Molecular phylogeny of Blastocystis isolates from wild rodents captured in Indonesia and Japan.

Blastocystis sp. is a common intestinal protist found worldwide in a variety of animals, including humans. Currently, 17 subtypes (STs) of Blastocystis isolates from mammalian and avian host species have been reported based on the small subunit ribosomal RNA gene (SSU rDNA). Among these, human Blastocystis were only identified among STs 1-9. Except ST9, all other STs comprised isolates from humans and other animal species. Entire sequence data of the SSU rDNA of nine Blastocystis isolates from laboratory rats or guinea pigs previously showed ST4, whereas Blastocystis isolates from wild rodents have not been addressed genetically. In this study, Blastocystis infection in wild rodents was surveyed in Indonesia and Japan, and 11 and 12 rodent Blastocystis parasites were obtained from Rattus exulans and R. novercious, respectively. All new Blastocystis isolates from wild rodents were identified as ST4 based on the SSU rDNA sequences. The best tree inferred with the entire sequences of the SSU rDNA of all ST4 isolates including 17 data registered in GenBank clearly showed monophyletic ST4A and ST4B clades. Although ST4 isolates from laboratory rats were separated into these two clades, all Blastocystis isolates from wild rodents in the present study were positioned into the clade ST4A and further separated into two sub-clusters within the clade ST4A according to the location of the host species. Considering the fact that laboratory rats were susceptible to both ST4A and ST4B, separation of the monophyletic sub-clusters of Blastocystis isolates from Indonesian Polynesian rats and Japanese brown rats may indicate the presence of geographical variations rather than a host-specific separation. In either way, the robust host preference to rodent species of ST4 Blastocystis was also confirmed.

Evidence of infection with Leptospira interrogans and spotted fever group rickettsiae among rodents in an urban area of Osaka City, Japan.

We examined 33 rodents captured in an urban area of Osaka City, Japan for IgG antibodies against Seoul virus, severe fever with thrombocytopenia syndrome virus, hepatitis E virus, Leptospira interrogans, Yersinia pestis, spotted fever, typhus and scrub typhus group rickettsiae. We found that 3 (9.1%) and 1 (3.0%) of the 33 rodents had antibodies against L. interrogans and spotted fever group rickettsiae, respectively. DNAs of leptospires were detected from 2 of the 3 seropositive rodents, but DNA of rickettsia was not detected. Phylogenetic analysis and multiple locus sequence typing revealed that the 2 leptospires were L. interrogans belonging to a novel sequence type. There is a potential risk for acquiring rodent-borne zoonotic pathogens even in cities in developed countries.

Novel mutations in K13 propeller gene of artemisinin-resistant Plasmodium falciparum.

We looked for mutations in the Plasmodium falciparum K13 propeller gene of an artemisinin-resistant parasite on islands in Lake Victoria, Kenya, where transmission in 2012-2013 was high. The 4 new types of nonsynonymous, and 5 of synonymous, mutations we detected among 539 samples analyzed provide clues to understanding artemisinin-resistant parasites.

Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum.

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Infectivity of Cryptosporidium andersoni Kawatabi type relative to the small number of oocysts in immunodeficient and immunocompetent neonatal and adult mice.

Cryptosporidium andersoni is a protozoan parasite found in many countries that invades the stomachs of primarily adult cattle. Unlike the isolates of C. andersoni in cattle from other countries, C. andersoni isolates from Japanese cattle can infect mice and were identified as a novel type and later defined as C. andersoni Kawatabi type. The biological characteristics of C. andersoni Kawatabi type have not yet been well documented. In the present study, we assess the infectivity of this type isolate in mice with different immune competence status and age. We found that inoculation of more than 1×10(4) oocysts is needed to establish infection in mature mice irrespective of immune status. All of the infected immunocompetent mice recovered after a patent period of approximately 20days. In immunodeficient mice, the pre-patent period was prolonged compared with that of 1×10(6) oocysts, but the pattern and the maximum shedding measured by the number of oocysts per day were almost identical. In neonatal immunocompetent and immunodeficient mice, inoculation with 1×10(4) to 10(5) oocysts was also needed to establish infection. Our results indicate that there is a threshold of oocysts needed to establish patent infection in the acidic conditions of the stomach.

Construction and analysis of full-length cDNA library of Cryptosporidium parvum.

