Niichiro Abe

A survey of Blastocystis sp. in livestock, pets, and zoo animals in Japan

The prevalence of Blastocystis sp. was examined in fecal samples collected from cattle, pigs, dogs, and a variety of zoo animals (primates, carnivores, herbivores, pheasants, and ducks) by direct observation of fresh fecal suspensions or cultured materials, using light microscopy. The cattle and pigs were randomly sampled from 11 and 12 commercial farms, respectively, located in the western region of Japan. The dog material used in this study was obtained from pets housed in an animal shelter in the city of Osaka. Zoo animals were chosen based on housing conditions that minimized the possibility of intra-zoo transmission of the organism. The prevalence rate among the groups varied greatly. A high prevalence of infection was observed in the farm animal group, ranging from 95% (58/61) in pigs to 71% (39/55) in cattle, whereas the dog fecal samples were completely free of the organism. Prevalence of the organism in the zoo animal were 85% (29/34) in primates, 80% (8/10) in pheasants, 56% (9/16) in ducks, and 0% (0/58) in various carnivores and herbivores. Among the zoo animals infected with Blastocystis, eight species of primates, eight species of pheasants, and four species of ducks were confirmed as new hosts. Since Blastocystis organisms isolated from various animals were morphologically indistinguishable from Blastocystis hominis by light microscopy, further genomic studies are required for analysis of the zoonotic potential or etiological significance of these isolates.

Zoonotic genotypes of Blastocystis hominis detected in cattle and pigs by PCR with diagnostic primers and restriction fragment length polymorphism analysis of the small subunit ribosomal RNA gene.

Twenty two Blastocystis isolates from cattle and pigs were genotyped by PCR using diagnostic primers, and the homology among isolates was then confirmed by restriction fragment length polymorphism (RFLP) analysis of the small subunit ribosomal RNA gene. Two distinct genotypes, subtypes 1 and 3, were detected in one and two of the ten isolates from cattle, and three and one of the 12 isolates from pigs, respectively. The RFLP profiles of all isolates designated as subtype 1 and subtype 3 were identical to those of known ribodemes 1 and 2, respectively. We found that 31.8% (7/22) of isolates examined were zoonotic genotypes of Bastocystis hominis. However, 68.2% (15/22) of isolates were not amplified with any of the diagnostic primers, and the profiles of these isolates were different from any ribodemes of B. hominis reported previously. This study shows that Blastocystis organisms in cattle and pigs are a potential source of human infection.

Molecular characterization of Blastocystis isolates from primates

Twelve Blastocystis isolates from primates were analyzed genetically by polymerase chain reaction (PCR) using diagnostic primers and PCR-restriction fragment length polymorphism (RFLP) of SSUrDNA. Two distinct genotypes, subtype 1 and a variant of subtype 1, were detected in two and six of the 12 isolates, respectively. The RFLP profiles of the isolates designated as subtype 1 were identical to the profile of ribodeme 1. The RFLP profiles of the six isolates designated as variants of subtype 1 were different from the profile of the variant of subtype 1 from a human reported previously. The other four isolates were not amplified with any diagnostic primers, but three of them showed the same RFLP profiles as ribodeme 6. This study was the first genomic analysis of Blastocystis isolates from primates, and showed the genetic similarity between the isolates from primates and the genotypes of Blastocystis hominis. However, it was unclear whether the isolates examined were zoonotic or not. Therefore, it is necessary to reveal the phylogenetic relationships between the isolates from primates and the multiple genotypes of B. hominis.

Molecular characterization of Blastocystis isolates from birds by PCR with diagnostic primers and restriction fragment length polymorphism analysis of the small subunit ribosomal RNA gene.

Abstract. Seven Blastocystis isolates from birds were genotyped by polymerase chain reaction (PCR) using diagnostic primers, and the homology among isolates was then confirmed by restriction fragment length polymorphism (RFLP) analysis of the small subunit ribosomal RNA gene. A zoonotic genotype, subtype 4, was detected in three isolates by PCR, but the RFLP profiles of these isolates differed from those of the subtype 4 reported previously from a human. None of the other four isolates were amplified with any subtype-specific primer sets, and the profiles of these isolates were different from any ribodemes of Blastocystis hominis reported previously. In the present study, the seven isolates from birds were classified into three genetically distinct genotypes. Therefore, birds appear to be infected with multiple genotypes. It is unclear whether the isolates examined in the present study are zoonotic or not. Therefore, it is important to determine the phylogenetic relationships between unknown genotypes from birds and the multiple genotypes of B. hominis.

Molecular epidemiology of noroviruses detected in seasonal outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan, from 1996-1997 to 2008-2009.

In seasons from 1996–1997 through 2008–2009, noroviruses (NoVs) were detected in 505 outbreaks (71%) of nonbacterial gastroenteritis in Osaka City, Japan using molecular diagnosis with reverse transcription (RT)‐PCR or real‐time RT‐PCR. The occurrences of NoV‐associated outbreaks were related with the cold season during November–March (85.3%), and occasionally small epidemics of NoVs occurring during April–June were observed. Oyster‐associated outbreaks were dominant transmission modes (25–61.1%) before the 2003–2004 season, and decreased (5–20.5%) from the 2003–2004 season, although outbreaks attributable to food‐borne transmission (except for oysters) and person‐to‐person contact increased from the 2003–2004 season. The NoV strains were characterized into genotypes based on sequence analysis of partial capsid regions. Genotyping analyses identified at least 30 genotypes (12 in genogroup I [GI] and 18 in genogroup II [GII]) of NoV. The most common genotype was GII.4 (44.6%), followed in order by GII.3, GII.6, GII.2, and GII.5. The number of GII.4 NoVs increased greatly from the 2003–2004 season, eventually comprising a large share among the NoV‐ associated outbreaks (97.4%) of the 2006–2007 season. Occasional increased prevalence of genotypes other than GII.4 was observed during this study period. This study showed the appearance, spread, and disappearance of various genotypes and the change of NoV epidemic in a limited geographic region. Continuous NoV molecular surveillance is important for understanding NoV infections and for improving measures for their control and prevention. J. Med. Virol. 82:2097–2105, 2010.

