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PubMed1993

Pathology of symptomatic microsporidial (Encephalitozoon hellem) bronchiolitis in the acquired immunodeficiency syndrome: a new respiratory pathogen diagnosed from lung biopsy, bronchoalveolar lavage, sputum, and tissue culture.

Encephalitozoon hellem is a recently described microsporidian associated with an expanding spectrum of clinical presentations in patients with the acquired immunodeficiency syndrome (AIDS). It is morphologically similar to Encephalitozoon cuniculi, a microsporidian infection of mammals and some avians, and their differentiation rests on biochemical and antigenic analyses. This report describes a patient previously diagnosed with keratoconjunctivitis due to E hellem who subsequently was found to have respiratory tract microsporidiosis by sputum cytology. He subsequently developed pulmonary symptoms and a left lower lobe interstitial infiltrate. A bronchoalveolar lavage and transbronchial biopsy revealed microsporidial bronchiolitis, and the etiologic agent was identified as E hellem using an immunofluorescent antibody technique. Lavage fluid was successfully cultured in monkey kidney cells, and cultivated E hellem organisms were studied using immunohistochemistry as well as scanning and transmission electron microscopy. The pathologic features of this newly described cause of protozoal bronchiolitis, the role of immunofluorescent antibody examination and in vitro tissue culture for species-specific diagnosis, and the significance of microsporidial pulmonary infections in AIDS patients are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Isolation and characterization of a new human microsporidian, Encephalitozoon hellem (n. sp.), from three AIDS patients with keratoconjunctivitis.

A new human microsporidian was isolated from cultures of Madin-Darby canine kidney cells incubated with conjunctival scrapings or corneal tissues from three AIDS patients with keratoconjunctivitis. The three isolates were morphologically similar to Encephalitozoon cuniculi. The spores averaged 1 x 1.5-2.0 microns, had six to eight polar filament coils, displayed monokaryotic nuclei, and possessed relatively thick endospores with irregularly shaped exospores. Organisms developed within a parasitophorous vacuole. By SDS-PAGE analysis, the three isolates appeared to be identical but were different from E. cuniculi. Identical banding patterns on Western blots of the three isolates were expressed by each patient's serum. By Western immunoblotting, murine antisera to E. cuniculi reacted to several antigens of the new AIDS-related microsporidian, whereas murine antisera bound weakly to Nosema corneum. The name Encephalitozoon hellem (n. sp.) is proposed to identify this new human microsporidian.

Acquired Immunodeficiency Syndrome↗
PubMed1975

Fine structure of Encephalitozoon cuniculi from rabbits, mice and hamsters.

Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is approximately 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.

Animals↗
PubMed1978

Some factors influencing the in vitro infectivity and replication of Encephalitozoon cuniculi.

Rabbit Encephalitozoon cuniculi were propagated in vitro using rabbit choroid plexus (RCP) cells. The organisms reached maximum titer and numbers by 15 days. The source and in vitro passage level of RCP cells moderately influenced the sensitivity of the cells to infection. Cells less than 1 week old were significantly less sensitive than older cells. A moderate increase in infectivity for RCP cells was demonstrated with increasing organism passage level in vitro. Rabbit E. cuniculi were not affected by penicillin-streptomycin or gentamicin in the culture medium. The organism survived more than 9 days in buffer at 37 C and least 24 days at 4 and 20 C. Storage at -70 C or in liquid nitrogen was successful for at least 6 months. Encephalitozoon cuniculi survived 60 but not 120 min at 56 C. They were killed after 10 min of autoclaving and by 2% (v/v) Lysol, 10% (v/v) formalin and 70% (v/v) ethyl alcohol. The organisms survived at least 24 h at pH 9 or pH 4 and were not affected by sonication, freezing and thawing, or distilled water but lost significant infectivity after 24 h in CsCl or 40% (w/v) sucrose.

Animals↗
PubMed1979

Humoral immune response to natural infection with Encephalitozoon cuniculi in rabbits.

Sera from 4 generations of a family of rabbits having a natural Encephalitozoon cuniculi infection were investigated. Antibodies were demonstrated in a litter of newborn rabbits and in another litter 11 days old. Histoloigcal examination of the brains and kidneys of these animals failed to reveal lesions attributable to the disease. Sera from a further litter of 2 rabbits, taken at weekly intervals from 4 to 42 weeks of age, revealed a low initial antibody titre which regressed to an undetectable level. After an interval, titres rose and then plateaued to the end of the period of observation. Encephalitozoon cuniculi infections in the dams of all the litters were confirmed by antibody assay and by histology. Parallel titration of samples treated and not treated with 2-mercaptoethanol showed that the india-ink immunoreaction demonstrates only immunoglobulin G.

