MORPHOLOGIC OBSERVATION AND FIRST MOLECULAR CHARACTERIZATION OF ENCYCLOMETRA JAPONICA (DIGENEA: ENCYCLOMETRIDAE) IN THE DIGESTIVE TRACT OF WILD TIGER KEELBACKS (RHABDOPHIS TIGRINUS) IN JAPAN.

Encyclometra japonica Yoshida and Ozaki, 1929 is a fluke that parasitizes the digestive tract of snakes. When the species was first reported, it was originally characterized on the basis of morphologic features, namely, the characteristics of the 2 testes located obliquely in the anterior-posterior direction, and the position of the ventral sucker one-fourth to one-third from the anterior extremity. Thereafter, more specimens with other morphologic variations were reported, and a new morphologic feature for species discrimination was proposed. However, the proposal is uncertain, and the criteria for morphologic discrimination between E. japonica and other Encyclometra species remain debatable. Thus, morphologically discriminable characteristics on the basis of a sufficient number of specimens are required, and molecular methods for species identification that allow for a more objective assessment need to be established. Therefore, in the present study, nucleotide sequences of 18S, 28S ribosomal deoxyribonucleic acid and cytochrome c oxidase subunit I (COI) of E. japonica were determined along with detailed morphologic observations for the first time. Key characteristics were observed in all specimens, whereas some morphologic variations were observed in the symmetry of the cecum. The COI sequences, which are known to be variable genetic regions, were identical among all specimens; therefore, all of them were considered the same species. This result clarifies the high morphologic variation in E. japonica. The 18S and 28S sequences were 99.78% and 99.11-99.19% similar to those of Encylometra colubrimurorum. The high degree of homology between these genes indicates that E. japonica and E. colubrimurorum are closely related but independent species. The present data will be used for the identification of E. japonica and to evaluate the relationships within the genus Encyclometra.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Detection and molecular characteristics of Rhytidodoides sp. (Digenea: Rhytidodidae) from the gall bladder of green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan.

Trematodes of the genus Rhytidodoides are parasitic in marine turtles. Of the already known species, Rhytidodoides similis Price, 1939, occurs especially in the gall bladder. In this study, we surveyed 73 green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan, and detected Rhytidodoides sp. from the gall bladders of 18 turtles. A detailed morphological analysis revealed that the forebody of Rhytidodoides sp. differed slightly in shape from that of R. similis. There has been no information on DNA sequences of the family Rhytidodidae. A molecular phylogeny based on 28S rDNA sequences of Rhytidodoides sp. and related taxa suggested that the Rhytidodidae is sister to the other families of Echinostomatoidea. The intraspecific diversity of Rhytidodoides sp. was examined by using DNA sequences of mitochondrial cytochrome c oxidase subunit 1 gene (COI). The population genetic features of the COI haplotypes demonstrated that Rhytidodoides sp. is highly diverse in the Ogasawara Islands. The DNA sequences determined in this study will contribute to the species identification of congeners and the taxonomic reconsideration of the Echinostomatoidea.

Morphological observation and first molecular characterization of Grassenema procaviae Petter, 1959 (Cosmocercoidea: Atractidae) in the stomach of Cape hyrax (Procavia capensis) raised in a zoo in Japan.

The superfamily Cosmocercoidea comprises three families: Cosmocericidae, Kathlaniidae and Atractidae. Information on the nucleotide sequences of the Cosmocercoidea is quite limited, and the molecular classification of the whole superfamily has been slow to progress. The genus Grassenema of the family Atractidae is a parasitic nematode group that occurs in the digestive tract of hyraxes and includes three species: Grassenema procaviae, G. dendrohyraci, and G. hyracis. The type species of the genus, G. procaviae, was isolated from the digestive tract of Cape hyraxes (Procavia capensis) and has the potential to cause gastric ulcers. Although G. procaviae is a common parasite of Cape hyraxes, no genetic information for the parasite is currently available. In this study, we obtained the first genomic sequences of G. procaviae and performed detailed morphological observations. Furthermore, molecular phylogenetic analysis was performed, and the taxonomic position of the parasite was evaluated using 18S and 28S rDNA sequences. Those data will be useful for molecular identification of G. procaviae and future phylogenetic analysis within the Atractidae.