Detection and genetic characterization of human enteric viruses in oyster‐associated gastroenteritis outbreaks between 2001 and 2012 in Osaka City, Japan

Enteric viruses are an important cause of viral food‐borne disease. Shellfish, especially oysters, are well recognized as a source of food‐borne diseases, and oyster‐associated gastroenteritis outbreaks have on occasion become international occurrences. In this study, 286 fecal specimens from 88 oyster‐associated gastroenteritis outbreaks were examined for the presence of 10 human enteric viruses using antigenic or genetic detection methods in order to determine the prevalence of these infections. All virus‐positive patients were over 18 years old. The most common enteric virus in outbreaks (96.6%) and fecal specimens (68.9%) was norovirus (NoV), indicating a high prevalence of NoV infection associated with the consumption of raw or under‐cooked oysters. Five other enteric viruses, aichiviruses, astroviruses, sapoviruses, enteroviruses (EVs), and rotavirus A, were detected in 30.7% of outbreaks. EV strains were characterized into three rare genotypes, coxsackievirus (CV) A1, A19, and EV76. No reports of CVA19 or EV76 have been made since 1981 in the Infectious Agents Surveillance Report by the National Infectious Diseases Surveillance Center, Japan. Their detection suggested that rare types of EVs are circulating in human populations inconspicuously and one of their transmission modes could be the consumption of contaminated oysters. Rapid identification of pathogens is important for the development of means for control and prevention. The results of the present study will be useful to establish an efficient approach for the identification of viral pathogens in oyster‐associated gastroenteritis in adults. J. Med. Virol. 86:2019–2025, 2014.

The detection of a novel type of Cryptosporidium andersoni oocyst in cattle in Japan

Cryptosporidium muris, found in rodents and cattle, has been recognized as a valid species. However, this organism from cattle was recently separated from C. muris infecting rodents based on molecular data and a transmission study. As a consequence, it has been proposed as a new species, C. andersoni. More recently, C. andersoni, which has infectivity to rodents, was detected in cattle in Japan, where it has been designated as a novel type. However, isolates from cattle in Japan have not been analyzed genetically, and therefore it remains unclear whether a novel type of C. andersoni is distributed widely in Japan. In the present study, we detected Cryptosporidium oocysts from cattle reared in a different area than those examined previously in Japan. These were identified by molecular analysis and experimental transmission. The 18S ribosomal RNA gene sequence of the isolate examined was identical with that of C. andersoni reported previously, and the isolate was successfully transmitted to severe combined immunodeficiency mice. Therefore, the isolate from cattle examined in the present study was identified as a novel type of C. andersoni. Our data suggests that it is widespread in cattle in Japan.

Cryptosporidium Avian Genotype III as a Possible Causative Agent of Chronic Vomiting in Peach-Faced Lovebirds (Agapornis roseicollis)

Abstract In the present study, Cryptosporidium oocysts were found, by light microscopy, in 37 fecal samples of peach-faced lovebirds (Agapornis roseicollis). Cryptosporidium avian genotype III was isolated in 13 of the 37 infected birds by sequence analysis of the small subunit ribosomal RNA and the actin genes. All of the birds showed chronic vomiting and weight loss with enlargement of isthmi, narrowed proventricular lumens, and thickened proventricular walls radiographically. Cryptosporidium parasites were found only in the ductal epithelium of the proventricular glands in three of the tissue samples provided for necropsy. To date, there have been no reports concerning the pathogenicity, nor the location, of avian genotype III in avian hosts. Our report confirms, for the first time, the presence of avian genotype III in peach-faced lovebirds in Japan and also reveals the location in the avian host.

Genomic Polymorphism Among Blastocystis Isolates and Development of PCR-Based Identification of Zoonotic Isolates

B1astocysti.r horninis is known as the most common protozoan parasite of the human intestinal tract [9]. The species name, Blustrtcystis horninis is generally used for isolates from humans, although many B. hominis-like organisms have been isolated from various animals. These isolates have been designated as B. hominis, Blastocystis sp. or newly proposed species because there were no consensus for species designation for newly isolated Blastocystis from nonhuman hosts. On the other hand, several zoonotic strains were identified from a chicken and a guinea pig [6,10], and animal handlers had a significantly higher than normal rate of B. horninis infection [7] suggesting that Blastocystis in a variety of animals are potential sources of human infections. Therefore, it is important to characterize the zoonotic potential of Blastocystis found in animals. Recently we developed several sequence-tagged site (STS) primers developed from random amplified polymorphic DNAs. These amplified only distinct subtypes that corresponded to phylogenetically different clades (as inferred by the small subunit rRNA genes, SSU rDNA) [4,11-131, the STS primers can be used as a tool for typing Blastocystis populations. This PCR-based technique was recently reported to be a practical tool for typing B. horninis isolates from humans and animals and for detection of some zoonotic genotypes from animals [1-3,11-14]. In this study Blastocystis isolates from a variety of animals were analyzed with STS primers and compared with the genotypes of human B. horninis.

Epidemic of Genotype GII.2 Noroviruses during Spring 2004 in Osaka City, Japan

ABSTRACT Between March and May 2004, a GII.2 genotype norovirus strain caused an epidemic of acute gastroenteritis in Osaka, Japan. Phylogenetic analysis showed that this strain was distinct from all other GII.2 strains detected in Osaka City between April 1996 and March 2005.