A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies.

In vitro assessment of anticryptosporidial efficacy and cytotoxicity of adenosine analogues using a SYBR Green real-time PCR method.

OBJECTIVES: The aims of this study were to provide a cost-effective and valuable method for evaluating drug efficacy against Cryptosporidium parvum using a quantitative SYBR Green real-time PCR (qPCR) and to assess the efficacy of adenosine analogues as drug templates. METHODS: C. parvum HNJ-1 strain growing in human ileocaecal adenocarcinoma cells was employed as an in vitro culture system. To normalize the DNA extraction efficiency, a specific plasmid was added to each sample before DNA purification; the genomic DNA of infected cells was quantified by qPCR using specific primers to confirm drug efficacy and cytotoxicity. To determine the mechanism of action, enzymatic inhibition analyses were conducted using C. parvum S-adenosyl-l-homocysteine hydrolase (CpSAHH) recombinant protein. RESULTS: The dose-dependent growth inhibition of C. parvum was confirmed; 50% effective concentrations of neplanocin A (NPA) and 2-fluoroadenosine (2FA) were 139 µM and 0.842 µM, respectively. Cytotoxicity evaluation showed that the 50% growth inhibition concentration of 2FA was 1.18 µM; NPA did not exhibit any cytotoxicity up to 200 µM. The screening system revealed the specific but marginal efficacy of NPA and showed 2FA to be cytotoxic. Recombinant CpSAHH inhibition analyses showed that NPA competitively inhibited CpSAHH activity (K(i )= 0.395 µM), whereas 2FA did not. CONCLUSIONS: This novel qPCR system confirmed not only drug efficacy against C. parvum but also cytotoxicity to host cells. Moreover, since the SYBR Green method is cost effective, it could therefore be used in a wide variety of clinical and research-oriented applications of Cryptosporidium analysis.

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan.

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.

Effect of low pH on the morphology and viability of Cryptosporidium andersoni sporozoites and histopathology in the stomachs of infected mice.

The genus Cryptosporidium includes many common parasites infecting animals and humans, and is a major cause of diarrheal illness worldwide. The biology of gastric Cryptosporidium spp., including replication in the stomach, has not been well documented. This study evaluated the viability of Cryptosporidium andersoni sporozoites in gastric environments after excystation and examined the endogenous development and histopathological changes in the stomachs of infected mice, using a novel type of C. andersoni. Sporozoites were affected by low pH (61.6% viability after 3h at pH2.0). Electron microscopy revealed developmental parasites on the gastric foveolae but not on the surface of the gastric mucosa. Histopathological examinations at 1, 2, 4 and 12 weeks p.i. uncovered three different lesions. The gastric mucosa of foveolae filled with parasites was extended and the amount of neutral mucopolysaccharide at the mucosal surface was decreased with the first type of lesion. The gastric mucosa was atrophied, some gastric glands were disrupted and the amount of acid mucopolysaccharide at the mucosal surface was increased with the second type. Finally, the gastric mucosa was slightly extended and goblet cells were present in the gastric mucosa, indicating intestinal metaplasia, in the third type. No parasites were detected in these areas with increased acidic mucin and indications of metaplasia. The results suggest that C. andersoni parasites could not survive in acidic environments for a long period before invading host cells and preferentially develop in neutral sites of the gastric mucosa, resulting in histopathological changes and chronic shedding of oocysts.

Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan.

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.

Cryptosporidium parvum in Korea: prevalence in individuals residing in three major river valleys and genetic characteristics of the isolates.

Cryptosporidiosis is a diarrheal illness caused by apicomplexa parasite Cryptosporidium spp. In this study, to examine the overall infection status of Cryptosporidium spp. in individuals residing in southern parts of Korea, eight counties around Yeongsan, Seomjin and Nakdong River valleys was surveyed. The investigation was carried out from April to October 2005. A total of 9,498 stool samples were collected from individuals. Stool samples were analyzed for modified acid-fast stains, and DNA fragment extracted from positive samples was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for 18S rRNA polymorphic region. Oocysts of Cryptosporidium spp. were detected in 239 specimens (2.5%) by a modified acid-fast stain. Infection rate was not significantly different between male (2.2%) and female (2.8%) individuals examined (P>0.05). In the infection rate by age, totally 1-9 (4.8%) and 80< (3.7%) age group were shown to the highest, and there was shown to significant differences (P<0.05). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 18S rRNA gene from 51 isolates showed that all the isolates were identified as C. parvum. Our data collectively suggested that C. parvum infection is prevalent in the studied areas of Korea and more comprehensive nation-wide epidemiological studies are needed to elucidate the infection status of Cryptosporidium infection in Korea.