Infection by and Molecular Features of Learedius learedi (Digenea: Schistosomatoidea) in Green Sea Turtles (Chelonia mydas) on the Ogasawara Islands, Japan.

Learedius learedi Price, 1934 , is a blood fluke found in sea turtles, and the adult fluke parasitizes the cardiovascular system of the host. In this study we surveyed 46 green sea turtles, Chelonia mydas, on the Ogasawara Islands, Japan, and blood flukes were detected in the heart and blood vessels of 26 turtles. The flukes were identified as L. learedi based on a detailed morphological description. In addition, molecular identification and characterization of the parasite were performed. The nucleotide sequences of nuclear internal transcribed spacer 2 (ITS2) regions were almost identical to those of L. learedi reported previously, but not to those of Hapalotrema spp., which is the closest related genus. The nucleotide sequences of the 28S ribosomal DNA region formed a single clade with those of the reference L. learedi in the phylogenetic tree, but not with those of Hapalotrema spp. Therefore, the nucleotide sequences of ITS2 and 28S are robust markers for distinguishing L. learedi from other species. The nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) region were analyzed to evaluate the genetic variations in L. learedi. The COI haplotypes revealed the extremely high genetic diversity of the species as well as the host turtles on the Ogasawara Islands. The haplotype frequency in the mitochondrial DNA of the green sea turtles on the Ogasawara Islands is known to be significantly different from those in other Pacific rookeries. Although the number of analyzed flukes is small in this study, no haplotype was close to that in other areas; on the basis of the data, we hypothesized that L. learedi differentiated along with the host turtles on the Ogasawara Islands.

First report of Paragonimus skrjabini miyazakii metacercariae in Geothelphusa dehaani (Sawagani) occurring in Iwate Prefecture, Japan.

Paragonimiasis is an important food-borne zoonosis caused by Paragonimus flukes and is endemic to western Japan. However, there have been few epidemiological studies in the Tohoku district of northeastern Japan. In this study, Paragonimus metacercariae (mc) was detected in Geothelphusa dehaani (Japanese freshwater crab or Sawagani) in Iwate Prefecture. Out of the 207 Sawagani collected from 35 localities, 12 individuals from six localities were infected with Paragonimus mc. The mc were identified as P. skrjabini miyazakii based on the sequences of nuclear ribosomal internal transcribed spacer 2 and mitochondrial cytochrome c oxidase I. This is the first report of P. s. miyazakii mc infection in Sawagani in Iwate Prefecture.

Nuclear and mitochondrial DNA analysis reveals that hybridization between Fasciola hepatica and Fasciola gigantica occurred in China.

The well-known pathogens of fasciolosis, Fasciola hepatica (Fh) and Fasciola Gigantica (Fg), possess abundant mature sperms in their seminal vesicles, and thus, they reproduce bisexually. On the other hand, aspermic Fasciola flukes reported from Asian countries, which have no sperm in their seminal vesicles, probably reproduce parthenogenetically. The aim of this study was to reveal the origin of aspermic Fasciola flukes. The nuclear single copy markers, phosphoenolpyruvate carboxykinase and DNA polymerase delta, were employed for analysis of Fasciola species from China. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the Fh/Fg type, which includes DNA fragments of both F. hepatica and F. gigantica. China can be regarded as the cradle of the interspecific hybridization because F. hepatica and F. gigantica were detected in the northern and southern parts of China, respectively, and hybrids flukes were distributed between the habitats of the two species. The Chinese origin was supported by the fact that a larger number of mitochondrial NADH dehydrogenase subunit 1 (nad1) haplotypes was detected in Chinese aspermic Fasciola populations than in aspermic populations from the neighbouring countries. Hereafter, 'aspermic' Fasciola flukes should be termed as 'hybrid' Fasciola flukes.

