidium parvum is a zoonotic pathogen composed of genetically distinct but morphologically identical genotypes. Recent molecular study indicates that dogs may transmit the cattle genotype, which is known to be pathogenic to humans. Although large-scale studies of Cryptosporidium infection in dogs have been performed in several countries, the isolates were not accurately identified because of the lack of a method for molecular analysis. It is important to identify the isolates harbored in dogs, which come in close contact with humans, in order to control human cryptosporidiosis. The aim of the present study was to calculate the prevalence of Cryptosporidium infection in dogs in Osaka city, Japan, and to characterize the isolates molecularly. The prevalence was determined to be 9.3% (13/140) by PCR. All isolates were found to be Cryptosporidium canis (previously known as the dog genotype), which is thought to be non-pathogenic in humans, based on the sequencing of diagnostic fragments. These results indicate that PCR-based diagnostic methods are a useful tool for the diagnosis and molecular epidemiology of Cryptosporidium infection in dogs, and that dogs living in Osaka are not a significant reservoir for human cryptosporidiosis. It is unclear why C. canis is dominant in dogs. Further study is required to understand this partial parasitism.

Animals↗