Molecular survey of Enterocytozoon bieneusi in a Japanese porcine population.

Enterocytozoon bieneusi is an emerging and clinically significant enteric human pathogen that is mainly associated with chronic diarrhea. It has been identified in a variety of wild, domestic, and companion mammals and birds. This pathogen is genetically diverse and is composed of over 80 host-specific and zoonotic genotypes. Pigs are considered one of the main reservoirs of this pathogen given its high reported prevalence in pigs, and pigs may harbor zoonotic and pig-specific genotypes. Therefore, genotyping of isolates from pigs and other animals is essential for the control of E. bieneusi infection. In Japan, it remains unclear whether this pathogen is present in the porcine population. In the present study, we examined 30 fecal samples from pigs reared on 6 farms in western Japan. Ten pigs (33%) were found to be positive when assessed by polymerase chain reaction. The genotypes were varied and were found to be animal specific (EbpA, H, PigEBITS5) and zoonotic (D, EbpC) in genotype. The observation that pigs in Japan appear to be infected with zoonotic and animal genotypes of E. bieneusi raises questions about the prevalence of this infection among the human population in Japan.

Coprological survey of parasitic infections in pigs and cattle in slaughterhouse in Osaka, Japan.

A coprological survey was performed at a slaughterhouse in Osaka, Japan, from 2004 to 2007 on 129 pigs reared in 8 prefectures, and on 213 cattle reared in 21 prefectures. Eimeria spp., Trichuris suis, Ascaris suum and Metastrongylus spp. infections were found in 52 (40.3%), 32 (24.8%), 19 (14.7%) and 3 pigs (2.3%), respectively, while Eimeria spp., Capillaria bovis and Trichuris sp. infections were detected in 163 (76.5%), 15 (7.0%) and 8 cattle (3.8%), respectively. Our results suggest that environmentally resistant oocysts and eggs of parasites could be widespread at the farms examined.

Molecular evidence of Enterocytozoon bieneusi in Japan.

Enterocytozoon bieneusi is an emerging and clinically significant enteric pathogen in humans associated mainly with chronic diarrhea. It has been found in a variety of wild, domestic and companion mammals and birds. To date, epidemiological surveys of E. bieneusi infection in humans, other mammals and birds have been performed in more than 21 countries in Africa, the Americas, Australasia and Europe. In Asia E. bieneusi has been found in India, Thailand, Vietnam and Korea, but it has been quite unclear whether this pathogen is present in Japan. In the present study, we examined 149 DNAs extracted from 45 human (9 of them HIV-positive) and 104 animal fecal samples by PCR. Two dogs and a cat were positive and their genotypes were found to be dog specific and zoonotic (genotype K) types, respectively. Present study is the first record of E. bieneusi in Japan.

Expression of interferon gamma and proinflammatory cytokines in the cecal mucosa of rats experimentally infected with Blastocystis sp. strain RN94-9.

Blastocystis hominis is a zoonotic intestinal protozoan parasite whose pathogenic potential is still controversial. The aim of the present study was to clarify the pathogenicity of Blastocystis parasites in rats. Oral inoculation with 1 x 10(5) cysts of Blastocystis sp. strain RN94-9 in rats resulted in chronic infection in the cecum at least until 4 weeks after infection. Histological examination revealed neither mucosal sloughing nor inflammatory cell infiltration but showed a slight but significant increase in goblet cell numbers in the cecal mucosa 1-3 weeks post-infection. Differential staining of acidic and neutral mucins by the alcian blue-periodic acid-Schiff method showed that the predominantly increased cells were neutral mucin(+) but not acidic mucin(+) goblet cells. Reverse transcription real-time polymerase chain reaction studies demonstrated significant upregulation of the expression of interferon-gamma, interleukin (IL)-12, and tumor necrosis factor alpha, but not IL-6 or granulocyte-macrophage colony-stimulating factor, in the cecal mucosa at 2 and/or 3 weeks post-infection. The induction of local host responses, including mild goblet cell hyperplasia, and significant upregulation of type-1 and proinflammatory cytokines, suggest that Blastocystis sp. strain RN94-9 is a weakly pathogenic organism that could elicit proinflammatory as well as protective responses in local tissues.