Collection methods of trematode eggs using experimental animal models.

Although observing the eggs of human parasitic helminth is essential for medical education in parasitology, opportunities for collection of the eggs are limited. Collection of the eggs using experimental animal models is needed for a sustainable supply. The metacercariae of three trematode species, Paragonimus westermani, Clonorchis sinensis and Metagonimus yokogawai, were collected from the second intermediate hosts: freshwater crabs and fishes, which were obtained using online shopping in Japan, and inoculated to experimental animal rat and dog. Consequently, eggs of the three trematode species were obtained abundantly from the feces of the animals. The eggs are being used for student training in several Japanese universities. In this article, we introduce the collection procedures for trematode eggs.

Effects of Tritrichomonas muris on the mouse intestine: a proteomic analysis.

Although Tritrichomonas muris is a common parasite often detected in experimental animals including mice, its pathogenesis in host animals remains unclear. Proteomics can be used to specifically analyze biochemical host-parasite interaction and immune responses of the host to parasites. However, proteomics have not yet been applied to T. muris studies. Here, the effects of T. muris on the host were analyzed by proteomics. We found that 10 different proteins were expressed in T. muris-infected mice intestines compared with non-infected intestines. The identified proteins represented several functions mainly related to stress, immune response, metabolism and signal transduction. The results suggest that T. muris infection may affect processes that are acclimatizing to the environmental changes caused by the infection in the mouse intestine.

Molecular characterization of Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola sp. in China based on nuclear and mitochondrial DNA.

Parthenogenic Fasciola forms as well as bisexual Fasciola hepatica and Fasciola gigantica in mainland China have been identified on the basis of their spermatogenesis and genotypes in nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase I (NDI). The Chinese aspermic Fasciola would include forms originating in interspecific hybrids between F. hepatica and F. gigantica, since they showed the genotype of ITS1-Fh/Fg that had mixed sequences of the two Fasciola species or heterogeneous genotypes in ITS1 and NDI. Additionally, there were Chinese aspermic flukes in which the sequences of ITS1 and NDI genotypes completely coincided with those in aspermic forms from Japan, Korea, and Vietnam, suggesting that the aspermic forms from these four countries are offspring with a common provenance. The Fh-C4 haplotype in NDI was detected in both aspermic specimens and F. hepatica, indicating that aspermic forms showing the haplotype might come into existence in China. The ratio of body length and width in aspermic Fasciola specimens showed intermediate values between those of F. hepatica and F. gigantica.

Paragonimiasis in Cameroon: molecular identification, serodiagnosis and clinical manifestations.

Paragonimiasis is a common parasitic zoonosis in Cameroon and neighbouring countries in Western Africa. Serum, sputum and faecal samples were collected in an endemic area of South West Province, Cameroon, after administration of a questionnaire to identify individuals with appropriate symptoms and histories. Microscopic examination revealed eggs in sputum from 16 people, but none in any faecal sample. These 16 were among the 25 and 26 people, respectively, positive by ELISA and by immunoblot using Paragonimus africanus crude antigens. Copro-DNA detection was attempted using 23 faecal samples (18 from sputum egg-negative and five from sputum egg-positive individuals). Copro-DNA was detected in four of the five sputum egg-positive individuals. These results strongly suggest that: (1) serology is much more sensitive than sputum examination for diagnosis of paragonimiasis; and (2) a copro-DNA test may be more sensitive than a microscopic search for eggs in faeces. Molecular sequence data from ITS2 and cox1 genes confirmed that adult worms experimentally raised in cats were P. africanus and that eggs from sputum or other worm products from human faeces also belonged to this species. Based on these results, 26 of 168 persons (15.5%) were diagnosed as suffering from paragonimiasis.

