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PubMed2006

Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Animals↗
PubMed2006

Natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens (Acari: Ixodidae).

The present paper reports the occurrence of natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens. Engorged females of ticks, collected from a naturally B. caballi-infected horse, were incubated at 27 degrees C and relative humidity over 83%. After a 6-day incubation period, Giemsa-stained smears prepared from hemolymph were examined microscopically under oil immersion. B. caballi infected ticks were dissected and samples of midgut tissue were examined by transmission electron microscopy, through which free sporokinetes were seen in the cytoplasm of gut epithelial cells. In addition, Encephalitozoon-like microsporidia were observed inside the parasitophorous vacuoles in the same cell in which sporokinetes of B. caballi were found and also in some neighbour cells. They presented different morphological stages, suggesting a sequential phases of development.

Animals↗
PubMed1993

Pathologic features and immunofluorescent antibody demonstration of ocular microsporidiosis (Encephalitozoon hellem) in seven patients with acquired immunodeficiency syndrome.

We studied the clinicopathologic features of seven patients with acquired immunodeficiency syndrome (AIDS) and ocular microsporidiosis. All patients had decreased levels of CD4-positive cells (mean, 26/ml3) and ocular symptoms; five had bilateral punctate epithelial keratopathy, one had intermittent red eyes with conjunctivitis, and one had red eyes only. Light and electron microscopy of corneal and conjunctival biopsy and cytologic specimens and intact globes disclosed microsporidia belonging to the genus Encephalitozoon. Because E. cuniculi and E. hellem, the two species of the Encephalitozoon genus, are morphologically identical, an immunofluorescent antibody technique was used for species identification. In all seven patients, the agent was identified as E. hellem. Pathologic examination of globes obtained after autopsy disclosed E. hellem infection to be restricted to the corneal and conjunctival epithelium. We studied methods for the routine diagnosis of ocular microsporidiosis in patients with AIDS, including the role of immunofluorescent antibody staining.

AIDS-Related Opportunistic Infections↗
PubMed2001

Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.

ATP-Binding Cassette Transporters↗
PubMed1997

Pulmonary microsporidiosis due to Encephalitozoon hellem in a patient with AIDS.

The microsporidian Encephalitozoon hellem is being reported with increasing frequency in HIV-positive subjects, as an agent of disseminated microsporidiosis without involving the gastrointestinal tract. We describe a case of pulmonary microsporidiosis in a 27-year-old Italian man with AIDS who developed fever, cough, and dyspnea. A chest X-ray showed multiple bilateral pulmonary opacities and mediastinal lymph-node enlargement. Stained smears of bronchoalveolar lavage sediment showed oval structures consistent with microsporidian spores. Viral, bacterial and fungal cultures were repeatedly negative, whereas microsporidia were successfully cultured in human and bovine fibroblast cell lines. Analysis of electron micrographs indicated that the isolate belonged to the genus Encephalitozoon. Based on further immunological, biochemical and molecular studies it was characterized as E. hellem. Even though a temporary improvement with albendazole therapy was noticed, the patient deteriorated clinically and died of severe respiratory distress.

AIDS-Related Opportunistic Infections↗
PubMed2002

First isolation and characterisation of Encephalitozoon cuniculi from a free-ranging rat (Rattus norvegicus).

The microsporidian species Encephalitozoon cuniculi can infect a wide variety of mammals including man. It is a common parasite in rabbits and several sporadic infections in laboratory rats have been described. Based on molecular data three E. cuniculi strains have been identified. Here we describe the first in vitro propagation of E. cuniculi, which was isolated from a free-ranging rat (Rattus norvegicus). The rat was one of three seropositive animals among 23 rats captured in the city of Zurich. The new isolate was further characterised as strain II ("mouse"-strain) based on the rDNA internal transcribed spacer sequence. Western blot analysis of this isolate revealed slight differences to other available strain II isolates originating from laboratory mice and farmed blue foxes. The new isolate caused disseminated infection in liver and lung upon oral inoculation of Brown Norway (BN) rats and was transmitted to sentinel rats. This rat-adapted isolate will be valuable to study the pathogenesis of Encephalitozoon infections in the rat model.

