Ancylostoma ceylanicum, novel etiological agent for traveler's diarrhea-report of four Japanese patients who returned from Southeast Asia and Papua New Guinea.

BACKGROUND: Countries in the Southeast Asia region have a high prevalence of soil-transmitted helminth, such as roundworm, whipworm, and hookworms [Ancylostoma duodenale, Necator americanus, Ancylostoma ceylanicum]. Recent molecular-based surveys have revealed that A. ceylanicum, a zoonotic hookworm, is likely the second most prevalent hookworm species infecting humans in that part of the world, while others have noted that this infection is an emerging public health risk not only for indigenous people but also for visitors from other countries. CASE PRESENTATION: We recently encountered four cases of A. ceylanicum infection in Japanese individuals who returned from Southeast Asia and Papua New Guinea. Case 1 was a 25-year-old male who stayed in a rainforest in Malaysia for 4 weeks, where he developed abdominal pain and diarrhea in the third week. Eleven adult worms (five males, six females) were expelled after treatment with pyrantel pamoate and identified as A. ceylanicum based on morphological characteristics and DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Case 2 was a 26-year-old male who spent 2 years as an overseas cooperation volunteer for agriculture in Papua New Guinea. He did not note any symptoms at that time, though eggs were detected in feces samples at a medical check-up examination after returning. Although collection of adult worms was unsuccessful, DNA analysis of the eggs for cox1 and the ribosomal internal transcribed spacer (ITS)-1 and ITS-2 genes demonstrated that they were A. ceylanicum. Case 3 was a 47-year-old male who spent 1 month in a rural village in Lao People's Democratic Republic and began suffering from watery diarrhea from the third week. A total of nine adult worms (three males, six females) were collected by endoscopic procedures and following treatment with pyrantel pamoate. Morphological examination and molecular analyses of the cox1 gene showed that they were A. ceylanicum. Case 4 was a 27-year-old male who participated in group travel to India for 5 days. Three weeks after returning, he developed abdominal pain and diarrhea. Hookworm eggs were found in feces samples and developed into larvae in culture, which were identified as A. ceylanicum based on molecular analysis of the cox1 gene. Eosinophilia was observed in all of the cases prior to treatment. CONCLUSIONS: A. ceylanicum should be recognized as an important etiologic pathogen of hookworm diseases in travelers to countries in the Southeast Asia and West Pacific Ocean regions.

Functional Connectivity Analysis of NIRS Data under Rubber Hand Illusion to Find a Biomarker of Sense of Ownership.

The self-identification, which is called sense of ownership, has been researched through methodology of rubber hand illusion (RHI) because of its simple setup. Although studies with neuroimaging technique, such as fMRI, revealed that several brain areas are associated with the sense of ownership, near-infrared spectroscopy (NIRS) has not yet been utilized. Here we introduced an automated setup to induce RHI, measured the brain activity during the RHI with NIRS, and analyzed the functional connectivity so as to understand dynamical brain relationship regarding the sense of ownership. The connectivity was evaluated by multivariate Granger causality. In this experiment, the peaks of oxy-Hb on right frontal and right motor related areas during the illusion were significantly higher compared with those during the nonillusion. Furthermore, by analyzing the NIRS recordings, we found a reliable connectivity from the frontal to the motor related areas during the illusion. This finding suggests that frontal cortex and motor related areas communicate with each other when the sense of ownership is induced. The result suggests that the sense of ownership is related to neural mechanism underlying human motor control, and it would be determining whether motor learning (i.e., neural plasticity) will occur. Thus RHI with the functional connectivity analysis will become an appropriate biomarker for neurorehabilitation.

Functional connectivity analysis of brain hemodynamics during rubber hand illusion.

