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Strongyloides is a genus of parasitic nematodes of vertebrates comprising approximately 50 documented species, each with various host ranges. Among these, three species (S. stercoralis, S. fuelleborni, and S. cebus) are known to infect primate hosts. S. fuelleborni typically infects non-human primates in the Old World. To complement the existing information on the global genetic structure of this species, we conducted a genotyping study of S. fuelleborni samples collected from rhesus macaques in Myanmar, Japanese macaques in Japan, and some zoo-kept primates. This study identified a novel haplotype group in isolates from the Myanmar rhesus macaques. Subsequently, we obtained the complete or nearly complete mitochondrial genome sequences of S. fuelleborni, S. cebus (Strongyloides of New World monkeys), and S. vituli (Strongyloides of cattle). Phylogenetic analysis based on concatenated mitochondrial protein sequences of various Strongyloides species indicated a close relationship between S. fuelleborni, S. vituli and S. papillosus (Strongyloides in sheep and cattle). S. cebus is quite distantly related to both S. fuelleborni and S. stercoralis, which led to the hypothesis that the three primate Strongyloides species evolved independently as parasites of primates.

Schistosomiasis remains a worldwide public health problem, especially in sub-Saharan Africa. The World Health Organization targets the goal for its elimination as a public health problem in the 2030 Neglected Tropical Diseases (NTDs) Roadmap. Concerted action and agile responses to challenges will be necessary to achieve the targets. Better diagnostic tests can accelerate progress towards the elimination by monitoring disease trends and evaluating the effectiveness of interventions; however, current examinations such as Kato-Katz technique are of limited power to detect light-intensity infections. The point-of-care circulating cathodic antigen (POC-CCA) test shows a higher sensitivity compared to the reference standard, Kato-Katz technique, but it still lacks sufficient sensitivity with low infection intensity. In this study, we examined antibody reactions against recombinant protein antigens; Schistosoma mansoni serine protease-inhibitor (SmSerpin) and RP26, by enzyme-linked immunosorbent assay (ELISA) in plasma samples with light-intensity infection. The sensitivity using the cocktail antigen of recombinant SmSerpin and RP26 showed 83.7%. The sensitivity using S. mansoni soluble egg antigen (SmSEA) was 90.8%, but it showed poor specificity (29.7%), while the cocktail antigen presented improved specificity (61.4%). We conclude that antibody detection to the SmSerpin and RP26 protein antigens is effective to detect S. mansoni light-intensity infections. Our study indicates the potential of detecting antibody against recombinant protein antigens to monitor the transmission of schistosomiasis in low endemicity contexts.

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases., Author summary Parasitic helminths have evolved smart strategies to thrive in diverse hosts. For example, parasitic helminths secrete various immunomodulators in the host to establish successful tissue migration to their reproductive niche and chronic parasitism. Identification and functional analyses have revealed these immunomodulators may have potential therapeutic effects in the treatment of immune-related diseases. However, few immunomodulators from parasitic helminths have been identified and analyzed to date. In this study, we determined that venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic nematode Strongyloides venezuelensis, bound to receptor for advanced glycation end products (RAGE), a host pro-inflammatory receptor, which downregulated RAGE-mediated inflammatory responses. S. venezuelensis larvae successfully migrated to their niche owing to the anti-inflammatory functions of venestatin. Venestatin could provide a novel therapeutic target for the treatment of RAGE-mediated inflammatory diseases, such as Alzheimer’s disease, rheumatoid arthritis, asthma, ulcerative colitis, and diabetes.

Backgrounds Primary infection with Toxoplasma gondii during pregnancy can pose serious health problems for the fetus. However, the epidemiological status of toxoplasmosis among reproductive-aged population in Myanmar is largely unknown. Although luciferase immunoprecipitation system (LIPS) assays for serodiagnosis of toxoplasmosis was developed mostly using mouse infection model, had not been tested by using field-derived human samples. Methods A total of 251 serum samples were collected from reproductive-aged women, residing in Shwegyin township, Bago region, Myanmar and analyzed with a commercial ELISA kit, as well as in-house LIPS assays. Results The overall seroprevalence for Toxoplasma gondii infection by the commercial ELISA was 11.5%. No clear risk factor was identified except for being in the younger age group (15–30 years old). Overall, LIPS assays showed low sensitivity when the commercial ELSA was used as a reference test. Conclusion We identified the epidemiological situation of toxoplasmosis in some rural communities in Myanmar. The data obtained here will serve as a primary information for the effort to reduce toxoplasmosis in this region. Although looked promising in the previous experiments with mouse infection model, we found that the reported LIPS procedures need further improvements to increase the sensitivities.

Strongyloides stercoralis is a parasitic nematode and a major pathogen responsible for human strongyloidiasis. The presence of this species in the dog population has led to an interest in studying the phylogenetic relationships among Strongyloides spp. in carnivore hosts. In the present study, Strongyloides spp. from various carnivore hosts (raccoon, Japanese badger, Siberian weasel, raccoon dog, masked palm civet, and domestic cat) were sought. Except for civets, Strongyloides spp. were identified in all host species. Based on 18S rDNA sequences, nine OTUs (operational taxonomy units) were identified. Molecular phylogenetic analyses using 18S[sbnd]28S rDNA and mitochondrial cox1 (cytochrome c oxidase subunit 1) sequences clustered them into two groups. The first group (named the stercoralis/procyonis group) was comprised of six OTUs and occurred in cats, raccoon dogs, raccoons (S. procyonis), Siberian weasels, and Japanese badgers and included S. stercoralis from humans and dogs. The second group (named the planiceps group) was made up of Strongyloides spp. from raccoon dogs (two OTUs) and one OTU from Siberian weasels. Subsequent analysis using almost the full-length nucleotide sequences of protein-coding genes in their mitochondrial genomes placed Strongyloides spp. of cats in a sister taxon position to S. stercoralis, whereas S. procyonis from raccoons was more distantly related to them. The presence of Strongyloides spp. from various carnivore hosts, which are close relatives of S. stercoralis, suggests this group of Strongyloides (the stercoralis/procyonis group) essentially evolved as parasites of carnivores, although more data on Strongyloides spp. from primate hosts are needed.