Animals↗
PubMed1979

The prevalence of Encephalitozoon antibodies in dogs and an evaluation of the indirect fluorescent antibody test.

Fifteen dogs which were serologically negative to the IFA test against Encephalitozoon developed antibodies in their sera following experimental infection. Six other dogs which were not tested prior to infection had titres to Encephalitozoon after experimental infection. In a sequential study in four dogs, antibodies first appeared between 32 and 39 days after infection. These results show that the IFA test would be suitable for epidemiological studies. Fifty serum samples collected from two kennels in which encephalitozoonosis had been confirmed showed an IFA test positive prevalence rate of 70%. In 220 serum samples submitted for various clinial pathological examinations the prevalence rate of antibodies to Ebcephalitozoon was 18%.

Animals↗
PubMed1979

Canine encephalitozoonosis in kennels and the isolation of Encephalitozoon in tissue culture.

The protozoan Encephalitozoon was isolated in primary kidney cultures from dogs originating from three separate outbreaks of encephalitozoonosis in kennels. The disease was characterized by a fading syndrome in young puppies with nervous signs developing in some cases. It was not possible to reproduce the clinical disease with cultured organisms in either normal dogs or dogs immunosuppressed with methylprednisolone. The organisms were, however, reisolated in primary culture from two immunosuppressed dogs but not from other experimentally infected dogs. The freezing of Encephalitozoon organisms in liquid nitrogen is described.

Animals↗
PubMed1980

A survey of serum antibodies to Encephalitozoon cuniculi in breeding rabbits and their young.

Screening of a rabbit breeding colony by the India-ink immunoreaction test revealed that 73% of the animals had antibodies to Encephalitozoon cuniculi. The study also indicated the passive transmission of antibodies from infected dams to their offspring. Ninety-five percent of the rabbits born to seropositive dams had maternal antibodies to Encephalitozoon cuniculi up to the age of 4 weeks with titers ranging from 1:25 to 1:800. After elimination of maternally transferred antibodies, about 50% of the young rabbits seroconverted; and from the age of 8--10 weeks, serum antibody titers over 1:800 were detectable. When these animals were tested again at 14 weeks, most of them had titers of 1:1600 or higher.

Animals↗
PubMed2004

A comparison of homologous genes encoding aminopeptidases among bird and human Encephalitozoon hellem isolates and a rabbit E. cuniculi isolate.

Encephalitozoon cuniculi and E. hellem are often recognized as the agents of human microsporidiosis, but less than optimal therapy is available for treatment. The identification of enzymes critical to the parasitic life cycle is an important step in finding targets for potential drug development. Aminopeptidase gene sequences were obtained from cDNA and gDNA from avian and human E. hellem isolates and from a rabbit E. cuniculi isolate. At the amino acid level, the aminopeptidase sequences from the E. hellem human and bird isolates share >99% identity and are nearly 70% identical with the E. cuniculi sequence. Conserved HEXXH and GAMEN motifs classify the predicted aminopeptidase in the MA clan of the M1 family. The obtained aminopeptidase gene sequences are likely homologous to the previously reported E. cuniculi glutamyl aminopeptidase. The conservation of this aminopeptidase between species and divergence from mammalian aminopeptidases indicate that this enzyme may be a valid target for drug therapy.

Amino Acid Motifs↗
PubMed2004

The genes encoding cAMP-dependent protein kinase catalytic subunit homologues of the microsporidia Encephalitozoon intestinalis and E. cuniculi: molecular characterisation and phylogenetic analysis.

A gene encoding a protein kinase was identified by homology-based PCR amplification in Encephalitozoon intestinalis, a microsporidian parasite pathogenic to humans, and its orthologue has been identified by database mining in the genome of the related species E. cuniculi, whose sequence has been recently published. Phylogenetic analysis revealed that the proteins encoded by these genes are homologues of the cAMP-dependent protein kinase catalytic subunits (PKAc). Southern blot analysis indicated that the EiPKAc gene is present in two copies in the E. intestinalis genome, whereas the E. cuniculi orthologue (EcPKAc) is a single copy gene. RT-PCR data showed that the EiPKAc gene is expressed in at least one of the intracellular stages during infection of the mammalian host cell by E. intestinalis.

Amino Acid Sequence↗
PubMed2003

Chlorine inactivation of spores of Encephalitozoon spp.