Embodied cognition has been eagerly studied in the recent neuroscience research field. In particular, hand ownership has been investigated through the rubber hand illusion (RHI). Most of the research measured the brain activities during the RHI by using EEG, fMRI, etc., however, near-infrared spectroscopy (NIRS) has not yet been utilized. Here we attempt to measure the brain activities during the RHI task with NIRS, and analyze the functional connectivity so as to understand the relationship between NIRS features and the state of embodied cognition. For the purpose, we developed a visuo-tactile stimulator in the study. As a result, we found that the subjects felt illusory experience showed significant peaks of oxy-Hb in both prefrontal and premotor cortices during RHI. Furthermore, we confirmed a reliable causality connection from right prefrontal to right premotor cortex. This result suggests that the RHI is associated with the neural circuits underlying motor control. Therefore, we considered that the RHI with the functional connectivity analysis will become an appropriate model investigating a biomarker for neurorehabilitation, and the diagnosis of the mental disorders.

In vivo imaging of intestinal helminths by capsule endoscopy.

This review examines the use of digestive endoscopy to visualize intestinal helminths. The infections caused by these parasites are responsible for high levels of morbidity and mortality. These helminths can be visualized using gastroduodenal endoscopy, endoscopic retrograde cholangiopancreatography, and colonoscopy. Endoscopic examination of the small bowel is limited by its considerable length and its distance from the mouth and anus. Since capsule endoscopy (CE) was first reported in 2000, it has been established as a noninvasive modality for the investigation of the gastrointestinal tract. CE is used as a first-line tool for imaging various small-bowel diseases, mainly obscure gastrointestinal bleeding and Crohn's disease. Since the Food and Drug Administration (FDA) approved CE in 2001, the indications for its use have expanded widely. For example, CE can be used to visualize the in vivo kinetics of intestinal helminths. If the current trends in technological development continue, CE will become more widely used to facilitate the diagnosis and treatment of helminth infections in the near future.

Mucosal trapping and degradation of Nippostrongylus brasiliensis occurs in the absence of STAT6.

Hookworms represent a major infectious burden globally, especially in developing countries. The murine hookworm Nippostrongylus brasiliensis is normally cleared in a manner dependent on IL-13, IL4-R and STAT6 signalling. Here we have used STAT6-deficient animals to model a non-resistant population and describe 2 novel STAT6-independent processes for the clearance of N. brasiliensis. During primary infection STAT6-/- animals are able to clear gut-dwelling N. brasiliensis by a mechanism involving the trapping and degradation of worms in the gut mucosa. Here, a previously undescribed STAT6-independent up-regulation of Relm-ß was observed which correlated with the mucosal trapping and degradation of worms. Previous studies have indicated that during secondary infection STAT6 deficient animals fail to expel adult worms and remain susceptible to re-infection and long-term colonization of the gut. We report here that an initial partially protective response occurs early upon re-infection in the absence of STAT6, and that a late-phase protective secondary response arises in the gut of STAT6-deficient mice leading to the clearance of the majority of N. brasiliensis, through their trapping and death in the mucosal layer of the lower region of the small intestine. These findings show that there are a number of redundant effector pathways which act to reduce worm burden in the gut which can be activated by mechanisms that do not work through the dominant STAT6 signalling pathway and may be useful as targets for future vaccination strategies against resistant hookworm strains.

Mucin-Related Molecular Responses of Bronchial Epithelial Cells in Rats Infected with the Nematode Nippostrongylus brasiliensis.

Although mucins are essential for the protection of internal epithelial surfaces, molecular responses involving mucin production and secretion in response to various infectious agents in the airway have not been fully elucidated. The present study analysed airway goblet cell mucins in rats infected with the nematode Nippostrongylus brasiliensis, which migrates to the lungs shortly after infection. Goblet cell hyperplasia occurred in the bronchial epithelium 3-10 days after infection. The high iron diamine-alcian blue staining combined with neuraminidase treatment showed that sialomucin is the major mucin in hyperplastic goblet cells. Immunohistochemical studies demonstrated that goblet cell mucins were immunoreactive with both the major airway mucin core peptide, Muc5AC, and the major intestinal mucin core peptide Muc2. Reverse transcription real-time PCR studies demonstrated upregulation of gene transcription levels of Muc5AC, Muc2, the sialyltransferase St3gal4, and the resistin-like molecule beta (Retnlb) in the lungs. These results showed that nematode infection induces airway epithelial responses characterised by the production of sialomucin with Muc5AC and Muc2 core peptides. These mucins, as well as Retnlb, might have important roles in the protection of mucosa from migrating nematodes in the airway.