Hymenolepis diminuta is a parasitic tapeworm of the rat small intestine and is recognized as a useful model for the analysis of cestode-host interactions. In this study, we analyzed factors affecting the biomass of the tapeworm through use of rat strains carrying genetic mutations, namely X-linked severe combined immunodeficiency (xscid; T, B and NK cells deficiency), nude (rnu; T cell deficiency), and mast cell deficient rats. The worm biomass of F344-xscid rats after infection with 5 cysticercoids was much larger than control F344 rats from 3 to 8 weeks. The biomass of F344-rnu rats was also larger than the controls, but was intermediate between F344-xscid and control rats. These observations demonstrated that host immunity can control the maximal tapeworm biomass, i.e., carrying capacity, of the rat small intestine. Both T cell and other immune cells (B and NK cells) have roles in determining the carrying capacity of tapeworms. Total worm biomass and worm numbers in mast cell deficient rats (WsRC-Ws/Ws) were not significantly different from control WsRC-+/+ rats after 3 and 6 weeks of primary infection. Mast cell deficient rats displayed reinfection resistance for worm biomass but not worm expulsion. These findings suggest that the mast cell has a role for controlling the biomass of this tapeworm in reinfection alone, but does not affect the rate of worm expulsion. Overall, our findings indicate that the mast cell is not a major effector cell for the control of the carrying capacity of tapeworms. The identity of the major effector cell remains unknown., ファイル公開：2019-08-01

Strongyloides is a genus of parasitic nematodes of vertebrates that contains over 50 species, each with a variable host range. A recent molecular phylogenetic analysis on this genus showed that Strongyloides spp. from various carnivore hosts form a strongly supported clade together with Strongyloides stercoralis, a major pathogen of humans and dogs (named the “stercoralis/procyonis group”). In the present study, we obtained DNA sequencing data of Strongyloides sp. isolated from an imported meerkat (Suricata suricatta). Based on the phylogenetic analysis, we considered this a new member of the stercoralis/procyonis group. This study represents the first isolation and molecular characterization of a Strongyloides species from hosts belonging to the family Herpestidae (mongooses and meerkat). However, whether the meerkat serves as a natural host of this Strongyloides species remains to be investigated.

The secretory EF-hand Ca++-binding proteins act as calcium signaling molecules for control of cell functions, but those proteins from parasitic helminths are poorly understood. Here, we have identified and characterized an EF-hand Ca++-binding protein from the rodent nematode, Strongyloides venezuelensis, termed 'venestatin', which is highly conserved in Strongyloides spp. Canonical two EF-hand domains and a signal peptide are present in venestatin. A gel mobility shift assay and Ruthenium red staining indicated that the recombinant venestatin possesses binding ability with Ca++ ions. Endogenous venestatin was seemingly localized in the hypodermis and gut of the worms and was found in the excretory-secretory products. Quantitative reverse transcription-PCR data showed that venestatin-specific transcript was upregulated in the parasitic stages of S. venezuelensis, and the upregulation occurred promptly after larval invasion through the host's skin, but not in the case of in vitro incubation. Immunization of mice with recombinant venestatin caused a 55% reduction in larval migration to the lungs, and lung hemorrhaging was mild compared with non-immunized groups, suggesting that anti-venestatin sera may interfere with larval migration from skin to lung. Our results suggest that venestatin is secreted from the hypodermis and gut of S. venezuelensis, and has pivotal roles in larval migration.

The development and application of next-generation sequencing (NGS) have enabled comprehensive analyses of the microbial community through extensive parallel sequencing. Current analyses of the eukaryotic microbial community are primarily based on polymerase chain reaction amplification of 18S rRNA gene (rDNA) fragments. We found that widely-used 18S rDNA primers can amplify numerous stretches of the bacterial 16S rRNA gene, preventing the high-throughput detection of rare eukaryotic species, particularly in bacteria-rich samples such as faecal material. In this study, we employed in silico and NGS-based analyses of faecal samples to evaluated the existing primers targeting eukaryotic 18S and 28S rDNA in terms of avoiding bacterial read contamination and improving taxonomic coverage for eukaryotes, with a particular emphasis on parasite taxa. Our findings revealed that newly selected primer sets could achieve these objectives, representing an alternative strategy for NGS.

Proliferative sparganosis is one of the most bizarre and mysterious parasitic diseases ever described. The causative parasite is Sparganum proliferum, which is a pseudophyllidean cestode distinct from Spirometra tapeworms. Here we overview this rare but fascinating disease with the all original case reports on human patients published in the last 115 years. Proliferative sparganosis is clearly divided into two disease types, cutaneous and internal proliferative sparganosis. Cutaneous type starts with a skin eruption caused by the dermal invasion of a sparganum. Skin lesion progresses to larger areas of the body if left untreated. Various internal organs and body wall can be eventually affected. The clinical symptoms of patients in this group are very similar to each other. Molecular data suggest that cutaneous proliferative sparganosis is caused by S. proliferum of which genetic variation is limited, regardless of the time or localities of the emergence of patients. Internal proliferative sparganosis, on the other hand, is much more heterogeneous. Some cases show aggressive infection in internal organs, while others show only restricted lesions. Some of the cases that had been cited as proliferative sparganosis in the past literature were removed from the list, because they were judged as cyclophyllidean tapeworm infections. DNA sequencing is mandatory for the definite diagnosis of proliferative sparganosis. The Venezuelan strain of S. proliferum is maintained in experimental mice in Japan, which is fully prepared for the experimental study with advanced technologies in modern molecular biology.

Chickens are considered to act as paratenic hosts for agents, Toxocara canis, T. cati and Ascaris suum; which cause ascarid larva migrans syndrome (ascarid LMS) in humans. In addition, they are the definitive host for Ascaridia galli, considered not to be infective for humans. All ascarid parasites can have a high homology of antigenicity, leading to cross-reactivity in serodiagnostic assays. This study was conducted to establish a procedure for the serological detection of those roundworm infections in chickens. Twenty-five male Julia chickens were divided into five groups (n = 5); T. canis-, T. cati-, Ascaris suum- and Ascaridia galli-infected, and an uninfected control group. In Ascaris suum-soluble worm antigen preparation (As-SWAP) ELISA, all infected groups showed an elevation of anti-ascarid antibodies, indicating the usefulness of As-SWAP as a screening antigen for the detection of ascarid infections. For infecting species identification, T. canis-excretory/secretory (Tc-ES) and Ascaris suum-ES (As-ES) antigen ELISA were conducted by serial dilution sera. Toxocara spp.-infected sera showed stronger binding to Tc-ES than As-ES, while Ascaris suum and Ascaridia galli-infected sera bound to As-ES more strongly than Tc-ES. To discriminate between Ascaris suum and Ascaridia galli infection, sera were pre-incubated with Ascaridia galli-SWAP antigen and applied to Tc-ES and As-ES ELISAs. In this pre-adsorbed ES antigen ELISAs, only the Ascaris suum infected group showed positive binding to As-ES, resulting from the adsorption of cross-reactive antibodies in Ascaridia galli-infected sera. Finally, anti-Toxocara specific antibodies were confirmed by Tc-ES western blot (WB). Toxocara spp.-infected sera showed toxocariasis-specific band pattern in Tc-ES WB, while no specific band appeared on any strip incubated with Ascaris suum, Ascaridia galli-infected and uninfected sera. In conclusion, the serodiagnostic assays evaluated in this study are useful for the detection of ascarid infections in chickens.