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Animals↗
PubMed1998

The serological surveillance of several groups of patients using antigens of Encephalitozoon hellem and E. cuniculi antibodies to microsporidia in patients.

This study was undertaken to attempt to identify correlations between microsporidial seroprevalence data in man, clinical diseases and groups of people at the risk of HIV/AIDS infection. Groups of patients were selected according to the predilection of members of the genus Encephalitozoon for nervous and kidney tissue. Female prostitutes and alcohol and intravenous drug abusers were selected as groups at risk of HIV/AIDS infections. A total of 401 samples of human sera were examined for the presence of antimicrosporidial IgG antibodies by ELISA test with a titre of 600 considered borderline positivity. The highest occurrence of antimicrosporidial antibodies was found in the groups of alcohol abusers (16% from 43 patients), intravenous drug abusers (11% from 9 patients) and prostitutes (10% from 80 women) for E. cuniculi antigen and in the groups of psychiatric patients (14% from 44 patients), malaria patients (11% from 38 patients) and alcohol abusers (7% from 43 patients) for E. hellem antigen. The occurrence of specific antibodies of the six examined diagnostic units (glomerulonephritis chronica, pyelonephritis chronica, schizophrenia, dementia, multiple sclerosis and cerebral stroke) was statistically significant only in patients with pyelonephritis chronica and dementia (p < 0.05). No cases of microsporidial infection were found among the female prostitutes by parasitological examination, although one case of giardiasis was identified. Sera of patients with high anti-E. cuniculi and anti-E. hellem antibodies (titres in ELISA of 600 and above) were confirmed by Western blot using E. cuniculi and E. hellem polypeptides, respectively. These results suggest that the examined patients could show residual antibodies from past or latent infections.

Animals↗
PubMed2001

The microsporidian Encephalitozoon.

Encephalitozoon cuniculi is an attractive model system for amitochondriate intracellular eukaryotic parasites. It is characterized by a very small genome (below 3 Mbp) and a unique invasion apparatus. Furthermore, as an infectious agent, it is important in human and veterinary medicine. The compactness of its genome involves the reduction of rDNA sequences as well as of some protein-coding genes and intergenic regions. Its highly differentiated apparatus to penetrate the host cell, an extrusome-like polar tube, is composed of novel proteins and may permit various pathways of infestation. Completion of the systematic E. cuniculi sequencing project should provide an important reference system for the comparative genomics of amitochondriate and mitochondriate parasites. Further analysis of orphan genes should help to identify factors that are responsible for its intracellular parasitic way of life.

Animals↗
PubMed2000

Occurence of subtelomeric rearrangements in the genome of the microsporidian parasite Encephalitozoon cuniculi, as revealed by a new fingerprinting procedure based on two-dimensional pulsed field gel electrophoresis.

In Microsporidia, mitochondria-lacking eukaryotic intracellular parasites, genomic comparisons were so far based on molecular karyotyping. The mammal-infecting species Encephalitozoon cuniculi is characterized by a very low haploid genome size (approximately 2.8 Mbp) and rather high karyotype variability. Recently, we developed a two-dimensional pulsed field gel electrophoresis (2-D PFGE) fingerprinting technique useful for constructing a restriction map fo the genome of a mouse E. cuniculi isolate (karyotype variant A). The so-called karyotype and restriction display 2-D PFGE (KARD-PFGE) protocol involved 1-D chromosome separation, digestion with a rare cutter, Klenow radiolabeling of genomic DNA and 2-D separation of restriction fragments followed by autoradiography. In order to assess its suitability for detecting polymorphic loci in E. cuniculi, we applied KARD-PFGE with either BssHII or Mlul digestion to genome analysis of two rabbit isolates representative of two different karyotype variants (A and C). The 2-D spot pattern of the rabbit isolate variant A is identical to the reference mouse isolate but differs greatly from the rabbit isolate variant C. Chromosomal restriction fragment length polymorphisms (RFLPs) provide strong evidence for homologous chromosomes and frequent DNA rearrangements within subtelomeric regions just upstream of the dispersed rDNA units closely associated with each chromosomal end.

Animals↗
PubMed2006

Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (microsporidia): a reference map for proteins expressed in late sporogonial stages.

The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.

Amino Acids↗
PubMed1998

Encephalitozoon intestinalis: humoral responses in interferon-gamma receptor knockout mice infected with a microsporidium pathogenic in AIDS patients.