Molecular identification of Oesophagostomum and Trichuris eggs isolated from wild Japanese macaques.

Natural habitat fragmentation and reducing habitat quality have resulted in an increased appearance of Japanese macaques, Macaca fuscata (Gray, 1870), in suburban areas in Japan. To investigate the risk of zoonotic infections, a coprological survey of helminth eggs passed by wild Japanese macaques was carried out in 2009 and 2010 in Shiga Prefecture, Japan. Microscopic examination found helminth eggs in high prevalence, and nucleotide sequencing of DNA extracted from the eggs identified Oesophagostomum cf. aculeatum and Trichuris trichiura. A fecal culture also detected infective larvae of Strongyloides fuelleborni. These zoonotic nematodes pose a potential health issue to local people in areas frequented by Japanese macaques.

Validity of the bear tapeworm Diphyllobothrium ursi (Cestoda: Diphyllobothriidae) based on morphological and molecular markers.

The bear tapeworm Diphyllobothrium ursi is described based upon the morphology of adult tapeworms recovered from the brown bear (Ursus arctos middendorffi) and larval plerocercoids found in sockeye salmon (Oncorhynchus nerka) from Kodiak Island in Alaska in 1952. However, in 1987 D. ursi was synonymized with Diphyllobothrium dendriticum, and the taxonomic relationship between both species has not subsequently been revised. In this study mitochondrial cytochrome c oxidase subunit 1 gene (cox1) sequences of holotype and paratype D. ursi specimens that had been preserved in a formalin-acetic acid-alcohol solution since the time the species was initially described approximately 60 yr ago were analyzed. Molecular and phylogenetic analysis of the cox1 sequences revealed that D. ursi is more closely related to D. dendriticum than it is to Diphyllobothrium nihonkaiense and Diphyllobothrium latum. In addition to molecular evidence, differences in the life cycle and ecology of the larval plerocercoids between D. ursi and D. dendriticum also suggest that D. ursi is a distinct species, separate from D. dendriticum and D. nihonkaiense, and also possibly from D. latum .

Molecular and histological identification of the acanthocephalan Bolbosoma cf. capitatum from the human small intestine.

Acanthocephalans of the genus Bolbosoma are intestinal parasites of marine mammals with a lifecycle similar to that of anisakid nematodes. Several cases of Bolbosoma infection in humans have been reported, but no species identification has been made. Here, we report a case of Bolbosoma infection, in which the worm was found in histological sections of the partially resected small intestine of a Japanese man. Morphological features of the worm reconstructed from serial sectioning indicated that the worm was most likely to be a sexually immature female of Bolbosoma capitatum. DNA extraction from paraffin-embedded sections and ITS1-5.8S rRNA-ITS2 sequencing showed that this species formed a monophyletic group with Bolbosoma nipponicum, and was clearly distinguishable from Corynosoma spp. or Polymorphus spp. These results may provide a reference for identifying and characterizing unknown acanthocephalans found in histological sections.

Anisakis simplex sensu stricto and Anisakis pegreffii: biological characteristics and pathogenetic potential in human anisakiasis.

