Ultrasensitive malaria detection system for Anopheles mosquito field surveillance using droplet digital PCR.

Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques. A total of 152 mosquitoes from six anopheline species were collected and first analyzed by a standard 18S rRNA nested-PCR analysis for malaria parasite which yielded negative results in all collected mosquitoes. Later, ddPCR was used and could detect simian malaria parasite DNA, i.e. Plasmodium cynomolgi, in 25 collected mosquitoes. And this is the first report of simian malaria parasite DNA detection in Anopheles sawadwongporni. This finding proves that ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles mosquitoes and can expand our understanding of the zoonotic potential of malaria transmission between monkeys and humans.

Histone H3.3 variant plays a critical role on zygote-to-oocyst development in malaria parasites.

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes.

Prevalence and metronidazole resistance of Trichomonas vaginalis among Japanese women in 2021.

Objectives: Trichomonas vaginalis is the most prevalent sexually transmitted parasite worldwide. However, no surveillance system exists to monitor T. vaginalis cases and drug resistance in Japan. Methods: Cervical cytology vaginal swabs were collected from women with and without suspected symptoms of T. vaginalis infection; these swabs were used for the detection of T. vaginalis, human papillomavirus (HPV), and Candida albicans using specific polymerase chain reaction. Clinical isolates of T. vaginalis were subjected to metronidazole susceptibility tests using the previously reported minimal lethal concentration (MLC) and newly established half-maximal inhibitory concentration (IC50) values. Results: The prevalence of T. vaginalis in the study population was 4.2% (5/119; 95% confidence interval [Cl], 1.5-9.7). Additionally, asymptomatic infection constituted 60% (3/5) of all cases of T. vaginalis infection. All T. vaginalis-positive patients were coinfected with HPV but not C. albicans. Five clinical T. vaginalis isolates showed metronidazole susceptibility, which was evaluated using MLC values. The quantitative IC50 values revealed that two of these clinical isolates exhibited a decreased metronidazole susceptibility. Conclusion: This is the first study to demonstrate the prevalence of T. vaginalis in Japanese women. The IC50 values of metronidazole against T. vaginalis enabled the precise and quantitative evaluation of metronidazole-susceptible T. vaginalis.

Human Gut Microbiota and Its Metabolites Impact Immune Responses in COVID-19 and Its Complications.

BACKGROUND & AIMS: We investigate interrelationships between gut microbes, metabolites, and cytokines that characterize COVID-19 and its complications, and we validate the results with follow-up, the Japanese 4D (Disease, Drug, Diet, Daily Life) microbiome cohort, and non-Japanese data sets. METHODS: We performed shotgun metagenomic sequencing and metabolomics on stools and cytokine measurements on plasma from 112 hospitalized patients with SARS-CoV-2 infection and 112 non-COVID-19 control individuals matched by important confounders. RESULTS: Multiple correlations were found between COVID-19-related microbes (eg, oral microbes and short-chain fatty acid producers) and gut metabolites (eg, branched-chain and aromatic amino acids, short-chain fatty acids, carbohydrates, neurotransmitters, and vitamin B6). Both were also linked to inflammatory cytokine dynamics (eg, interferon γ, interferon λ3, interleukin 6, CXCL-9, and CXCL-10). Such interrelationships were detected highly in severe disease and pneumonia; moderately in the high D-dimer level, kidney dysfunction, and liver dysfunction groups; but rarely in the diarrhea group. We confirmed concordances of altered metabolites (eg, branched-chain amino acids, spermidine, putrescine, and vitamin B6) in COVID-19 with their corresponding microbial functional genes. Results in microbial and metabolomic alterations with severe disease from the cross-sectional data set were partly concordant with those from the follow-up data set. Microbial signatures for COVID-19 were distinct from diabetes, inflammatory bowel disease, and proton-pump inhibitors but overlapping for rheumatoid arthritis. Random forest classifier models using microbiomes can highly predict COVID-19 and severe disease. The microbial signatures for COVID-19 showed moderate concordance between Hong Kong and Japan. CONCLUSIONS: Multiomics analysis revealed multiple gut microbe-metabolite-cytokine interrelationships in COVID-19 and COVID-19related complications but few in gastrointestinal complications, suggesting microbiota-mediated immune responses distinct between the organ sites. Our results underscore the existence of a gut-lung axis in COVID-19.

Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.

Live Vaccination with Blood-Stage Plasmodium yoelii 17XNL Prevents the Development of Experimental Cerebral Malaria.

In our work, we aim to develop a malaria vaccine with cross-strain (-species) protection. C57BL/6 mice infected with the P. berghei ANKA strain (PbA) develop experimental cerebral malaria (ECM). In contrast, ECM development is inhibited in infected mice depleted of T cells. The clinical applications of immune-cell depletion are limited due to the benefits of host defense against infectious diseases. Therefore, in the present study we attempted to develop a new method for preventing ECM without immune cell depletion. We demonstrated that mice inoculated with a heterologous live-vaccine of P. yoelii 17XNL were able to prevent both ECM and lung pathology and survived longer than control mice when challenged with PbA. Live vaccination protected blood-organ barriers from PbA infection. Meanwhile, live vaccination conferred sterile protection against homologous challenge with the P. yoelii 17XL virulent strain for the long-term. Analysis of the immune response induced by live vaccination showed that cross-reactive antibodies against PbA antigens were generated. IL-10, which has an immunosuppressive effect, was strongly induced in mice challenged with PbA, unlike the pro-inflammatory cytokine IFNγ. These results suggest that the protective effect of heterologous live vaccination against ECM development results from IL-10-mediated host protection.

Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator.

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

Long-term acrylamide exposure exacerbates brain and lung pathology in a mouse malaria model.

The consumption of dietary acrylamide (ACR), a carcinogen, results in the dysfunction of various organs and the immune system. However, the impact of ACR exposure on the progression of infectious diseases is unknown. This study investigated the effect of ACR on the progression of malaria infection using a mouse model of malaria. C57BL/6 mice were continuously treated with ACR at a dose of 20 mg/kg bodyweight/day for six weeks (long-term exposure) or phosphate-buffered saline (PBS). Next, the mice were infected with the rodent malaria parasite, Plasmodium berghei NK65 (PbNK). Parasitemia and survival rate were analyzed in the different treatment groups. Magnetic resonance imaging (MRI) and histopathological analyses were performed to evaluate the effect of ACR exposure on the morphology of various organs. Long-term ACR exposure exacerbated PbNK-induced multiorgan dysfunction. MRI and histopathological analysis revealed signs of encephalomeningitis and acute respiratory distress syndrome in the PbNK-infected long-term ACR exposure mice, which decreased the survival rate of mice, but not in the PbNK-infected long-term PBS exposure group. These findings enhance our understanding of the impact of ACR on the progression of infectious diseases, such as malaria.

Author Correction: CD8+ T cell activation by murine erythroblasts infected with malaria parasites.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blood-cerebrospinal fluid barrier: another site disrupted during experimental cerebral malaria caused by Plasmodium berghei ANKA.