Traditionally, Strongyloides nematode infecting cattle had been thought to be a single species, S. papillosus. Surprisingly, Eberhardt et al. in 2008 reported two, rather than one Strongyloides species infected cattle, with one being S. papillosus and the other S. vituli. However, there was no subsequent report to support their findings. In July 2018, a case of a sudden death of a calf believed to be due to heavy infection with S. papillosus at a dairy farm in Miyazaki Prefecture, Japan, was reported. One month after the initiation of a deworming program to prevent further sudden deaths, fecal specimens from 24 cattle housed in the same barn were examined. Eight samples were positive for Strongyloides eggs. For species determination, the nucleotide sequences of 18S rDNA (small subunit ribosomal DNA gene), rpl-10 (ribosomal protein L10 gene), and mitochondrial (mt) cox1 (cytochrome c oxidase subunit 1 gene) were obtained. Typing data for all three marker genes indicated the presence of both species, S. papillosus and S. vituli, in the fecal samples. To our knowledge, this study is the first to support the original report by Eberhardt et al. regarding the sympatric existence of S. papillosus and S. vituli in cattle, and to report the presence of S. vituli in Japan. Interestingly, phylogenetic analyses of both rpl-10 and mt cox1 sequences indicated a closer genetic relationship of S. vituli with S. venezuelensis (Strongyloides of rats) than with S. papillosus, shedding light on the speciation history of Strongyloides nematodes.

Paragonimiasis, or lung fluke disease, is a typical food-borne parasitic zoonosis caused by infection with trematodes belonging to the genus Paragonimus. More than 50 species of Paragonimus have been reported throughout the world, of which seven valid species infect humans, an estimated one million people annually worldwide. Among the seven species, P. westermani, P. heterotremus, and P. skrjabini/P. s. miyazakii, distributed in Asia, are the most important species as the cause of paragonimiasis. Humans acquire infection through the ingestion of raw, pickled or undercooked freshwater crustaceans, 2nd intermediate hosts, or consuming raw meat of wild boar or deer, paratenic hosts. Infections often occur clustered in foci where dietary habits allow transmission of the parasites. Paragonimiasis typically causes a subacute to chronic inflammatory disease of the lungs. The symptoms, including chronic cough, chest pain, dyspnea and hemoptysis, mimic those of tuberculosis and lung cancer. Serologic tests are commonly used for the diagnosis of paragonimiasis, and Praziquantel is the treatment of choice. In this review, the current status of Paragonimus and paragonimiasis in Asia is outlined based on the latest information and findings. We also summarize current trends of paragonimiasis in Japan, which is one of the most endemic area of paragonimiasis in the world, for the better understanding and control of paragonimiasis.

© The Author(s)., [Background]: Parasites excrete and secrete a wide range of molecules that act as the primary interface with their hosts and play critical roles in establishing parasitism during different stages of infection. Strongyloides venezuelensis is a gastrointestinal parasite of rats that is widely used as a laboratory model and is known to produce both soluble and insoluble (adhesive) secretions during its parasitic stages. However, little is known about the constituents of these secretions., [Results]: Using mass spectrometry, we identified 436 proteins from the infective third-stage larvae (iL3s) and 196 proteins from the parasitic females of S. venezuelensis. The proteins that were secreted by the iL3s were enriched with peptidase activity, embryo development and the oxidation-reduction process, while those of the parasitic females were associated with glycolysis, DNA binding (histones) and other unknown functions. Trypsin inhibitor-like domain-containing proteins were identified as the main component of the adhesive secretion from parasitic females. An absence of secretion signals in many of the proteins indicated that they are secreted via non-classical secretion pathways., [Conclusions]: We found that S. venezuelensis secretes a wide range of proteins to establish parasitism. This includes proteins that have previously been identified as being involved in parasitism in other helminths as well as proteins that are unique to this species. These findings provide insights into the molecular mechanisms underlying Strongyloides parasitism., This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant numbers 26292178, 16H04722 and 15K14503, and AMED 18fk0108009h0003

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.

Understanding parasite diversity has important implications in several research fields, including ecology, evolutionary biology, and epidemiology. Here, we introduce a novel method to assess the biodiversity of parasites-especially those in the host alimentary tract-using an 18S rDNA-based metagenomic approach. The method is easy and quick compared to conventional methods, and does not require dissections of host bodies or identification skills for various parasite species. The use of a "next generation sequencer" in this method allows us to perform the assessment in a high throughput manner, which will increase our knowledge of parasite diversity.

Ascarid nematodes, Ascaris suum, Toxocara canis and Toxocara cati, are the most important causative species of larva migrans syndrome (LMS) in humans. Although the diagnosis of ascarid LMS is generally based on serological tests, specific serological tests for A. suum infection have not been fully developed. In the present study, the sensitivity and specificity of three A. suum antigen preparations, i.e., the somatic adult worm antigen (As-SWAP), larval excretory-secretory (ES) antigens derived from infective L3 (AsiL3-ES) and larval ES from tissue migratory L3 (AsmL3-ES), were evaluated for the serodiagnosis of A. suum infection in enzyme-linked immunosorbent assay (ELISA). We found that all A. suum antigen preparations showed positive reaction to all sera from A. suum-infected mice, while only AsmL3-ES obtained 100 % detection of anti-A. suum antibodies in human visceral ascarosis patients. Comparing the reactivity of each A. suum antigen, sera from both A. suum-infected mice and human patients bound to AsiL3-ES significantly weaker than As-SWAP and AsmL3-ES. Moreover, the OD450 values of ELISA with the A. suum antigen preparations and T. canis larval ES antigen (TciL3-ES) were compared in order to discriminate between ascarosis and toxocarosis. Linear discriminant analysis showed that diagnosis based on TciL3-ES and AsmL3-ES ELISA gave the most reliable result for the discrimination of infecting species. In conclusion, the application of AsmL3-ES antigen in ELISA can be recommended for the serodiagnosis of A. suum infection in humans.

