Hisao Yoshikawa

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 subtypexa03 and it varied from 41.7% to 92.3%. The second most common genotype among four populations was either subtypexa01 (7.7–25.0%) or subtype 4 (10.0–22.9%). Subtypexa02, subtypexa05, and/or subtypexa07 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 subtypexa01 or subtypexa03 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.

Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore

Blastocystis is an enteric protozoan parasite commonly found in humans and animals. Phylogenetic and genotypic analyses have shown that Blastocystis exhibits extreme genetic diversity, and humans are host to a number of zoonotic isolates. In the present study, the prevalence of Blastocystis in 276 stool samples from a hospital in Singapore was examined, and for the first time, riboprinting using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genetic diversity of the Blastocystis isolated from the Singapore population. The prevalence rate was determined to be 3.3% (9/276), and Blastocystis displaying two main ribotypes were isolated. As a comparison, we performed PCR-RFLP using two different published methodologies, and both methods allowed the isolates to be divided into two distinct groups based on their riboprint patterns. According to a recently proposed classification scheme, 78% (7/9) of the isolates were of subtype 3, while 22% (2/9) were subtype 1. The predominance of subtype 3 in an urbanized city state such as Singapore is in agreement with the idea that subtype 3 is a genotype of human origin.

Fecal-oral transmission of the cyst form of Blastocystis hominis in rats.

The infectivity of two Blastocystis hominis strains, RN94-9 and NIH:1295:1, was examined in 3-week-old SPF Wistar rats. The NIH:1295:1 strain, originally isolated from a guinea pig, was only able to infect rats via intracecal inoculation of the cultured organisms, while the RN94-9 strain, originally isolated from a laboratory rat, was able to infect rats by oral inoculation of the cultures due to the presence of a cystic form in the in vitro culture. Since many cysts were discharged in the feces of the infected rats, the infectivity of the concentrated cysts was compared between the two strains. Successful oral infection was observed in rats inoculated with 1×102–1×106 cysts of the RN94-9 and NIH:1295:1 strains. The infectivity of the ten cysts varied in the three experiments of ten rats, being 20–100% and 30–100% in the RN94-9 and NIH:1295:1 strains, respectively. When an uninfected normal rat was housed with five experimentally inoculated rats, the normal rat became infected, demonstrating the fecal-oral transmission of the cyst form of this parasite. These results show that the Wistar rat is an ideal host for the propagation of strains RN94-9 and NIH:1295:1 of B. hominis, and demonstrate that the cyst form is the only transmissible form of this parasite.

Infectivity of Blastocystis isolates from chickens, quails and geese in chickens

The infectivity of six Blastocystis isolates obtained from two domestic chickens, two Japanese quails and two domestic geese, were examined in 1-week-old male chicks. All six isolates were able to infect the chicks via the intracecal inoculation of 1×106xa0cells of cultured organisms. Since the infected chicks discharged many cysts in their feces, the infectivity of the concentrated cysts in chicks was compared among three isolates from different bird species. The CK86-1 and QQ93-3 isolates, which were obtained from a chicken and a quail, respectively, were successfully infected in chicks by orally inoculating with 1×102–1×106 cysts. On the other hand, the AC03-1 isolate from a goose required more cysts to infect the chicks, from 1×103 cysts to 1×106 cysts. In addition, when an uninfected normal chick was housed with five experimentally inoculated chicks with cysts of the QQ93-3 isolate, the normal chick became infected, indicating the fecal–oral transmission of the cyst form among the birds. These results show that the transmission of Blastocystis infection occurs easily between the same or different bird species. Therefore, the proposal of new Blastocystis species on the basis of different avian host species is problematic.

Ultrastructural and Phylogenetic Studies on Blastocystis Isolates from Cockroaches

ABSTRACT. Four Blastocystis isolates from cockroaches were established and these isolates were morphologically confirmed as Blastocystis organisms by light and/or electron microscopy. As these isolates were morphologically indistinguishable from Blastocystis isolated from other animals, phylogenetic analyses were conducted using their small subunit ribosomal RNA genes. A analyses of these sequences with previously reported ones that had been classified into nine Blastocystis clades indicated the presence of a new clade that comprised only Blastocystis organisms from cockroaches (clade X). A clade comprised of amphibian and reptilian Blastocystis organisms (clade IX) was located at the basal position of the Blastocystis tree together with the common ancestor of Proteromonas and Protoopalina, clade X emerged after the divergences of these two basal clades and its branching position was clearly supported by bootstrap analysis.