Cerebral malaria is one of the most severe pathologies of malaria; it induces neuro-cognitive sequelae and has a high mortality rate. Although many factors involved in the development of cerebral malaria have been discovered, its pathogenic mechanisms are still not completely understood. Most studies on cerebral malaria have focused on the blood-brain barrier, despite the importance of the blood-cerebrospinal fluid barrier, which protects the brain from peripheral inflammation. Consequently, the pathological role of the blood-cerebrospinal fluid barrier in cerebral malaria is currently unknown. To examine the status of the blood-cerebrospinal fluid barrier in cerebral malaria and malaria without this pathology (non-cerebral malaria), we developed a new method for evaluating the permeabilization of the blood-cerebrospinal fluid barrier during cerebral malaria in mice, using Evans blue dye and a software-assisted image analysis. Using C57BL/6J (B6) mice infected with Plasmodium berghei ANKA strain as an experimental cerebral malaria model and B6 mice infected with P. berghei NK65 strain or Plasmodium yoelii as non-cerebral malaria models, we revealed that the permeability of the blood-cerebrospinal fluid barrier increased during experimental cerebral malaria but not during non-cerebral malaria. We observed haemorrhaging in the cerebral ventricles and hemozoin-like structures in the choroid plexus, which is a key component of the blood-cerebrospinal fluid barrier, in cerebral malaria mice. Taken together, this evidence indicates that the blood-cerebrospinal fluid barrier is disrupted in experimental cerebral malaria, whereas it remains intact in non-cerebral malaria. We also found that P. berghei ANKA parasites and CD8+ T cells are involved in the blood-cerebrospinal fluid barrier disruption in experimental cerebral malaria. An understanding of the mechanisms underlying cerebral malaria might help in the development of effective strategies to prevent and manage cerebral malaria in humans.

Potential and Limitations of Cross-Protective Vaccine against Malaria by Blood-Stage Naturally Attenuated Parasite.

Human malaria vaccine trials have revealed vaccine efficacy but improvement is still needed. In this study, we aimed to re-evaluate vaccination with blood-stage naturally attenuated parasites, as a whole-organism vaccine model against cross-strain and cross-species malaria, to establish a better vaccination strategy. C57BL/6 mice controlled blood-stage Plasmodium yoelii 17XNL (PyNL) within 1 month of infection, while mice with a variety of immunodeficiencies demonstrated different susceptibilities to PyNL, including succumbing to hyperparasitemia. However, after recovery, survivors had complete protection against a challenge with the lethal strain PyL. Unlike cross-strain protection, PyNL-recovered mice failed to induce sterile immunity against Plasmodium berghei ANKA, although prolonged survival was observed in some vaccinated mice. Splenomegaly is a typical characteristic of malaria; the splenic structure became reorganized to prioritize extra-medullary hematopoiesis and to eliminate parasites. We also found that the peritoneal lymph node was enlarged, containing activated/memory phenotype cells that did not confer protection against PyL challenge. Hemozoins remained in the spleen several months after PyNL infection. Generation of an attenuated human blood-stage parasite expressing proteins from multiple species of malaria would greatly improve anti-malaria vaccination.

CD8+ regulatory T cells are critical in prevention of autoimmune-mediated diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic ß-cells are destroyed. Intestinal helminths can cause asymptomatic chronic and immunosuppressive infections and suppress disease in rodent models of T1D. However, the underlying regulatory mechanisms for this protection are unclear. Here, we report that CD8+ regulatory T (Treg) cells prevent the onset of streptozotocin -induced diabetes by a rodent intestinal nematode. Trehalose derived from nematodes affects the intestinal microbiota and increases the abundance of Ruminococcus spp., resulting in the induction of CD8+ Treg cells. Furthermore, trehalose has therapeutic effects on both streptozotocin-induced diabetes and in the NOD mouse model of T1D. In addition, compared with healthy volunteers, patients with T1D have fewer CD8+ Treg cells, and the abundance of intestinal Ruminococcus positively correlates with the number of CD8+ Treg cells in humans.

IL-33 is essential to prevent high-fat diet-induced obesity in mice infected with an intestinal helminth.