An 11-year-old boy living in Otsu City, Shiga Prefecture, Kansai Region, Western Honshu, Japan had zoonotic onchocercosis. The patient developed a painful swelling on the little finger of his left hand. The worm detected in the excised mass had external transverse ridges but did not have inner striae in the cuticle. On the basis of the parasite's histopathological characteristics, the causative agent was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species of the filarial parasite was confirmed by sequencing the cox1 gene of the parasite. The Japanese wild boar Sus scrofa leucomystax is a definitive host for O. dewittei japonica, which is then transmitted by blackflies as the vector to humans. The current case described occurred in the Kansai Region, Western Honshu, where such infections were previously not reported.

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100 pg to 100 ag and found to be 10 fg, 1 fg and 100 fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.

Recently, reports of delayed hemolytic anemia after treatment with artemisinin and its derivatives have emerged. Here we report two cases of delayed hemolytic anemia in a patient with severe falciparum malaria after treatment with oral artemether-lumefantrine (AL). The first patient, a 20-year-old Japanese male student, was diagnosed with falciparum malaria and was administered AL. As having a high parasitemia rate (20.6%) was the only severe malaria criterion met in this case and his general condition was stable, we continued with AL treatment. Despite disappearance of malarial parasites after 4 days of AL administration, a persistent fever remained. On days 13 and 16, a diagnosis of hemolytic anemia was made (lactate dehydrogenase [LDH]: 1,466 U/L, hemoglobin [Hb]: 7.2 g/dL). A blood smear at that time revealed no parasites. He recovered naturally from delayed hemolysis. The second patient, a 27-year-old Japanese female student, was diagnosed with falciparum malaria (parasitemia: 4.5%) and treated initially with oral quinine hydrochloride and doxycycline. The following day, parasitemia increased to 7.9% and oral AL was initiated. She was discharged on day 4 after achieving parasite clearance and afebrility. However, on day 5, fever (body temperature > 38°C) recurred, and on day 11, a diagnosis of hemolytic anemia was made (LDH: 712 U/L, Hb: 8.8 g/dL). A follow-up confirmed that her condition improved gradually. AL treatment of severe malaria can cause delayed hemolytic anemia. Patients should be followed up for up to 4 weeks to detect signs of hemolysis and provide appropriate symptomatic treatment.

SUMMARYStrongyloides venezuelensis is a parasitic nematode that infects rodents. Although Strongyloides species described to date are known to exhibit parthenogenetic reproduction in the parasitic stage of their life cycle and sexual reproduction in the free-living stage, we did not observe any free-living males in S. venezuelensis in our strain, suggesting that the nematode is likely to depend on parthenogenetic reproduction. We confirmed by cytological analysis that S. venezuelensis produces eggs by parthenogenesis during the parasitic stage of its life cycle. Phylogenetic analysis using nearly the full length of 18S and D3 region of 28S ribosomal RNA gene suggested that S. venezuelensis is distantly related to another rodent parasite, namely Strongyloides ratti, but more closely related to a ruminant parasite, Strongyloides papillosus. Karyotype analysis revealed S. venezuelensis reproduces with mitotic parthenogenesis, and has the same number of chromosomes as S. papillosus (2n = 4), but differs from S. ratti (2n = 6) in this regard. These results, taken together, suggest that S. venezuelensis evolved its parasitism for rodents independently from S. ratti and, therefore, is likely to have a different reproductive strategy.

Strongyloides venezuelensis is one of some 50 species of genus Strongyloides, obligate gastrointestinal parasites of vertebrates, responsible for strongyloidiasis in humans and other domestic/companion animals. Although S. venezuelensis has been widely used as a model species for studying human/animal strongyloidiasis, the sequence information for this species has been quite limited. To create a more comprehensive catalogue of expressed genes for identification of genes potentially involved in animal parasitism, we conducted a de novo sequencing analysis of the transcriptomes from four developmental stages of S. venezuelensis, using a Roche 454 GS FLX Titanium pyrosequencing platform. A total of 14,573 contigs were produced after de novo assemblies of over 2 million sequencing reads and formed a dataset "Vene454". BLAST homology search of Vene454 against proteome and transcriptome data from other animal-parasitic and non-animal-parasitic nematode species revealed several interesting genes, which may be potentially related to animal parasitism, including nicotinamide phosphoribosyltransferase and ferrochelatase. The Vene454 dataset analysis also enabled us to identify transcripts that are specifically enriched in each developmental stage. This work represents the first large-scale transcriptome analysis of S. venezuelensis and the first study to examine the transcriptome of the lung L3 developmental stage of any Strongyloides species. The results not only will serve as valuable resources for future functional genomics analyses to understand the molecular aspects of animal parasitism, but also will provide essential information for ongoing whole genome sequencing efforts in this species.

Paragonimiasis is a typical food-borne parasitic disease, endemic in most parts of Asia, with sporadic case reports from American and African countries. The major source of infection is undercooked freshwater crab or crayfish, though consumption of wild boar meat is also responsible for the infection in Japan, because wild boar is a paratenic host for Paragonimus westermani. Recently, living juveniles of P. westermani were isolated from muscle of a sika deer, Cervus nippon, in Japan, raising the possibility that venison has been another source of infection. In order to clarify the potential contribution of venison consumption to the occurrence of paragonimiasis, we analysed dietary histories of those paragonimiasis patients in whose diagnoses we were involved between 2001 and 2015. Among 380 patients, freshwater crab had been consumed by 208 patients, wild boar meat by 190, and wild deer meat by 76 patients before the onset of the disease. Overall contribution of wild deer meat was estimated to be 6.8% to 20.0%, although in Oita and Gifu Prefectures, where a substantial proportion of patients had consumed raw venison, the contribution of venison consumption was much higher (27.5 to 62.1% and 42.1 to 78.9% in Oita and Gifu Prefectures, respectively). We demonstrated P. westermani-specific antibodies in the sera of 4 out of 160 sika deer from Gifu Prefecture, strongly suggesting that these deer were infected with P. westermani.

