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PubMedDate not supplied

Primers designed for amplification of Echinococcus multilocularis DNA amplify the DNA of Encephalitozoon-like spores in the polymerase chain reaction.

Microsporidian spores were developed from cells which were grown in vitro from a human liver lesion which was due to larval Echinococcus multilocularis. The microsporidian spores developed in the same fashion as an Encephalitozoon cuniculi. The Encephalitozoon-like spores were completely separated on Percoll gradients. The separated spores contained DNA capable of amplification by two different primer sets designed for the polymerase chain reaction (PCR) of E. multilocularis DNA. However, the cell DNA from which microsporidium developed was thoroughly insensitive to the PCR using the E. multilocularis primer sets. The results strongly suggested that Encephalitozoon should be taken into consideration, when DNA isolated from larval E. multilocularis is analyzed.

Animals↗
PubMedDate not supplied

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.

Animals↗
PubMed2004

In vitro effects of resveratrol on the viability and infectivity of the microsporidian Encephalitozoon cuniculi.

Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 microM showed significant sporicidal activity, and at 10 to 50 microM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 microM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of Encephalitozoon infections.

Animals↗
PubMed2000

Developmental expression of a tandemly repeated, glycine- and serine-rich spore wall protein in the microsporidian pathogen Encephalitozoon cuniculi.

Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculi with the aid of a monoclonal antibody, and the corresponding gene, SWP1, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1 is a single-copy gene that is also present in the genomes of Encephalitozoon intestinalis and Encephalitozoon hellem as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

Amino Acid Sequence↗
PubMed1994

Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection.

Microsporidia are primitive, spore-forming, mitochondria-lacking, eukaryotic protozoa that are obligate intracellular parasites. They are known to parasitize almost every group of animals including humans. Recently, microsporidia have increasingly been found to infect patients with AIDS. Five genera (Encephalitozoon, Enterocytozoon, Nosema, Septata, and Pleistophora) of microsporidia are known to infect humans. Enterocytozoon organisms cause gastrointestinal disease in a majority of AIDS patients with microsporidiosis. However, a smaller, but an expanding, number of patients with AIDS are being diagnosed with ocular and disseminated infection with Encephalitozoon hellem. Although microsporidial spores can be identified in clinical samples by a staining technique such as one with Weber's chromotrope stain, identification to the species level is dependent on cumbersome and time-consuming electron microscopy. We have recently isolated and established in continuous culture several strains of E. hellem from urine, bronchoalveolar lavage, and sputum samples from AIDS patients with disseminated microsporidiosis. We developed polyclonal and monoclonal antibodies and PCR primers to a strain of E. hellem that can be used successfully to identify E. hellem from other species of microsporidia either in clinical specimens or in cultures established from clinical specimens. Since patients infected with Encephalitozoon spp. are known to respond favorably to albendazole, identification of the parasite to the species level would be invaluable in the treatment of disseminated microsporidiosis.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Detection by an immunofluorescence test of Encephalitozoon intestinalis spores in routinely formalin-fixed stool samples stored at room temperature.

Of the several microsporidia that infect humans, Enterocytozoon bieneusi is known to cause a gastrointestinal disease whereas Encephalitozoon intestinalis causes both a disseminated and an intestinal disease. Although several different staining techniques, including the chromotrope technique and its modifications, Uvitex 2B, and the quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples, they do not identify the species of microsporidia. A need for an easily performed test therefore exists. We reevaluated 120 stool samples that had been found positive for microsporidia previously, using the quick-hot Gram-chromotrope technique, and segregated them into two groups on the basis of spore size. We also screened the smears by immunofluorescence microscopy, using a polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity with bacteria, yeasts, or other structures in the stool samples was seen. Additionally, the numbers of spores that fluoresced in seven of these samples were substantially smaller than the numbers of spores that were present in the stained smears, indicating that these samples were probably derived from patients with mixed infections of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae or with Enterocytozoon bieneusi spores in feces, we concluded that an immunofluorescence test using this serum is a good alternative for the specific identification of E. intestinalis infections.