Intestinal helminthes induce immunosuppressive responses as well as type 2 immunity. Their suppressive properties are intended to regulate inflammatory diseases such as allergies and autoimmune diseases. This study evaluated whether helminthic infections suppress obesity, a chronic inflammatory state, using an intestinal nematode, Heligmosomoides polygyrus (Hp). Infection with Hp at the same time as feeding a high-fat diet (HFD) prevented weight gain, dyslipidaemia and glucose intolerance observed in uninfected obese mice. Immunologically, Hp infection skewed M1 macrophages to M2 macrophages and induced type 2 innate lymphoid cells in adipose tissues. The expression of interleukin (IL)-33, a potent initiator of type 2 responses, was also increased in association with uncoupled protein 1 (UCP1). To further investigate the anti-obesity effects of IL-33 in mice infected with Hp, IL-33-deficient mice were fed the HFD and infected with Hp. These mutant mice rapidly gained weight compared with wild-type mice, indicating the anti-obesity effect of IL-33. In the absence of IL-33, the rapid increase in weight was not prevented, and type 2 responses and UCP1 expression were not observed even during Hp infection. These results suggested that the suppression of obesity by Hp is dependent on IL-33.

Suppression of systemic lupus erythematosus in NZBWF1 mice infected with Hymenolepis microstoma.

Intestinal helminths induce immune suppressive responses thought to regulate inflammatory diseases including allergies and autoimmune diseases. This study was designed to evaluate whether helminthic infections suppress the natural development of systemic lupus erythematosus (SLE) in NZBWF1 mice. Infection of NZBWF1 SLE-prone mice with two nematodes failed to establish long-lasting settlement. However, the Hymenolepis microstoma (Hm) rodent tapeworm successfully established long-term parasitization of NZBWF1 mice and was used to evaluate the suppressive effects of helminth infection. Ten-month-old NZBWF1 mice developed symptoms including autoantibody generation, proteinuria, glomerular histopathology, and splenomegaly, but mice infected with Hm at 2 months of age did not show any clinical signs. Furthermore, infection with Hm reduced lymphocyte activation and increased regulatory T cells in the spleen and mesenteric lymph nodes. These results indicate that infection with Hm protects NZBWF1 mice from naturally developing SLE and suggest that pathological immunity is attenuated, presumably because of the induction of regulatory T cells.

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development.

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.

Fluctuations of Spleen Cytokine and Blood Lactate, Importance of Cellular Immunity in Host Defense Against Blood Stage Malaria Plasmodium yoelii.

Our previous studies of protective immunity and pathology against blood stage malaria parasites have shown that not only CD4+ T cells, but also CD8+ T cells and macrophages, are important for host defense against blood stage malaria infection. Furthermore, we found that Plasmodium yoelii 17XNL (PyNL) parasitizes erythroblasts, the red blood cell (RBC) precursor cells, which then express MHC class I molecules. In the present study, we analyzed spleen cytokine production. In CD8+ T cell-depleted mice, IL-10 production in early stage infection was increased over two-fold relative to infected control animals and IL-10+ CD3- cells were increased, whereas IFN-γ production in the late stage of infection was decreased. At day 16 after PyNL infection, CD8+ T cells produced more IFN-γ than CD4+ T cells. We evaluated the involvement of the immunoproteasome in induction of immune CD8+ T cells, and the role of Fas in protection against PyNL both of which are downstream of IFN-γ. In cell transfer experiments, at least the single molecules LMP7, LMP2, and PA28 are not essential for CD8+ T cell induction. The Fas mutant LPR mouse was weaker in resistance to PyNL infection than WT mice, and 20% of the animals died. LPR-derived parasitized erythroid cells exhibited less externalization of phosphatidylserine (PS), and phagocytosis by macrophages was impaired. Furthermore, we tried to identify the cause of death in malaria infection. Blood lactate concentration was increased in the CD8+ T cell-depleted PyNL-infected group at day 19 (around peak parasitemia) to similar levels as day 7 after infection with a lethal strain of Py. When we injected mice with lactate at day 4 and 6 of PyNL infection, all mice died at day 8 despite demonstrating low parasitemia, suggesting that hyperlactatemia is one of the causes of death in CD8+ T cell-depleted PyNL-infected mice. We conclude that CD8+ T cells might control cytokine production to some extent and regulate hyperparasitemia and hyperlactatemia in protection against blood stage malaria parasites.