Animals↗
PubMed2003

Overt fatal and chronic subclinical Encephalitozoon cuniculi microsporidiosis in a colony of captive emperor tamarins (Saguinus imperator).

The course of an infection with the microsporidian Encephalitozoon cuniculi in a colony of captive emperor tamarins (Saguinus imperator) is described. In two litters, the infection was associated with overt disease and death of all infants. Immunohistochemistry for E. cuniculi showed generalized infections, and histopathologic evaluation revealed systemic vasculitis and disseminated mixed inflammatory cell infiltration with and without necrosis in several organs. Serologically, some of the juvenile animals presented with high titres for Encephalitozoon, while the adults had low titres. The E. cuniculi "dog strain" was identified by molecular means for the first time in Europe. The origin of the infection appeared to be a pair of breeding adults that originated from the US. Our data suggest that the organism persisted over years in the colony, and that subclinically infected animals most likely were involved in perpetuating the infection. Efforts should be made to ascertain if this microorganism is present in other captive populations of this endangered monkey species and to prevent its further spreading.

Animals↗
PubMed2001

Resistance to Encephalitozoon intestinalis is associated with interferon-gamma and interleukin-2 cytokines in infected mice.

The understanding of the immunopathology of infections caused by microsporidia has pinpointed the importance of T cell-mediated immunity. The immunopathology caused by the interesting protozoan parasite Encephalitozoon intestinalis, a microsporidium pathogenic in man, is not clearly understood. In this study, we demonstrate that a specific cellular immune response is implicated in the control of microsporidiosis infection in mice. Interferon (IFN)-gamma receptor knockout mice (IFN-gamma R(o/o)) developed a chronic infection with E. intestinalis, whereas a transient infection developed in wild-type mice. Encephalitozoon intestinalis proteins induced proliferation of murine spleen and mesenteric lymph node cells collected from infected mice. The host response to microsporidia infection was regulated by a specific pattern of cytokine protection. Spleen cells derived from resistant 129 Sv/Ev mice inoculated with E. intestinalis secreted significant levels of gamma-interferon and interleukin-2 but cells from highly susceptible IFN-gamma R knockout mice secreted high levels of interleukin-4 (mostly between 2 and 4 weeks post infection). This is the first report in which a specific cellular immune response against E. intestinalis infection is presented.

Animals↗
PubMed1998

Asymptomatic respiratory tract microsporidiosis due to Encephalitozoon hellem in three patients with AIDS.

Microsporidia of the genera Enterocytozoon and Encephalitozoon have been identified as frequent causes of intestinal and disseminated infections, respectively, in patients with AIDS. Even though most subjects infected with these protozoa develop overt disease, simple colonization without illness may occur, as we observed in three severely immunosuppressed patients with AIDS. The parasites, recognized in and isolated from bronchoalveolar lavage sediment specimens, were characterized as Encephalitozoon hellem. Colonization of the bronchial tree was temporary, and treatment with albendazole was not needed to clear the infection.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Encephalitozoon (Septata) intestinalis: cytologic, histologic, and electron microscopic features of a systemic intestinal pathogen.

Encephalitozoon (Septata) intestinalis affects AIDS patients with CD4 counts <100/microL, causing intestinal and disseminated disease. It must be distinguished from the more common intestinal microsporidian, Enterocytozoon bieneusi, and from other microsporidia of extraintestinal tissues, such as Encephalitozoon hellem and E cuniculi, because clinical manifestations and treatment differ. In this report, the authors describe the diagnostic features of E intestinalis and illustrate all stages of its life cycle as exemplified by a case studied in detail. Spores can be detected by light microscopy in feces, urine, or nasal secretions, but not identified to species. A presumptive tissue diagnosis of E intestinalis can be made if 20 to 50 organisms 1.2-2.5 microm in diameter are seen within vacuoles in enterocytes. The diagnosis is substantiated if organisms also are present in stromal cells. On electron microscopy, the septate parasitophorous vacuole is pathognomonic. E bieneusi occurs only in intestinal and biliary epithelial cells, and never within a vacuole. E hellem and E cuniculi, which are not intestinal pathogens, may cause systemic infection but develop in a nonseptate vacuole.