AIDS-Related Opportunistic Infections↗
PubMed1997

Encephalitozoon hellem in budgerigars (Melopsittacus undulatus).

Microsporidiosis with concurrent megabacteriosis in budgerigar (Melopsittacus undulatus) chicks contributed to significant economic floss in a commercial pet bird aviary in Mississippi. Three budgerigar chicks, 1-2 weeks old, from the aviary were necropsied. Microscopic lesions in the chicks consisted of heavy infection of enterocytes with microsporidia (2/3; autolysis precluded critical evaluation of the intestine of chick No. 2), multifocal hepatic necrosis and inflammation with intralesional microsporidia (1/3), spherical clusters of microsporidia in the hepatic sinusoids in the absence of inflammation (1/3), and gastric megabacteriosis (3/3). The ultrastructure of the microsporidian spores was consistent with an Encephalitozoon species. The polymerase chain reaction and Southern blot analysis were used to identify the microsporidian as Encephalitozoon hellem, an organism that has only been identified in humans. Encephalitozoon hellem causes keratoconjunctivitis and respiratory infections in humans with acquired immunodeficiency syndrome. This report presents the first confirmed case of microsporidiosis in budgerigars. The finding of E. hellem in pet birds may be important in elucidating the epidemiology of human infections with this organism.

Animals↗
PubMed2003

Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis and Cryptosporidium parvum in water used for drinking.

Human enteropathogenic microsporidia (HEM), Cryptosporidium parvum, Cyclospora cayetanesis, and Giardia lamblia are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of HEM (Encephalitozoon intestinalis and Enterocytozoon bieneusi) and Cyclospora cayetanesis have not been fully elucidated due to lack of sensitive and specific environmental screening methods. The present study was undertaken with recently developed methods, to screen various water sources used for public consumption in rural areas around the city of Guatemala. Water concentrates collected in these areas were subjected to community DNA extraction followed by PCR amplification, PCR sequencing and computer database homology comparison (CDHC). All water samples screened in this study had been previously confirmed positive for Giardia spp. by immunofluorescent assay (IFA). Of the 12 water concentrates screened, 6 showed amplification of microsporidial SSU-rDNA and were subsequently confirmed to be Encephalitozoon intestinalis. Five of the samples allowed for amplification of Cyclospora 18S-rDNA; three of these were confirmed to be Cyclospora cayetanesis while two could not be identified because of inadequate sequence information. Thus, this study represents the first confirmed identification of Cyclospora cayetanesis and Encephalitozoon intestinalis in source water used for consumption. The fact that the waters tested may be used for human consumption indicates that these emerging protozoa may be transmitted by ingestion of contaminated water.

Animals↗
PubMed2004

Susceptibility of IFN-gamma or IL-12 knock-out and SCID mice to infection with two microsporidian species, Encephalitozoon cuniculi and E. intestinalis.

Susceptibility of three strains of immunodeficient mice to two related microsporidian species Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 and Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) was compared. While both, severe combined immunodeficient (SCID) and interferon-gamma knock-out (IFN-gamma KO) mice, succumbed to either intraperitoneal (i.p.) or peroral (p.o.) (natural) infection with both parasites, only i.p. infection with E. cuniculi killed interleukin-12 knock-out (IL-12 KO) mice. IFN-gamma KO mice died earlier than SCID mice. Adoptive transfer of naive splenocytes from IFN-gamma KO mice did not protect the SCID mice from a lethal infection with either of the Encephalitozoon species. However, reconstituted mice survived significantly longer (P<0.05), thus indicating the role of IFN-gamma produced by host NK cells in the development of mechanisms of anti-microsporidial protective immunity. Non-lethal outcome of the infection always correlated with the increase in CD8+ T lymphocyte subpopulation. Both E. intestinalis-infected IFN-gamma KO and IL-12 KO mice produced comparable levels of specific antibodies, suggesting that antibodies did not protect IFN-gamma KO mice from lethal infection.