Suppression of Obesity by an Intestinal Helminth through Interactions with Intestinal Microbiota.

Obesity is increasingly causing lifestyle diseases in developed countries where helminthic infections are rarely seen. Here, we investigated whether an intestinal nematode, Heligmosomoides polygyrus, has a suppressive role in diet-induced obesity in mice. Infection with H. polygyrus suppressed weight gain in obese mice, which was associated with increased uncoupling protein 1 (UCP1) expression in adipocytes and a higher serum norepinephrine (NE) concentration. Blocking interactions of NE with its receptor on adipocytes resulted in the failure to prevent weight gain and to enhance UCP1 expression in obese mice infected with H. polygyrus, indicating that NE is responsible for the protective effects of H. polygyrus on obesity. In addition to sympathetic nerve-derived NE, the intestinal microbiota was involved in the increase in NE. Infection with H. polygyrus altered the composition of intestinal bacteria, and antibiotic treatment to reduce intestinal bacteria reversed the higher NE concentration, UCP1 expression, and prevention of the weight gain observed after H. polygyrus infection. Our data indicate that H. polygyrus exerts suppressive roles on obesity through modulation of microbiota that produce NE.

Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate.

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.

Intestinal Inflammation-Mediated Clearance of Amebic Parasites Is Dependent on IFN-γ.

Intestinal amebiasis is a major cause of diarrhea. However, research on host-amebae interactions has been hampered owing to a lack of appropriate animal models. Recently, a mouse model of intestinal amebiasis was established, and using it, we reported that Entamoeba moshkovskii colonized the intestine in a manner similar to that of the pathogenic Entamoeba histolytica In this study, we evaluated the protective mechanisms present against amebae using this model. CBA/J mice infected with E. histolytica had a persistent infection without apparent symptoms. In contrast, E. moshkovskii-infected mice rapidly expelled the ameba, which was associated with weight loss, diarrhea, and intestinal damage characterized by apoptosis of intestinal epithelial cells (IECs). Expression of NKG2D on intestinal intraepithelial lymphocytes (IELs) and IFN-γ-producing cells in Peyer's patches were significantly induced after infection with E. moshkovskii but not with E. histolytica IFN-γ-deficient mice infected with E. moshkovskii showed no obvious symptoms. Notably, none of these mice expelled E. moshkovskii, indicating that IFN-γ is responsible not only for intestinal symptoms but also for the expulsion of amebae. Furthermore, apoptosis of IECs and expression of NKG2D on IELs observed in E. moshkovskii-infected mice did not occur in the absence of IFN-γ. In vivo blocking of NKG2D in mice infected with E. moshkovskii enabled ameba to survive longer and remarkably reduced apoptotic IECs. Our results clearly demonstrate a novel protective mechanism exerted by IFN-γ against intestinal amebae, including induction of cytotoxicity of IELs toward IECs.

A possible origin population of pathogenic intestinal nematodes, Strongyloides stercoralis, unveiled by molecular phylogeny.

Humans and dogs are the two major hosts of Strongyloides stercoralis, an intestinal parasitic nematode. To better understand the phylogenetic relationships among S. stercoralis isolates infecting humans and dogs and to assess the zoonotic potential of this parasite, we analyzed mitochondrial Cox1, nuclear 18S rDNA, 28S rDNA, and a major sperm protein domain-containing protein genes. Overall, our analyses indicated the presence of two distinct lineages of S. stercoralis (referred to as type A and type B). While type A parasites were isolated both from humans and dogs in different countries, type B parasites were found exclusively in dogs, indicating that the type B has not adapted to infect humans. These epidemiological data, together with the close phylogenetic relationship of S. stercoralis with S. procyonis, a Strongyloides parasite of raccoons, possibly indicates that S. stercoralis originally evolved as a canid parasite, and later spread into humans. The inability to infect humans might be an ancestral character of this species and the type B might be surmised to be an origin population from which human-infecting strains are derived.