Encephalitozoon cuniculi: Light and Electron Microscopic Evidence for Di-, Tetra-, and Octosporous Sporogony and a Note on the Molecular Phylogeny of Encephalitozoonidae

We demonstrate, based on the light, electron microscopic, and immunofluorescence studies carried out on two isolates of Encephalitozoon cuniculi established in culture, that E. cuniculi exhibits di-, tri-, tetra- and octosporous sporogony. We therefore propose that the generic characters of Encephalitozoon should be amended to include tetra-sporous sporogony as generic features. Additionally, the molecular phylogenetic analysis indicates that E. cuniculi, E. hellem, and E. (Septata) intestinalis form a cohesive group.     

Polar tube proteins of microsporidia of the family encephalitozoonidae

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.     

Ribosomal RNA Sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: Phylogenetic Construction and Structural Correspondence

ABSTRACT. The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced. The forward primer 530f (5'-GTGCCATCCAGCCGCGG-3') was in the small subunit rRNA (SSU-rRNA) and the reverse primer 580r (5'-GGTCCGTGTTTCAAGACGG-3') was in the large subunit rRNA (LSU-rRNA). We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU-rRNA genes from E. bieneusi and S. intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection. The higher sequence similarities demonstrated between S. intestinalis and E. cuniculi support the placement of S. intestinalis in the family Encephalitozoonidae. This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora. In addition, the highly variable region (E. coli numbering 590–650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving.     

Studies on ocular microsporidia.

Sera from six ocular microsporidiosis patients and eight individuals with no history of microsporidiosis were assayed by enzyme-linked immunosorbent assay (ELISA) and by Western blot immunodetection. Microsporidia used as antigen include Nosema corneum, Encephalitozoon hellem, Encephalitozoon cuniculi, and Nosema algerae. Three AIDS patients with known E. hellem infections displayed ELISA antibody titers to E. hellem ranging from 1:400 to 1:12,800. Two patients with unclassified microsporidial infections displayed highest antibody titers to N. algerae (1:1,600 and 1:3,200), a mosquito microsporidian which, reportedly, cannot infect man. A sixth patient with a known N. corneum infection displayed the same ELISA antibody titer (1:1,600) to all four microsporidia. Western blot patterns also were variable among the patient sera; however, the most intense and complex antibody-binding patterns corresponded with the higher ELISA antibody titers. Sera from eight HIV-seronegative individuals with no history of microsporidiosis reacted variably to the four microsporidia. These results suggest that diagnosis of microsporidiosis may depend upon direct detection of the organisms using species-specific antibodies or molecular probes rather than conventional serology.     

First detection and genotyping of human-associated microsporidia in pigeons from urban parks

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.     

Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia,Spain

In this survey we examined 87 domestic animal stool samples in order to detect the possible presence of microsporidia in animals in close contact with humans in Galicia (NW, Spain). The detection of Enterocytozoon bieneusi spores was confirmed in faecal samples from two dogs and one goat by polymerase chain reaction. None of the positive samples for microsporidia in the staining method were amplified with species-specific primers for Encephalitozoon intestinalis, E. hellem and E. cuniculi. Four rabbits faecal samples reacted with anti-E. cuniculi serum. Our results could indicate the importance of domestic animals as zoonotic reservoirs of microsporidial human infections.     

Effects of gamma radiation on viability of Encephalitozoon spores

Spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis harvested from cultured mammalian cells were suspended in deionized water, exposed to gamma irradiation at doses of 0-3.0 kGy, and then tested for infectivity by inoculating spores into monolayer cultures of Madin-Darby bovine kidney cells. The cultures were examined for developing microsporidia 4 days later. As the dosage level of radiation increased, corresponding decreases were observed in the number of developing microsporidia for all 3 species. For E. cuniculi and E. intestinalis, 100% inhibition of development was observed after exposure to 1.5 and 2.0 kGy, respectively. Although development of E. hellem was greatly inhibited (97.6% inhibition) after exposure to 3.0 kGy, complete inhibition was not obtained. These findings provide a baseline for investigating the dose levels required to render food products safe when kept under varying temperature, moisture, and other storage conditions.     

Rabbit intestinal xenograft model for human Encephalitozoon infections in mice.

BACKGROUND AND PURPOSE: The gastrointestinal tract is a common portal of entry for Encephalitozoon cuniculi, one of several microsporidial organisms emerging as opportunistic pathogens in immunocompromised humans. Although most human microsporidial pathogens can be propagated in vitro and in a variety of laboratory animals, an experimental animal system to specifically study intestinal uptake and systemic spread of these organisms does not exist. METHODS: Paired segments of near-term fetal rabbit small intestine were implanted subcutaneously into 25 athymic nude or 10 severe combined immune deficient mice. Five weeks after surgery, 65 xenografts were inoculated intraluminally with E. cuniculi (n = 14), E. intestinalis (n = 27), E. hellem (n = 20), or RK-13 cells (n = 2), or were left uninoculated (n = 2). RESULTS: Intestinal xenograft infection with E. cuniculi (n = 11), E. intestinalis (n = 17), and E. hellem (n = 18) was determined by light microscopy; control xenografts remained uninfected. Extraintestinal infection with E. cuniculi developed in host mouse brain, respiratory tract, spleen, salivary glands, and gastrointestinal tract (3 of 3 mice), and infection with E. intestinalis developed in the liver (8 of 15 mice). CONCLUSION: Intestinal xenografts provide a unique, sterile, and biologically relevant animal model system for studying host enterocyte/parasite interactions, mechanisms of microsporidial pathogenicity, antimicrosporidial chemotherapeutic agents, and immune effector mechanisms. This model provides evidence for persistent graft infection with three Encephalitozoon spp., and for intestinal spread of E. cuniculi and E. intestinalis from infected enterocytes in immunoincompetent mice.     

DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.     

Identification of Encephalitozoon and Enterocytozoon (microsporidia) spores in stool and urine samples obtained from free-living South American Coatis (Nasua nasua)

This study emphasizes the importance of free-living coatis as a potential source of microsporidian infection for humans living in large cities. We found 19 (31.7%) positive results among 60 fecal samples analyzed by PCR-based analysis and the Gram-Chromotrope staining technique (11.7% were positive for Encephalitozoon cuniculi, 6.7% for E. intestinalis, 6.7% for E. hellem, and 6.7% for Enterocytozoon bieneusi). Only 5 (8.4%) urine samples tested positive for E. cuniculi as assessed by the two techniques.     

Analysis of four human microsporidian isolates by MALDI-TOF mass spectrometry

Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000-8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.     

Infectivity of microsporidia spores stored in water at environmental temperatures

To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.     

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi,Encephalitozoon hellem,and Vittaforma corneae

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.     

In vitro and in vivo investigations of human microsporidia.

The numerous infections of microsporidia which have been diagnosed in patients with AIDS have revealed the potential of these organisms for establishing themselves when the immune status of the host is compromised. Two species of Encephalitozoon, E. cuniculi and E. hellem, have been diagnosed in man, the former infecting a variety of tissues, the latter restricted to the corneal and conjunctival epithelia. These species are morphologically indistinguishable even at the ultrastructural level but can be separated biochemically. Two human sera were found to react with equal intensity in the ELISA on spores of E. cuniculi and E. hellem purified from in vitro cultures, and gave similar binding patterns in Western blots on SDS-PAGE protein profiles of the two species. This has raised questions about the identity of Encephalitozoon infections diagnosed previously in man. The diagnosis of Enterocytozoon bieneusi, which infects the intestinal enterocytes of AIDS patients and is associated with chronic diarrhoea, requires observation of smears or sections of biopsies or specialist observation of stool preparations. In vitro cultures, which would facilitate the raising of specific antisera, have proved difficult to establish. In vitro and in vivo systems for assaying drugs for microsporidia have revealed that albendazole has a marked effect on parasite numbers and morphology but does not eliminate infection, which resurges when drug pressure is removed.     

Evidence of Actin in the Cytoskeleton of Microsporidia

Using transmission electron microscopy, immuno-electron microscopy, and biochemical techniques such as 2-D electrophoresis and immunoblotting, actin was found in all biological stages of the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi.     

Antigenic diversity of Encephalitozoon hellem demonstrated by subspecies-specific monoclonal antibodies

Encephalitozoon hellem is a unicellular, obligate intracellular microsporidian species detected and isolated in HIV-infected patients presenting with keratoconjunctivitis, sinusitis, tracheobronchitis, nephritis, cystitis, and disseminated infection. A total of 24 monoclonal antibodies were produced against E. hellem and characterized. The monoclonal antibodies were of the immunoglobulin (Ig) G and Ig M subclasses, and, when incorporated into indirect immunofluorescence and immunoblotting assays, reacted against 13 isolates of E. hellem originating from three geographic regions. These monoclonal antibodies did not react with one strain each of either Encephalitozoon intestinalis or Encephalitozoon cuniculi, demonstrating their specificity. Two monoclonal antibodies reacted with all karyotype B-E. hellem isolates but did not react with karyotype A-isolates from North America and the Netherlands, thus demonstrating antigenic diversity among E. hellem isolates. These results add to the increasing evidence for diversity among E. hellem, which therefore may be reclassified into subspecies.     

Encephalitozoon hellem in Two Eclectus Parrots (Eclectus roratus): Identification from Archival Tissues

ABSTRACT Members of the phylum Microspora are obligate, intracellular, single-celled parasites identified in a wide range of vertebrate and invertebrate hosts. Only a few cases of microsporidial infections have been documented in psittacine birds including peach-faced, masked, and Fischer's lovebirds ( Agapornis roseicollis, A. personata , and A. fischeri. respectively), budgerigars ( Melopsittacus undulatus ), and a double yellow-headed Amazon parrot ( Amazona ochrocephala ). Parasite identification has typically been limited to phylum or genus, and no avian species of microsporidia has clearly been described. In this report, microsporidia were identified in the kidney and intestine of a new host, the eclectus parrot ( Eclectus roratus ). Parasites were identified as Encephalitozoon hellem using morphologic, ultrastructural, and small subunit ribosomal RNA gene sequence data obtained from archived tissues. This parasite species was first identified in immunocompromised humans and may be a potential zoonotic pathogen. The epidemiology and prevalence of this parasite in humans and birds should be further explored.     

On the use of FTA technology for collection, archieving, and molecular analysis of microsporidia dna from clinical stool samples

The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates.     

Infectivity of microsporidia spores stored in seawater at environmental temperatures

To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.