Free-living infective larvae of Strongyloides nematodes fulfill a number of requirements for the successful infection. They need to endure a long wait in harsh environmental conditions, like temperature, salinity, and pH, which might change drastically from time to time. Infective larvae also have to deal with pathogens and potentially hazardous free-living microbes in the environment. In addition, infective larvae must recognize the adequate host properly, and start skin penetration as quickly as possible. All these tasks are essentially important for the survival of Strongyloides nematodes, however, our knowledge is extremely limited in any one of these aspects. In order to understand how Strongyloides infective larvae meet these requirements, we examined transcripts of infective larvae by randomly sequencing cDNA clones constructed from S. venezuelensis infective larvae. After assembling successfully sequenced clones, we obtained 162 unique singletons and contigs, of which 84 had been significantly annotated. Annotated genes included those for respiratory enzymes, heat-shock proteins, neuromuscular proteins, proteases, and immunodominant antigens. Genes for lipase, small heat-shock protein, globin-like protein and cytochrome c oxidase were most abundantly transcribed, though genes of unknown functions were also abundantly transcribed. There were no hits found against NCBI or NEMABASE4 for 37 (22.3%) EST out of the total 162 EST. Although most of the transcripts were not infective larva-specific, the expression of respiration related proteins was most actively transcribed in the infective larva stage. The expression of astacin-like metalloprotease, small heat-shock protein, S. stercoralis L3Nie antigen homologue, and one unannotated and 2 novel genes was highly specific for the infective larva stage.

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P

Eosinophils were examined for the capacity of attacking Strongyloides venezuelensis adult worms in the intestinal mucosa by using interleukin (IL)-5 transgenic mice. In IL-5 transgenic mice, most of the subcutaneously inoculated infective larvae were killed during migration, and only a few worms could reach the small intestine. When the same number of adult worms were surgically implanted in the small intestine of IL-5 transgenic and control mice, fecal egg output as well as the number of adult worms recovered from the intestine was significantly lower in IL-5 transgenic mice. In the intestinal mucosa of IL-5 transgenic mice, large number of eosinophils was present in the lamina propria even before adult worm implantation. The number of eosinophils increased significantly as early as 24 h after implantation and tripled by day 3, whereas mucosal eosinophilia remained low in wild-type mice. Most notably, eosinophils infiltrated into the intestinal epithelium and surrounded adult worms in IL-5 transgenic mice, which was never seen in wild-type control mice. However, IL-5 transgenic mice required the same period as normal mice to completely expel implanted adult worms. The amount of specific IgA as well as total IgA in the stool was high in IL-5 transgenic mice before adult worm implantation, and dropped rapidly after adult worm implantation. The present study suggests that eosinophils are capable of attacking adult nematodes in the intestinal epithelia, probably in conjunction with secretory IgA, although they are not enough for the complete worm expulsion.

Effects of Schistosoma mansoni infection on anti-tumor immunity were examined in CBF1 mice with ultraviolet-induced UVfemale1 fibrosarcoma cells. Although many laboratory established tumor cells had rejection mechanisms independent of CD4(+) T cells, we confirmed that CD4(+) cells had significant roles in rejection of UVfemale1 cells in the syngeneic CBF1 mice. When we prepared two CBF1 mouse groups, S. mansoni-infected and schistosome-free, the former group showed up-regulation of Th2-like response to UVfemale1 cells, whereas the latter group mice showed rather type 1-dominant patterns. Cytotoxic activity against UVfemale1 cells tested in vitro, which was attributed to CD8(+) cells, was significantly weaker in S. mansoni-infected mice compared with infection-free mice. In tumor challenge experiments in vivo, we observed that rapid and complete rejection of UVfemale1 cells required the presence of CD8(+) T cells. Under only CD4-depleted situation, survival of tumor cells in schistosome-free mice was prolonged up to 1 month or more. Under the presence of both CD4(+) and CD8(+) cells, S. mansoni infected mice rejected the challenged UVfemale1 cells as was seen in normal mice. However, when CD8(+) cells were depleted from S. mansoni-infected mice, inoculated UVfemale1 cells grew more rapidly than in infection-free mice. Our results suggest that functionally polarized cytokine patterns in schistosome-infected hosts promote rapid tumor growth.

One of the most common zoonotic helminth infections is caused by species in the genus Toxocara, particularly Toxocara canis and T. cati (Syn. T. mystax). However, their relative contribution to toxocarosis in humans remains largely unknown because causative larvae are seldom recovered and uncertainties regarding the validity of existing serological assays. In this study, we used sera from a pig model experimentally infected with T. canis and T. cati to evaluate whether a Western blot could discriminate between the two species. No proteins were observed that could be used as a diagnostic tool. In addition, a heterogenic protein pattern between individual hosts was found, which was most pronounced in the T. cati-infected pigs. There is therefore an urgent need to optimize and validate current methods or develop new species-specific serological methods in order to implement appropriate control measures.

Background: Zoonotic infections with Onchocerca species are uncommon, and to date only 25 clinical cases have been reported worldwide. In Japan, five previous zoonotic infections were concentrated in Oita, Kyushu (the southern island), with one previous case in Hiroshima in the western part of Honshu (the main island). The causative agent in Japan was identified as Onchocerca dewittei japonica Uni, Bain & Takaoka, 2001 from Japanese wild boars (Sus scrofa leucomystax Temminck, 1842). Here we report two infections caused by a female and male O. dewittei japonica, respectively, among residents of Hiroshima and Shimane Prefectures in the western part of Honshu. Methods: In both cases, nodules were surgically removed. The parasites in nodules were identified on the basis of their histopathological characteristics. Identification was confirmed by sequencing the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from worms in the tissues used in the histological preparations. Results: Case 1 was a 61-year-old woman from Hiroshima Prefecture who complained of a painful subcutaneous nodule on the back of her right hand. The causative agent was identified as a female O. dewittei japonica owing to transverse ridges on the cuticle and molecular analysis. Case 2 was a 78-year-old woman from Shimane Prefecture who had a painful nodule in the left temporal region. Histopathological characteristics and cox1 sequencing of the worm indicated that the causative agent was a male O. dewittei japonica. Conclusions: For Cases 1 and 2, we diagnosed the causative agents as a female and male O. dewittei japonica, respectively. These findings indicate the spread of a zoonosis caused by O. dewittei japonica in the western part of Honshu, where wild boars have recently extended their habitats because of decreased annual snowfall, unused rice fields and a decline in the number of hunters in Japan. The O. dewittei japonica infection rate among wild boars was reported as 78% in Shimane Prefecture, in the western part of Honshu. Therefore, in the near future, zoonotic onchocercosis is likely to occur in Honshu as well as Kyushu, where wild boars, blackfly vectors and humans share the same habitat.

Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity.

A large subunit of calpain, a calcium-activated neutral proteinase, fromSchistosoma japonicumwas cloned and expressed inEscherichia coli. When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed(P +T cells. Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-γ seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung. On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promotingS. japonicuminfection. Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number. Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects. Taken together, these results point toS. japonicumcalpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.

Maruyawa, H., Hatano, H., Kumagai, T., El-Malky, M., Yoshida, A., and Ohta, N. 2000. Strongyloides venezuelensis: Heparin-binding adhesion substances in immunologically damaged adult worms. Experimental Parasitology 95, 170–175. Immunologically damaged Strongyloides venezuelensis adult worms were examined for their mucosal invasion ability and secretion of heparin-binding adhesion substances. S. venezuelensis was expelled from male Wistar rats 4 to 5 weeks after infection. Four-week-old adult worms were smaller and had fewer eggs than 1-week-old adult worms. One-week-old, 4-week-old, and 5-week-old adult worms equally established in the recipient mouse intestine when surgically implanted. Adult worms of 4 and 5 weeks of age secreted adhesion substances as much as 1-week-old adult worms. There was no difference in the heparin-binding activities and the lectin-binding profile of adhesion substances among adult worms of different ages. The rate of secretion of adhesion substances from the mouth was also identical. Heparin-binding activities were detected in crude adult worm proteins; however, proteins of 5-week-old adult worms had weaker heparin-binding activities than those of 1-week-old adult worms. Western blotting revealed that a number of heparin-binding proteins were lost in 5-week-old adult worms. A heparin-binding protein of 42.0 kDa, which was consistently expressed in adult worms, was a possible component of heparin-binding adhesion substances which are secreted from the mouth.

Mice infected with Schistosoma japonicum were resistant to the intestinal nematode, Strongyloides venezuelensis. The numbers of adult S. venezuelensis recovered from mice were significantly decreased when infections were given from 6 weeks after S. japonicum infection. Larval recovery from the lungs showed that significant numbers of subcutaneously inoculated S. venezuelensis larvae were eliminated by 3 days in S. japonicum-infected mice (P

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

Fine binding specificities to Ascaris suum and A. lumbricoides antigens of the sera from patients with probable visceral larva migrans (VLM) due to A. suum infection were examined. Although multiple-dot enzyme-linked immunosorbent assay (ELISA) was found to be useful for the primary screening of patients, identification of the responsible species was sometimes difficult due to extensive cross reactions with other ascarid parasite antigens. Fine resolution to determine the causative pathogen was obtained by a rather classical Ouchterlony's double immunodiffusion test. The difference in the binding of the patients' sera to A. suum and A. lumbricoides antigens was also demonstrated by an inhibition ELISA. The patients' antibodies bound with higher avidity to the A. suum antigen than to the A. lumbricoides and Toxocara canis antigens. Combination of at least two different immunological assay methods is recommended for the diagnosis of VLM due to ascarid parasites.

Background Strongyloides venezuelensis has been used as a tool and model for strongyloidiasis research. Elimination of S. venezuelensis adult worms from mice has been particularly associated with proliferation and activation of intestinal mast cells and eosinophils. To date, the role of B-cells in the protective mechanism against adult Strongyloides infection in experimental animals has not been reported in the literature. Therefore, the present study was carried to investigate the role of B-lymphocytes in immunity against adult S. venezuelensis infection using mice with a targeted deletion of the JH locus. Methods JHD knockout mice with its wild-type Balb/c mice were infected by intra-duodenal implantation of adult S. venezuelensis. Fecal egg count, intestinal worm recovery, mucosal mast cells and eosinophils were counted. Results At day 11 post infection, parasites in wild-type mice stopped egg laying, while in JHD knockout mice parasites continued to excrete eggs until the end of the observation period, day 107. The higher number of parasite eggs expelled in the feces of JHD knockout infected mice was a consequence of higher worm burdens, which established in the small intestine of these animals. On the other hand worm fecundity was comparable in both groups of mice. Both B-cell-deficient mice and wild-type mice, showed an influx of mucosal mast cells and eosinophils. The absolute numbers in JHD knockout mice were lower than those seen in wild-type mice at day 11, but not to a level of significance. JHD knockout mice could not recover from infection despite the recruitment of both types of cells. Conclusion Our findings highlight a role of B cells in mucosal immunity against invasion of adult S. venezuelensis and in its expulsion. Therefore, we conclude that B-cells together with mucosal mast cells and eosinophils, contribute to immunity against adult S. venezuelensis by mechanism(s) to be investigated.

The clinical management of amebiasis is a growing concern, particularly among human immunodeficiency virus (HIV)-infected individuals who are predisposed to severe illness. Treatment with a luminal amebicide is strongly recommended following acute-stage treatment with a nitroimidazole. In 2004, the Japanese Research Group on Chemotherapy of Tropical Diseases introduced paromomycin, which was not nationally licensed, and offered it to a number of patients. From 2004 to 2011, 143 case records of amebiasis (123 with amebic colitis, 16 with amebic liver abscess, and 4 with both) in which patients were treated with paromomycin, mainly 1,500 mg/day for 9 or 10 days following metronidazole treatment, were submitted. Among 123 evaluable cases, 23 (18.7%) experienced possible adverse effects, the most common being diarrhea (17/123, 13.8%) and other gastrointestinal problems that were resolved after the completion or discontinuation of treatment. In addition, single cases of bloody stools associated with Clostridium difficile colitis, skin rash, and the elevation of liver enzymes were also reported, although the causal relationship was not clear. HIV infection did not appear to increase the incidence of adverse drug effects. Each of the 11 asymptomatic or mildly symptomatic amebic colitis cases became negative for stool cysts after paromomycin treatment. Paromomycin was shown to be safe and well tolerated, as well as effective in a special subset of amebic colitis cases.