IFN-gamma receptor knockout mice and wild-type mice were infected per os with Encephalitozoon intestinalis. Both groups developed an infection that was chronic in the mutant mice whereas it was only transient in wild-type mice. The infection of mutant mice was characterized by the continual shedding of spores in feces, splenomegaly, the enlargement of the biliary tract, and the occurrence of numerous nodules in the liver and in the small intestine wall. The humoral response was studied by ELISA, IFA, and Western blotting. ELISA titers of anti-E. intestinalis antibodies of IgG, IgM, and IgA isotypes were higher in IFN-gamma R0/0 mice than in wild-type mice and they increased in time after infection. Levels of IgG2a were inferior to those of IgG1 in mutant mice in contrast to wild-type mice. High levels of parasite specific antibodies were accompanied by an increase in type 2 cytokines (IL-4 and IL-10) secretion in the duodenum of IFN-gamma R0/0 mice. The E. intestinalis spore wall was recognized by IgM, IgG, and IgA from all infected mice whereas the extruded polar tube only reacted with IgG and IgA from IFN-gamma R0/0 mice after 45 days of the infection. IFN-gamma R0/0 mice IgG and IgA reacting with polar tube identified also a series of proteins which could be components of this structure. On the proteins recognized by all infected mice sera, two were first recognized by IgM at day 15 and then by IgG at day 30 in wild-type (WT) mice. The persistent reactivity of all proteins in mutant mice is consistent with the chronicity of the infection in these animals; in contrast, their resorption at day 30 in WT animals corroborates the transient character of the infection in these mice. The correlation between the evolution of the proteic pattern and the development of the infection provides evidence of the validity of this murine model to study human microsporidiosis. Indeed the reported results confirm the potential value of serological methods for diagnosing E. intestinalis infection in immunocompetent and in immunocompromised human subjects, for elucidating the age pattern of the microsporidiosis and also for identifying risk groups.

AIDS-Related Opportunistic Infections↗
PubMed2001

Encephalitozoon cuniculi (Microspora): characterization of a phospholipid metabolic pathway potentially linked to therapeutics.

Phospholipid metabolism of the microsporidian Encephalitozoon cuniculi, an obligate intracellular parasite, has been investigated. Labeled precursor incorporation experiments have shown that phosphatidylserine decarboxylase and phosphatidylethanolamine N-methyltransferase are more active in cells infected by E. cuniculi than in uninfected cells. In contrast, no difference was observed in the activity of Kennedy pathway's enzymes, the mammalian pathway. This suggests the occurrence in microsporidia of a bacteria- and fungi-typical pathway for phospholipid synthesis, which is supported by the identification of two genes implicated in this pathway, the cds gene encoding the key enzyme CDP-diacylglycerol synthase (E.C. 2.7.7.41) and the pss gene for CDP-alcohol phosphatidyltransferase. The pss gene could encode phosphatidylserine synthase (E.C. 2.7.8.8.), which catalyses the de novo synthesis of phosphatidylserine in bacteria and fungi. The complete CDP-diacylglycerol synthase messenger has been isolated and shows very short 5' and 3' untranslated regions. This is strong evidence for the functionality of a metabolic pathway which could be a potential target against microsporidia which infect humans.

Amino Acid Sequence↗
PubMed2000

Actin mediates Encephalitozoon intestinalis entry into the human enterocyte-like cell line, Caco-2.

Microsporidia are spore-forming obligate intracellular eucaryotes that parasitize eukaryotic cells. Encephalitozoon intestinalis (formerly Septata intestinalis) is a microsporidian species of emerging medical importance, responsible for chronic diarrhoea in immunocompetent patients and enteritis and systemic infections in HIV-1 infected patients. Infection of host enterocytes has been demonstrated in HIV-1-infected patients. However, the mechanisms of entry of E. intestinalis into host enterocytes have not been studied and remain hypothetically based on diacytosis, a model involving the injection of microsporidian sporoplasm through the polar tubule into the host cell. An electron microscopy based study recently challenged this hypothesis. We studied the entry of E. intestinalis into intestinal epithelial cells by infecting the human enterocyte-like cell line Caco-2. Entry was mediated by directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on E. intestinalis uptake, colocalization of E. intestinalis and F-actin and engulfment of E. intestinalis into Caco-2 cell protrusions. Confocal- and electron microscopy observations also suggested that after initial contacts through the posterior pole of the microsporidian spore, the basolateral surface of Caco-2 cells may be the portal of entry for E. intestinalis sporoplasm. Our observations allowed us to propose a new, actin-based model to describe the entry of microsporidia into enterocytes.

Actins↗