AIDS-Related Opportunistic Infections↗
PubMed1998

Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core.

Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.

Animals↗
PubMed2003

Disseminated encephalitozoon cuniculi infection in a Mexican kidney transplant recipient.

BACKGROUND: No cases of Encephalitozoon cuniculi infection have been reported in transplant patients. METHODS: A 42-year-old man received a renal transplant 8 months earlier because of terminal glomerulonephritis and was admitted with cough, fever, diarrhea, abdominal pain, and colon wall thickening. While under rapamycin (2 g/day), cyclosporine A (4.4 mg/kg/day), and prednisone (100 mg/day) therapy, he developed Banff grade IB graft rejection and was treated with methylprednisolone (1 g/day) for 3 days and oral prednisone (60 mg/d). RESULTS: Microbiologic studies were inconclusive, and biopsy specimens of ileum, colon, liver, and the grafted kidney revealed numerous gram-positive microsporidia spores. Parasitophorous vacuoles containing various developing stages of Encephalitozoon were seen. Immunofluorescence studies identified the etiologic agent as E. cuniculi. Albendazole therapy resulted in clinical improvement but no eradication after 10 months of follow-up. CONCLUSIONS: This report describes what is, to the authors' knowledge, the first case of disseminated E. cuniculi infection in a kidney transplant human immunodeficiency virus-negative patient from Mexico.

Adult↗
PubMedDate not supplied

Encephalitozoon cuniculi isolated from the urine of an AIDS patient, which differs from canine and murine isolates.

A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymorphic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi. However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi, with a band at 42-45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.

AIDS-Related Opportunistic Infections↗
PubMedDate not supplied

Calcium and hydrogen ion concentrations in the parasitophorous vacuoles of epithelial cells infected with the microsporidian Encephalitozoon hellem.

Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.

Animals↗
PubMedDate not supplied

Encephalitozoon-like organisms in patients with alveolar hydatid disease: cell culture, ultrastructure, histoimmunochemical localization and seroprevalence.

We found Encephalitozoon-like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis. The organisms developed in the same fashion as an Encephalitozoon cuniculi. The spores that developed in parasitophorous vacuoles were 2.0-2.6 x 1.1-1.5 microns; each contained a single nucleus and 4-5 polar tubule coils, closely resembling E. cuniculi in its ultrastructure. Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients. We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05-1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml. Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ. The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD). Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test. The Percoll-separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting. Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals.

Adolescent↗
PubMedDate not supplied

Nucleotide sequences of DNA fragments of Encephalitozoon cuniculi amplified by polymerase chain reaction with primers regarded as specific for Echinococcus.

Encephalitozoon-like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi, which was shown to have En. cuniculi-specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus, were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus-specific primer sets could amplify not only Ec. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus-specific primers-amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis.

Animals↗
PubMedDate not supplied

Fractionation of sporogonial stages of the microsporidian Encephalitozoon cuniculi by Percoll gradients.

Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK-13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll. Transmission electron microscopy and SDS-polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.

Animals↗
PubMedDate not supplied

Fluorescent in situ detection of Encephalitozoon hellem spores with a 6-carboxyfluorescein-labeled ribosomal RNA-targeted oligonucleotide probe.

A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonucleotide probe, HEL878F, designed to be complementary to the nucleic acid sequence 878-896, a highly variable segment of the 16S ribosomal RNA of E. hellem spores. The specificity of this probe for its ribosomal RNA target site was confirmed using RNA degradation, ribosomal RNA target site competition, and nucleotide base mismatch control probe assays. Furthermore, the specificity of the HEL878F oligonucleotide probe for E. hellem spores was established when it was evaluated on spores from all three species of the genus Encephalitozoon that had been seeded in reagent water and environmental water concentrates. The specificity of the HEL878F oligonucleotide probe was further corroborated when tested on algae, bacteria, and protozoa commonly found in environmental water. The study demonstrates the applicability of a fluorescent in situ hybridization assay using a species-specific fluorescent-labeled oligonucleotide probe for the detection of E. hellem spores in water samples.

Animals↗