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan.

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.

Decreased bodyweight without rebound and regulated lipoprotein metabolism by gymnemate in genetic multifactor syndrome animal.

OBJECTIVE: The aim of this work was to find obesity control method without rebound. In our previous studies, gymnemate extracted from Gymnema sylvestre, inhibited oleic acid absorption. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a genetic multifactor syndrome model, exhibits progressive overweight, hyperlipidemia and hyperglycemia. The effect of gymnemate on obesity in OLETF was investigated. METHODS: Three groups were divided (n=4-8): (1) OLETF-gymnemate, gymnema water extract (containing gymnemate) diet (62.5 g/kg) and water (2.5 g/kg) were supplied 2 weeks from 26-28 weeks, following it general diet and water were fed 3 weeks to observe if it rebound, (2) OLETF-control and (3) the counterpart Long-Evans Tokushima Otsuka rats as normal-control. RESULTS: With gymnemate treatment, the food and water intake were decreased about 1/3 and 2/3, along with body weight reduced 57.2+/- 6.4 and 75.5+/- 6.3 g during 1 and 2 weeks respectively. In the end of experiment (3 weeks after gymnemate withdrawal), the body weight was decreased to no significant difference with normal-control. The total cholesterol was decreased about 1/3, moreover LDL+VLDL (low-density and very-low-density lipoprotein) cholesterol decreased about 1/2. The proportion of HDL (high-density lipoprotein) cholesterol to the total cholesterol was increased. The serum triglyceride was decreased to the 1/4 of OLETF control. The level of serum cholesterol and triglyceride was no significant difference in gymnemate group with normal group. CONCLUSION: Supplementation with gymnemate promoted weight loss by its ability to reduce hyperlipidemia, which was no withdrawal rebound: an important discovery. Supplementation with gymnemate is a novel therapeutic tool for weight management, especially in multifactor syndrome.

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses.

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.

Molecular characterization of parthenogenic Fasciola sp. in Korea on the basis of DNA sequences of ribosomal ITS1 and mitochondrial NDI gene.

Nucleotide sequences of ribosomal internal transcribed spacer (ITS1) and mitochondrial NADH dehydrogenase I (NDI) gene were analyzed to genetically characterize aspermic Fasciola forms in Korea. From the difference in ITS1 sequences, Korean flukes were divided into 3 haplotypes represented by Kor1, Kor2 and Kor1/2, which had nucleotides identical to F. hepatica, F. gigantica and those overlapped between the two species, respectively. NDI sequences also showed that Korean flukes could be classified into 3 distinct haplotypes (Kor1: F. hepatica-type, Kor2a and Kor2b: F. gigantica-type). The sequences of Kor1 and Kor2a were 100% identical to those of the haplotypes Fsp1and Fsp2, respectively, which are major Fasciola forms in Japan. These findings strongly suggest that aspermic Fasciola forms in Korea and Japan originated from same ancestors and have recently spread throughout both countries.

Changes in extracellular neurotransmitters in the cerebrum of familial idiopathic epileptic shetland sheepdogs using an intracerebral microdialysis technique and immunohistochemical study for glutamate metabolism.

Intracerebral microdialysis combined with electroencephalographic recordings was performed on 4 dogs of a familial idiopathic epileptic Shetland sheepdog colony to identify the kinds of neurotransmitters responsible for seizure activity. Immunohistochemistry using glutamate (Glu), glutamate transporter (GLT-1 and GLAST), and glutamine synthetase (GS) antibodies was also carried out on the cerebrum of four familial dogs that died of status epilepticus (SE). High values for extracellular levels of Glu and aspartate (ASP) were detected in association with an increased number of spikes and sharp waves during hyperventilation in 3 of 4 the familial epileptic dogs. The values of other amino acids analyzed were not altered in any of the familial epileptic dogs. Immunohistochemically, Glu-positive granules were occasionally found in the perineuronal spaces of the cerebral cortex in 3 of the familial epileptic dogs that died of SE. Immunostains for GLT-1 antibody predominantly decreased in the cerebral cortex and lateral nucleus of the thalamus in all the dogs that died of SE, whereas there were no differences detected in immunolabellings for GLAST and GS antibodies between familial epileptic dogs and controls. These results suggest that an extracellular release of both Glu and Asp may play an important role in the occurrence of seizure activity in this epileptic colony, and that a decreased expression of astrocytic GLT-1 may be related to development of SE.