Anisakiasis is one of the most common fishborne helminthic diseases in Japan, which is contracted by ingesting the larvae of the nematode Anisakis spp. carried by marine fish. Anisakis simplex sensu stricto (s.s.) and A. pegreffii are the dominant species in fish caught offshore Japan. The present study aimed to identify the anisakid species infecting Japanese patients and determine whether there is any difference in the pathogenetic potential of A. simplex (s.s.) and A. pegreffii. In total, 41 and 301 Anisakis larvae were isolated from Japanese patients and chub mackerel (Scomber japonicus), respectively; these were subjected to molecular identification using polymerase chain reaction targeted at a ribosomal DNA internal transcribed spacer region. Chub mackerel larvae were further examined for survival in artificial gastric juice (pH 1.8) for 7 days and for invasiveness on 0.75% solid agar over a 24-h interval. All clinical isolates, including those of asymptomatic, acute, and chronic infections as well as those from the stomach, small intestine, colon, and stool, were identified as A. simplex (s.s.). Chub mackerel harbored A. simplex (s.s.) and A. pegreffii larvae, together with a few larvae of other anisakid species. A. simplex (s.s.) larvae from chub mackerel tolerated the artificial gastric juice better than A. pegreffii, with 50% mortality in 2.6 and 1.4 days, respectively. In addition, A. simplex (s.s.) penetrated the agar at significantly higher rates than A. pegreffii. These results show that A. simplex (s.s.) larvae have the potential to survive acidic gastric juice to some extent and penetrate the stomach, small intestine, or colon in infected humans.

Hsp12.6 expression is inducible by host immunity in adult worms of the parasitic nematode Nippostrongylus brasiliensis.

Heat shock proteins (Hsp) are a family of stress-inducible molecular chaperones that play multiple roles in a wide variety of animals. However, the roles of Hsps in parasitic nematodes remain largely unknown. To elucidate the roles of Hsps in the survival and longevity of nematodes, particularly at the 2 most critical stages in their lifecycle, the infective-L3 stage and adult stage, which is subjected to host-derived immunological pressure, we examined the temporal gene transcription patterns of Hsp12.6, Hsp20, Hsp70, and Hsp90 throughout the developmental course of the nematode Nippostrongylus brasiliensis by reverse transcriptase real-time PCR. Nb-Hsp70 and Nb-Hsp90 expression were observed throughout the nematode's lifecycle, while the expression of Nb-Hsp20 was restricted to adults. Interestingly, Nb-Hsp12.6 showed a biphasic temporal expression pattern; i.e., it was expressed in infective-L3 larvae and in adults during worm expulsion from immunocompetent rats. However, the activation of Nb-Hsp12.6 in adult worms was aborted when they infected permissive athymic-rnu/rnu rats and was only marginal when they infected mast-cell-deficient Ws/Ws rats, which exhibited a low response of rat mast cell protease (RMCP) II and resistin-like molecule (Relm)-ß expression compared to those observed in immunocompetent rats. Moreover, the activation of Nb-Hsp12.6 was reversed when adult worms were transplanted into the naive rat intestine. These features of Nb-Hsp12.6, the expression of which is not only stage-specific in infective-L3, but is also inducible by mucosal immunity in adults, have implications for the survival strategies of parasitic nematodes in deleterious environmental conditions both outside and inside the host.

Long fish tapeworm in the intestine: an in situ observation by capsule endoscopy.

We present the case of a patient who was referred to our hospital after she reported having passed a long, whitish object per rectum. Accordingly, capsule endoscopy was performed using the PillCam(®) SB video capsule. A tapeworm of the species Diphyllobothrium nihonkaiense was detected; it appeared to be freely floating and unfolded in the jejunum and sometimes tangled or irregularly folded in the ileum. The stretching of the strobila by strong peristalsis in the ileum may have resulted in the separation of the caudal portion of the strobila, which descended into the colon and was eventually passed per rectum.

Full-length sequence of mouse acupuncture-induced 1-L (aig1l) gene including its transcriptional start site.

We have been investigating the molecular efficacy of electroacupuncture (EA), which is one type of acupuncture therapy. In our previous molecular biological study of acupuncture, we found an EA-induced gene, named acupuncture-induced 1-L (Aig1l), in mouse skeletal muscle. The aims of this study consisted of identification of the full-length cDNA sequence of Aig1l including the transcriptional start site, determination of the tissue distribution of Aig1l and analysis of the effect of EA on Aig1l gene expression. We determined the complete cDNA sequence including the transcriptional start site via cDNA cloning with the cap site hunting method. We then analyzed the tissue distribution of Aig1l by means of northern blot analysis and real-time quantitative polymerase chain reaction. We used the semiquantitative reverse transcriptase-polymerase chain reaction to examine the effect of EA on Aig1l gene expression. Our results showed that the complete cDNA sequence of Aig1l was 6073 bp long, and the putative protein consisted of 962 amino acids. All seven tissues that we analyzed expressed the Aig1l gene. In skeletal muscle, EA induced expression of the Aig1l gene, with high expression observed after 3 hours of EA. Our findings thus suggest that the Aig1l gene may play a key role in the molecular mechanisms of EA efficacy.