Heme is an essential molecule for vast majority of organisms serving as a prosthetic group for various hemoproteins. Although most organisms synthesize heme from 5-aminolevulinic acid through a conserved heme biosynthetic pathway composed of seven consecutive enzymatic reactions, nematodes are known to be natural heme auxotrophs. The completely sequenced Caenorhabditis elegans genome, for example, lacks all seven genes for heme biosynthesis. However, genome/transcriptome sequencing of Strongyloides venezuelensis, an important model nematode species for studying human strongyloidiasis, indicated the presence of a gene for ferrochelatase (FeCH), which catalyzes the terminal step of heme biosynthesis, whereas the other six heme biosynthesis genes are apparently missing. Phylogenetic analyses indicated that nematode FeCH genes, including that of S. venezuelensis (SvFeCH) have a fundamentally different evolutionally origin from the FeCH genes of non-nematode metazoa. Although all non-nematode metazoan FeCH genes appear to be inherited vertically from an ancestral opisthokont, nematode FeCH may have been acquired from an alpha-proteobacterium, horizontally. The identified SvFeCH sequence was found to function as FeCH as expected based on both in vitro chelatase assays using recombinant SvFeCH and in vivo complementation experiments using an FeCH-deficient strain of Escherichia coli. Messenger RNA expression levels during the S. venezuelensis lifecycle were examined by real-time RT-PCR. SvFeCH mRNA was expressed at all the stages examined with a marked reduction at the infective third-stage larvae. Our study demonstrates the presence of a bacteria-like FeCH gene in the S. venezuelensis genome. It appeared that S. venezuelensis and some other animal parasitic nematodes reacquired the once-lost FeCH gene. Although the underlying evolutionary pressures that necessitated this reacquisition remain to be investigated, it is interesting that the presence of FeCH genes in the absence of other heme biosynthesis genes has been reported only for animal pathogens, and this finding may be related to nutritional availability in animal hosts.

Kinetic changes of eosinophil chemotactic factor (ECF) production from granulomas, splenic T-cells or mast cells were examined with reference to granuloma formation around newly deposited single eggs in Schistosoma japonicum -infected mice. The peri-ovular granulomas began to appear at around 5 weeks post-infection (p.i.). Their size reached a peak at 6 weeks and then decreased gradually. Up to 8 weeks p.i., eosinophils were the predominant cell type in the granulomas. ECF-release from isolated granulomas paralleled the size of granulomas. Circulating ECF-A, which was assumed to be derived from mast cells, was also detected 6 weeks afterwards in parallel with the level of specific IgE antibody level against egg antigens in the serum. The circulating ECF-A peaked at 8 weeks and decreased after 10 weeks. Spleen cells began to produce ECF specific to bone-marrow eosinophils began at 5 weeks p.i., reached a peak at 6 weeks and then decreased rapidly. On the other hand, the production of ECF specific to eosinophils obtained from the peritoneal cavity began at 6 weeks and decreased rapidly thereafter. These results suggest that various kinds of host-derived ECFs seem to contribute, in one way or an other, to the accumulation of eosinophils in and around granulomatons lesions. The possible role of these ECFs in eosinophil mobilization from the site of production to the inflamed site is discussed.

Ascarid larva migrans syndrome (ascarid LMS) is caused by ascarid roundworms including Ascaris suum. In East Asia, ascarid LMS has been considered as a food-borne disease in adulthood who has the dietary habitat of consuming raw or lightly cooked meat and organ meats. To evaluate the potential risk of A. suum infection from these foods, Ascaris specific real-time PCR was developed. The assay could constantly detect A. suum DNA up to 10 fg. Its specificity was confirmed by non-amplification with Toxocara canis and Toxocara cati DNA. A. suum DNA could be amplified from not only a single larva but also mouse liver spiked with a larva. Moreover, the assay could detect A. suum DNA in experimentally infected mouse liver, and showed higher sensitivity than a conventional digestion method. This real-time PCR assay would be useful for detecting the A. suum larval contamination in meat and organ meats. Practical applications It has been considered that one of the most important risk factors for ascarid LMS in humans is the consumption of raw or undercooked meat or organ meats of domestic animals infected with Ascaris suum. Thus, we developed the novel real-time PCR with the high sensitivity and specificity in order to specifically detect A. suum DNA from animal tissues. This assay can be applied to the meat inspection procedure for the identification of parasite larval contamination that may adversely impact on public health as well as on animal health and welfare.

Malaria remains an important health risk among travelers to tropical/subtropical regions. However, in Japan, only 2 antimalarials are licensed for clinical use - oral quinine and mefloquine. The Research Group on Chemotherapy of Tropical Diseases introduced atovaquone-proguanil in 1999, and reported on its excellent antimalarial efficacy and safety for treating non-immune patients with uncomplicated Plasmodium falciparum malaria (20 adult and 3 pediatric cases) in 2006. In the present study, additional cases of malaria were analyzed to confirm the efficacy and safety of this antimalarial drug. Fourteen adult and 2 pediatric cases of P. falciparum malaria and 13 adult cases and 1 pediatric case of P. vivax/ovale malaria were successfully treated with atovaquone-proguanil, including 3 P. falciparum cases in which the antecedent treatment failed. Two patients with P. vivax malaria were treated twice due to primaquine treatment failure as opposed to atovaquone-proguanil treatment failure. Except for 1 patient with P. falciparum malaria who developed a moderate liver function disturbance, no significant adverse effects were observed. Despite the intrinsic limitations of this study, which was not a formal clinical trial, the data showed that atovaquone-proguanil was an effective and well-tolerated therapeutic option; licensure of this drug in Japan could greatly contribute to individually appropriate treatment options.

We report the case of a 62-year-old man who developed eosinophilic pneumonia due to visceral larva migrans (VLM) that was possibly caused by Ascaris suum. The patient, a resident of the middle Kyushu area who was found of eating raw porcine liver, complained of dry cough without dyspnea. The chest radiography showed a migration of infiltrative shadow. Transbronchial lung biopsy of the right middle lobe revealed massive infiltration of eosinophils. The multi-dot enzyme-linked immunosorbent assay (ELISA) and microtiter plate ELISA showed positive results for A. suum; therefore, the patient was diagnosed with VLM caused by A. suum. The patient was administered albendazole (600 mg/day) for 28 days; he recovered successfully with no adverse effects except mild liver dysfunction. Several cases of VLM caused by A. suum have been reported in Japan, with a majority of the cases being reported in Kyushu. Careful history taking of the patient's area of residence and dietary habit is essential for the diagnosis of this parasitic disease with underestimated prevalence.