Adoptive Transfer↗
PubMed2006

[Frequency of microsporidial Encephalitozoon intestinalis in patients with diarrhea].

Encephalitozoon intestinalis is an obligate intracellular sporeforming protozoa belonging to the order Microsporidia of the phylum Microspora. In the year 1923 Levadetia et al., using transmision electron microscopy, for the first time, identified microspridial organism, Encphalitozon intestinalis as causing acute diarrhea. Encephalitozzon-like spores were also found in cerebrospinal fluid in rabits. Three species have been identified in order Encephalitozoon firstly named Sepatata intestinalis and later renamed in Encephalitizon spp. by Didier. Thereafter, numerous reports have sugested that microspridial infections may be responsible for many cases of diarrhea esspecialy in patients with AIDS. Only rarely have microsporidial infections been found in non-HIV infected individuals. The aim of our study is to detect microsporidian protozoal parasite Encephalitozoon intestinalis as a cause of acute diarrhea in non HIV-infectid patients in Slovenia.

Acute Disease↗
PubMed1994

Isolation and identification of Encephalitozoon hellem from an Italian AIDS patient with disseminated microsporidiosis.

Microsporidia are primitive mitochondria-lacking spore-forming eukaryotic protozoa that infect a wide variety of animals and also humans. Of the five genera (Encephalitozoon, Enterocytozoon, Septata, Nosema and Pleistophora) that cause infections in humans, Enterocytozoon bieneusi, Septata intestinalis, and Encephalitozoon hellem are being increasingly identified in patients with acquired immunodeficiency syndrome (AIDS). E. bieneusi causes gastrointestinal disease, S. intestinalis causes gastrointestinal and disseminated disease, and E. hellem causes ocular as well as disseminated disease. We have established in continuous culture a strain of microsporidia isolated from the urine and throat washings of an Italian AIDS patient and identified it as Encephalitozoon hellem, based on its ultrastructural morphology, antigenic pattern, and polymerase chain reaction-amplified small subunit ribosomal RNA. We believe that this is the first time that a strain of microsporidia has been isolated from the throat washings of a patient with microsporidiosis.

AIDS-Related Opportunistic Infections↗
PubMed1997

Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS.

Microsporidia spores, identified as Encephalitozoon cuniculi (CDC: V282), were isolated from the urine of a patient with acquired immunodeficiency syndrome and disseminated microsporidiosis, established in continuous culture on monkey kidney cells (E6), and antiserum was produced in rabbits. Immunoblot studies that used the patient serum and the rabbit sera against CDC:V282, Encephalitozoon hellem (CDC:0291:V213), and Encephalitozoon intestinalis (CDC:V297) revealed that CDC:V282 and the rabbit isolate of E. cuniculi (ECLD) reacted intensely with the patient's serum and the rabbit anti-CDC:V282, producing a number of bands ranging from 200 to 15 kDa. By contrast, the heterologous antigens (CDC:0291:V213 and CDCV297) reacted minimally. Both CDC:V282 and ECLD isolates of E. cuniculi reacted minimally with the rabbit anti-E. hellem and the rabbit anti-E. intestinalis sera. In the immunofluorescence test, performed on the lung biopsy section of the patient, the rabbit anti-CDC:V282 serum reacted extensively with the spores in the tissue section and produced bright apple green fluorescence. These studies demonstrated that the human (CDC:282) and the rabbit (ECLD) isolates of E. cuniculi were similar in their antigenic profiles but differed considerably from E. hellem and E. intestinalis, and that the patient's serum reacted specifically, strongly, and with equal intensity, with the 2 isolates of E. cuniculi.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Identification of the microsporidian Encephalitozoon hellem using immunoglobulin G monoclonal antibodies.