Fundamental study of expression-library immunization against Taenia taeniaeformis in rats.

Expression-library immunization has been proposed as an effective means to screen a large number of genes of the pathogen as candidate protective molecules. In this study, we examined the efficacy of expression-library immunization using a T. taeniaeformis rat model system. Total RNAs were isolated from the last 15 segments of adult T. taeniaeformis and poly A RNA was purified. cDNA library was produced using SuperScript Plasmid System, which contains a mammalian expression vector, pCMV*SPORT6. From about 3,500 clones examined, more than 800 clones were found to contain DNA fragments. About 200 clones were sequenced and the homology search was carried out. The blast search revealed that 29% of the expression genes were mitochondrial genes (rRNA; 17%, protein; 12%). Nuclear rRNA genes (10%), nuclear protein (9%) and genes from Escherichia coli were also detected. Forty-two percent of sequences did not show a significant similarity to any genes deposited in the public database. Rats were immunized with expression-library and injected orally with 1,000 T. taeniaeformis eggs. However the protective effect of expression-library vaccine was not confirmed.

Experimental fascioliasis in the rat-like hamster, Tscherskia triton, and other rodent hosts.

Experimental infection with Fasciola hepatica and parthenogenetic Fasciola sp. in laboratory animals have been conducted in rats and rabbits. Inoculation of less than 5 metacercariae into rat-like hamsters, Tscherskia triton, is sufficient to establish Fasciola infections. The prepatent period of F. hepatica and the parthenogenetic Fasciola sp. in T. triton was shorter than that in rats and rabbits, suggesting that T. triton is a suitable experimental model for these flukes. In contrast, F. gigantica infection in T. triton did not yield adult flukes; T. triton, is therefore, considered to be an unsuitable host for F. gigantica. The cotton rat, Sigmodon hispidus, was an unsuitable host for the parthenogenetic Fasciola sp.

Predominant maternal expression of the mouse Atp10c in hippocampus and olfactory bulb.

The human chromosome 15q11-q13 region is one of the most intriguing imprinted domains, and the abnormalities inherited are associated with neurological disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. Recently we have identified a novel maternally expressed gene, ATP10C, that encodes a putative aminophospholipid translocase within this critical region, 200 kb distal to UBE3A in an imprinted domain on human chromosome 15. ATP10C, with UBE3A, displayed tissue-specific imprinting with predominant expression of the maternal allele in the brain. In this study, we demonstrated that the mouse homologue, Atp10c/pfatp, showed tissue-specific maternal expression in the hippocampus and olfactory bulb, which overlapped the region of imprinted Ube3a expression. These data suggest that the imprinted transcript of Atp10c in the specific region of CNS may be associated with neurological disorders including AS and autism.

The rat-like hamster, Tscherskia triton, as a host for the parthenogenetic liver fluke Fasciola sp.

Metacercariae of parthenogenetic Fasciola sp. triploid were inoculated into the rat-like hamster Tscherskia triton. Flukes at various stages of growth were found in the bile ducts of all 8 (50%) animals that survived from 42 to 90 days. The body length to width ratio ranged from 1.8 to 2.9, and flukes with the highest ratio were passed 68 days after inoculation. Our results indicate that T. triton is a suitable host for experimental infection when induced by a small number of metacercariae (less than 5) of Fasciola sp.