C-type lectins (CTLs) are a group of proteins which bind to carbohydrate epitopes in the presence of Ca(2+), which have been described in a wide range of species. In this study, a cDNA sequence coding a putative CTL has been identified from the cDNA library constructed from the pig round worm Ascaris suum lung L3 (LL3) larvae, which was designated as A. suum C-type lectin-1 (As-CTL-1). The 510 nucleotide open reading frame of As-CTL-1 cDNA encoded the predicted 169 amino acid protein including a putative signal peptide of 23 residues and C-type lectin/C-type lectin-like domain (CLECT) at residue 26 to 167. As-CTL-1 was most similar to Toxocara canis C-type lectin-1 and 4 (Tc-CTL-1 and 4), and highly homologous to namatode CTLs and mammalian CTLs as well, such as human C-type lectin domain family 4 member G (CLECG4). In addition, As-CTL-1 was strongly expressed in tissue migrating LL3 and the L4 larvae, which were developmental larvae stages within the mammalian host. These results suggest that A. suum larvae might utilize As-CTL-1 to avoid pathogen recognition mechanisms in mammalian hosts due to it is similarity to host immune cell receptors.

A female of Onchocerca sp. was found to be the probable causative agent of a subcutaneous nodule in the left knee of a 70-year-old man in a rural area of Hiroshima Prefecture, Honshu, the main island of Japan. We compared the characteristics of the agent with the features of the four previously suspected species found in cattle and horses in various parts of the world, as well as O. lupi and O. jakutensis that were suspected or proved, respectively, in zoonotic cases in Europe. In addition, the morphologic characteristics of this parasite were compared with those of the four Onchocerca species found in wild animals in Japan. Based on such characteristics as the large triangle ridges, the considerable distance between any two adjacent ridges, and the absence of inner cuticular striae in the longitudinal sections, we found the causative agent in the present case to be identical to the female of Onchocerca dewittei japonica. All five previous cases of zoonotic onchocerciasis in Japan had been found in Oita, Kyushu, the main southern island. This human case caused by O. dewittei japonica suggests that zoonotic onchocerciasis is liable to occur in rural areas in Japan where wild boar, Simulium vectors, and humans overlap.

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.

Infective larvae of an intestinal nematode,Strongyloides venezuelensis, enter rodent hosts percutaneously, and migrate through connective tissues and lungs. Then they arrive at the small intestine, where they reach maturity. It is not known howS. venezuelensislarvae develop during tissue migration. Here we demonstrate that tissue invasion ability ofS. venezuelensislarvae changes drastically during tissue migration, and that the changes are associated with stage-specific protein expression. Infective larvae, connective tissue larvae, lung larvae, and mucosal larvae were used to infect mice by various infection methods, including percutaneous, subcutaneous, oral, and intraduodenal inoculation. Among different migration stages, only infective larvae penetrated mouse skin. Larvae, once inside the host, quickly lost skin penetration ability, which was associated with the disappearance of an infective larva-specific metalloprotease. Migrating larvae had connective tissue migration ability until in the lungs, where larvae became able to settle down in the intestinal mucosa. Lung larvae and mucosal larvae were capable of producing and secreting adhesion molecules.

The therapeutic effects of artesunate against experimental Schistosoma mansoni infection in mice were analyzed. Previous studies showed that artesunate is highly effective against S. japonicum infection, but the action of this drug against S. mansoni remained uncovered. The present study examines the optical conditions for artesunate against S. mansoni and evaluates the effects of inhibiting the sexual maturation of adult worms. Mice infected with S. mansoni were orally administered with artesunate according to different schedules. Four consecutive administrations of 300 mg/kg of artesunate at 2-week intervals conferred almost total protection without the development of pathological lesions in the liver. The significant reduction in the number of eggs produced by surviving worms and the status of egg maturation suggested that artesunate inhibits sexual maturation. Electron microscopy revealed that artesunate caused morphological damage, especially on the worm tegument. Artesunate was also very effective in iron-deficient mice. Furthermore, the efficacy of artesunate was equal to or better than that of artemether against S. japonicum infection. Considering that artemether is more toxic, artesunate is currently one of the most efficient drugs against immature S. mansoni.

The parasitic female of Strongyloides venezuelensis keeps invading the epithelial layer of the host intestinal mucosa. Upon invasion, it adheres to the surface of the intestinal epithelial cells with adhesion molecules secreted from the mouth. It has been demonstrated that S. venezuelensis are expelled from the intestine because mucosal mast cells inhibit the attachment of adult worms to the mucosal surface. In the present study, we generated specific antibodies against secreted adhesion molecules to investigate their function in vivo, because these molecules have been demonstrated only in vitro in spite of the importance in the infection processes. A mouse monoclonal antibody specific to S. venezuelensis adhesion molecules inhibited the attachment of adult worms to plastic dishes and the binding of adhesion molecules to rat intestinal epithelial cells. Immunohistochemical study revealed that adhesion molecules were produced by oesophageal glands and were continuously secreted in vivo to line the wall of the tunnels formed by adult worms in the intestinal mucosa. Our findings indicate that adhesion molecules play essential roles in the infection processes of S. venezuelensis in the host intestine.

In order to study the mucosal invasion of a rodent intestinal nematode in bird intestine, chicks were infected with the intestinal nematode of rodents, Strongyloides venezuelensis, by subcutaneous larva inoculation and adult worm implantation. No evidence was obtained for larvae reaching the lungs or the intestine after infective larva inoculation. Adult worms implanted in the small intestine invaded the mucosa and remained there at least for 24 h, whereas those implanted in the caecum were trapped by mucus, and did not invade the mucosa. Mucosal invasion of adult worms in the small intestine was confirmed by histological examination. The number of adult worms in the intestinal mucosal tissue dropped rapidly within the first 24 h, which was associated with infiltrating granulocytes around the worms. The present study suggests that S. venezuelensis adult worms are able to invade the intestinal tissue of chicks, which do not belong to the vertebrate class of its normal definitive host, but that they are eliminated rapidly by mucosal defense system of the bird.