Genetical survey of novel type of Cryptosporidium andersoni in cattle in Japan.

Previously, we reported that an isolate of novel type of Cryptosporidium andersoni detected in cattle in Japan contained Type A (identical to C. andersoni reported previously) and Type B (having a thymine nucleotide insertion unlike the Type A) genotypes in the 18S rRNA gene. Here, we conducted an extensive investigation of Cryptosporidium infections in adult cattle in Japan from 2004 to 2007. Consequently, Cryptosporidium sp. were detected in 12 of the 205 cattle examined (5.9%), and partial sequences of the Cryptosporidium oocyst wall protein (COWP) gene in all isolates were identical to those of the previously reported data for C. andersoni whereas two signals were observed in the sequence of the partial 18S rRNA gene in all the isolates. In transmission studies using five of the isolates, they all infected SCID mice. Modified multiplex PCR using DNA of a single oocyst isolated from the infected SCID mice revealed that the partial sequences in the 18S rRNA gene of 40-80% of 10 isolates were identical to the Type A genotype of C. andersoni and those of other samples were identical to the Type B genotype. These results suggested that the C. andersoni novel type is widespread in cattle throughout Japan, and have multiple copies (Types A and B) in the 18S rRNA gene.

Genotypic characterization of cryptosporidium oocysts isolated from healthy people in three different counties of Korea.

To investigate Cryptosporidium infection among healthy people, we collected stool samples from 150 healthy individuals in Gokseong, Muan, and Imshil Counties, southwest Korea, where neighbors on both an animal farm and a river respectively. In 12 of 150 samples, Cryptosporidium oocysts were detected by means of modified acid-fast staining. The bovine genotype, Cryptosporidium parvum, was identified by PCR/RFLP and 18S rRNA sequencing. C. parvum existed endemically in these areas, and the residents showed a relatively higher infection rate for C. parvum than that for C. hominis. Our results indicate that countermeasures against Cryptosporidium infection must be taken in these areas to ensure human health.

Detection of a mixed infection of a novel Cryptosporidium andersoni and its subgenotype in Japanese cattle.

Cryptosporidium oocysts were detected in the feces of cattle in Saga, Japan. Isolates were morphologically large. We attempted to identify the species or genotypes of the isolates by analyzing the partial sequences of the 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes, and measuring the infectivity in mice. The isolates showed 100% homology with Cryptosporidium andersoni in the COWP gene sequence and it could be transmitted to mice, but in the 18S rRNA gene, there was an additional signal in the ABI sequence chromatogram. To examine the additional signal, we analyzed both the 18S rRNA and the COWP gene sequences of a single oocyst passaged from mice using a modified multiplex PCR that was able to amplify both genes. As a result, it was revealed that two distinct genotypes (Types A and B) of a novel C. andersoni type existed in the 18S rRNA gene, whereas the COWP gene sequences of both oocysts were identical to C. andersoni. Although the sequence of the 18S rRNA gene of Type A was identical to that of C. andersoni, that of Type B had a thymine insertion and was not identical to any sequence registered with GenBank. Here we report that this is a new type of C. andersoni.

Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats.

Most Blastocystis hominis isolates from humans are believed to be potentially zoonotic. This is because B. hominis isolates found in a variety of other host species have been found to have identical or relatively similar genotypes to those found in human isolates. However, the transmission of human B. hominis isolates to other animals has not been confirmed experimentally. In this study, the infectivity associated with several unique human Blastocystis genotypes (subtypes 2, 3, 4 and 7) was therefore investigated by infecting chickens and rats with two isolates of each subtype experimentally. The results showed that one isolate of subtype 4 and one isolate of subtype 7 was capable of infecting both chickens and rats, while two isolates of subtype 2, another isolate of subtype 4, and another isolate of subtype 7 could only infect chickens. Conversely, two isolates of subtype 3 failed to infect either of the animals. These results confirmed that several genotypes from human isolates could infect chickens and/or rats, indicating that chickens and rats are suitable experimental animal models for studying the zoonotic potential of human Blastocystis isolates.