OBJECTIVE: Microsporidia isolated from clinical specimens so far have been identified to level of species by electron microscopy, indirect immunofluorescence (IIF), western blot (WB), and genetic analysis. Recent studies, however, indicate extensive serologic cross-reactions among microsporidian species involved in human disease. DESIGN AND SETTING: In this study, we used IIF and WB techniques to evaluate the reactivity of six different immunoglobulin G monoclonal antibodies (MAbs) raised against Encephalitozoon hellem with six isolates of E hellem that originated from patients with acquired immunodeficiency syndrome. A rabbit isolate of Encephalitozoon cuniculi, and an isolate of Encephalitozoon intestinalis, which was established in cultures from the urine of a patient with acquired immunodeficiency syndrome were also used for comparison. RESULTS: Five of the six antibodies, when analyzed by both IIF and WB assays, specifically identified six isolates of E hellem originating from three patients with acquired immunodeficiency syndrome. The sixth MAb, however, reacted with all of the E hellem isolates in the WB assay, but failed to react with them in the IIF assay. Using the IIF test, five of the six MAbs failed to react with E cuniculi and E intestinalis, even at a dilution of 1:50. The MAbs also did not react in the IIF test with Enterocytozoon bieneusi, Giardia, and Cryptosporidium. These MAbs did react with E cuniculi and E intestinalis in the WB assay, but the banding patterns were very different from those of E hellem, thus facilitating the identification of E hellem from the other microsporidia. The MAbs also reacted, in the IIF test, with E hellem spores in formalin-fixed tissue sections that were heated in a microwave oven. CONCLUSIONS: Identification of microsporidian agents to the species level is important. Since certain therapeutic agents (eg, fumagillin, albendazole) are efficacious in treating E hellem infections of the cornea, as well as urogenital and respiratory infections caused by E hellem, a quick and definitive identification of the organism is important so that successful therapy may be instituted. An IIF test using the MAbs described here would therefore be invaluable in the quick identification of this parasite.

Animals↗
PubMed1979

A simple method for the detection of antibodies to Encephalitozoon cuniculi in rabbits.

Rabbit antibodies against Encephalitozoon cuniculi were detected in an indirect microagglutination test using a bead substrate to which anti-rabbit immunoglobin G light and heavy chain antibodies were coupled. The test was positive using immune whole serum or F(ab)' and F(ab)'2 fragments of immunoglobin G but negative using the F(c) fragment. The reaction was blocked by saturating the beads with rabbit serum or by absorbing positive sera with excess Encephalitozoon cuniculi. The test provided a simple method to detect antibodies to Encephalitozoon cuniculi, did not require elaborate equipment and could be performed using frozen antigen.

Agglutination Tests↗
PubMedDate not supplied

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.

Animals↗
PubMed1992

Diagnosis of Encephalitozoon cuniculi infection by western blot and the use of cross-reactive antigens for the possible detection of microsporidiosis in humans.

Microsporidia are very primitive, eukaryotic, obligate, intracellular, protozoan parasites. Encephalitozoon cuniculi, a microsporidian originally described from a rabbit infection, has been described in humans as well as in many species of laboratory animals. We report the detection of E. cuniculi by Western blotting in a rabbit with torticollis that was obtained from an Encephalitozoon-free colony. Cross-reactivity of this serum was observed with antigens prepared from several genera of microsporidia. Identical Western blotting patterns were obtained with sera obtained from a rabbit immunized with E. cuniculi that was purified from tissue culture cells. In addition, we were able to demonstrate cross-reactivity between E. cuniculi rabbit antisera and Enterocytozoon bieneusi antigens by indirect immunofluorescent assay techniques in human intestinal biopsy samples. These cross-reactions between microsporidia may be useful in developing diagnostic tests for non-cultivatable microsporidia such as Enterocytozoon bieneusi.

Animals↗
PubMed1999

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.

Animals↗
PubMed2001

Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species.

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. helleri, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Animals↗