Ascariasis in Japan: is pig-derived Ascaris infecting humans?

Human ascariasis is caused by infection with the common roundworm Ascaris lumbricoides, although the pig roundworm Ascaris suum has also been reported to infect humans and develop into the adult stage. To elucidate whether pig-derived Ascaris infects humans in Japan, 9 Ascaris isolates obtained from Japanese patients and a further 9 Ascaris isolates of pig origin were analyzed to determine their internal transcribed spacer-1 sequences. Six of the 9 clinical isolates showed the Ascaris genotype which predominantly infects humans in endemic countries, while the other 3 clinical isolates and 9 pig-derived isolates showed the genotype predominant in pigs worldwide. These results suggest that at least some cases of human ascariasis in Japan are a result of infection with pig-derived Ascaris.

Diphyllobothriasis associated with eating raw pacific salmon.

The incidence of human infection with the broad tapeworm Diphyllobothrium nihonkaiense has been increasing in urban areas of Japan and in European countries. D. nihonkaiense is morphologically similar to but genetically distinct from D. latum and exploits anadromous wild Pacific salmon as its second intermediate host. Clinical signs in humans include diarrhea and discharge of the strobila, which can be as long as 12 m. The natural life history and the geographic range of the tapeworm remain to be elucidated, but recent studies have indicated that the brown bear in the northern territories of the Pacific coast region is its natural final host. A recent surge of clinical cases highlights a change in the epidemiologic trend of this tapeworm disease from one of rural populations to a disease of urban populations worldwide who eat seafood as part of a healthy diet.

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

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

Mitochondrial DNA divergence in populations of the tapeworm Diphyllobothrium nihonkaiense and its phylogenetic relationship with Diphyllobothrium klebanovskii.

Diphyllobothrium nihonkaiense [Y. Yamane, H. Kamo, G. Bylund, J.P. Wilkgren. Diphyllobothrium nihonkaiense sp. nov (Cestoda: Diphyllobothriidae)- revised identification of Japanese broad tapeworm. Shimane J Med Sci 1986;10:29-48.] and Diphyllobothrium klebanovskii [I.V. Muratov, P.S. Posokhov. Causative agent of human diphyllobothriasis - Diphyllobothrium klebanovskii sp. n. Parazitologiia. 1988;22:165-170.] are two major species of human diphyllobothriasis in Japan and Far East Russia, respectively, but their taxonomical relationship remains unclear. In this study, we analysed the DNA sequences of 16 clinical isolates of D. nihonkaiense from Japanese people, 3 isolates of D. klebanovskii from a bear in Kamchatka, and 4 clinical isolates of D. klebanovskii from native Udygeyci people in Russia, as well as 4 plerocercoids from Oncorhynchus spp. 18S rDNA and internal transcribed spacer 1 (ITS1) sequences from D. nihonkaiense and D. klebanovskii showed a high level of similarity, indicating synonymy of the two species. Analyses of mitochondrial DNA (mtDNA) sequence polymorphisms in the cox1 and nad3 genes of D. nihonkaiense (D. klebanovskii) revealed two deeply divergent lineages, A and B, with genetic distances (Kimura-2 parameter) of 0.018-0.022. Furthermore, the distinct monophyletic groupings of cox1 haplotypes corresponded to the distinct monophyletic groupings of nad3 haplotypes. The two lineages were neither distinguished by morphological features nor defined by the localities of the samples. These results suggest that the two morphologically cryptic lineages have diverged and coexisted over a long period of time.