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PubMed1986

The infectivity of Encephalitozoon cuniculi in vivo and in vitro.

The infectivity of Encephalitozoon cuniculi grown in cell cultures was determined in cultured cells and in wild and domestic rabbits. The ratio of the total to tissue culture viable count was 1,300 (median of seven determinations). The mean ratio of intact spore count to total count, as determined by electron microscopy was 0.12. Although variation between infectivity experiments was large, the median animal infective dose contained 51 FFU (cell culture focus-forming units) for wild rabbits (Oryctolagus cuniculus) and 40 FFU for domestic rabbits. These two infectivities were not statistically different.

Animals↗
PubMed1992

Ultrastructure of the development of a species of Encephalitozoon cultured from the eye of an AIDS patient.

Human fibroblast cell cultures inoculated with microsporidia-infected corneal scrapings from an AIDS patient were fixed in situ and examined by scanning and transmission electron microscopy. The parasite grew prolifically and all developmental stages were observed. Meronts underwent binary fission and the daughter cells transformed into elongate, chain-like sporonts that eventually separated into sporoblasts. The formation of components of the mature spores is described. The parasite, a species of Encephalitozoon, underwent development both in the cytoplasm and within a parasitophorous vacuole, distinguishing it from the morphologically similar species E. cuniculi and E. hellem, both of which have been described from lesions in the human eye and have been reported to develop exclusively within a parasitophorous vacuole.

Acquired Immunodeficiency Syndrome↗
PubMed2002

The homeobox genes of Encephalitozoon cuniculi (Microsporidia) reveal a putative mating-type locus.

Homeobox genes have been found in animals, fungi, and plants. Recently, the complete genomic sequence of Encephalitozoon cuniculi has become available and it was shown that this Microsporida species is related to fungi. Given this close relatedness to fungi, I have searched the genome of E. cuniculifor homeobox genes. There are 12 homeobox genes as well as one STE12 orthologue. The large number of homeobox genes when compared to the annotations, which only list one, suggests that possibly other smaller genes may have been overlooked in the published analysis and E. cuniculi may have more than 1,997 genes. Sequence and phylogenetic analyses show that the E. cuniculi homeobox genes also fall into two distinct groups, with two TALE and ten typical homeobox genes being present. Like in the mating-type locus of yeast and other fungi, one TALE and one typical homeobox are found in close proximity of each other on the chromosome, suggesting that Microsporidia also contain a mating-type locus.

Amino Acid Sequence↗
PubMed1998

Renal Encephalitozoon (Septata) intestinalis infection in a patient with AIDS. Post-mortem identification by means of transmission electron microscopy and PCR.

We describe the occurrence of renal Encephalitozoon (Septata) intestinalis infection in a 35-year-old AIDS patient who died with disseminated tuberculosis. The patient did not complain of specific symptoms involving the kidney or lower urinary tract during life, but at autopsy, light microscopic examination of the kidney revealed numerous small round or oval bodies in the tubules and tubular cell cytoplasm that were interpreted as intracellular protozoa. Transmission electron microscopy of tissue retrieved from paraffin-embedded samples identified these organisms as microsporidia belonging to the Encephalitozoonidae family, but did not allow definitive identification of the species of infecting parasite. This was made possible only by means of Southern blot hybridization after the polymerase chain reaction, which recognized the micro-organism as E. intestinalis.

AIDS-Related Opportunistic Infections↗
PubMed2002

Role of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon intestinalis infection.

Severe combined immunodeficient (SCID) mice reconstituted with spleen cells from naive adult BALB/c mice were completely resistant to peroral infection with Encephalitozoon intestinalis (Calli, Kotler and Orenstein, 1993) Canning, Field, Hing and Marriott, 1994, whereas control, non-reconstituted SCID mice succumbed to the infection. The role of T-lymphocyte subpopulations in the protection against peroral E. intestinalis infection was studied in adoptive transfer experiments using SCID mice. SCID mice reconstituted with both CD4+ and CD8+ T-lymphocyte-depleted splenocytes succumbed to the peroral route of infection. In contrast, SCID mice reconstituted with either CD4+-depleted or CD8+ T-lymphocyte-depleted splenocytes completely resolved the infection. This indicates that CD4+ and CD8+ T-lymphocyte subpopulations play a substantive role in protection against peroral infection with the microsporidian, E. intestinalis.

Adoptive Transfer↗
PubMed2002

A single genotype of Encephalitozoon intestinalis infects free-ranging gorillas and people sharing their habitats in Uganda.

Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.

Animals↗
PubMed2006

Humoral response of chicken infected with the microsporidium Encephalitozoon hellem.

Chicken (Gallus gallus) were used as the experimental model for study of immune response against the microsporidium Encephalitozoon hellem (Didier et al., J Inf Dis 163:617-621, 1991) infection in birds. Two-day-old chicken were infected perorally or intraperitoneally with a dose of 10(7) spores of E. hellem. The anti-E. hellem immunoglobulin (Ig)A, IgY, and IgM antibody responses in sera and dropping sample extracts were determined by enzyme-linked immunosorbent assay. Results have shown specific antibody production in sera and intestinal secretions of infected birds. Chicken inoculated perorally developed the lowest antibody response. Microsporidian spores were not identified in the smears from cloacal swab samples of individual chicken. Intestinal segment cultures of perorally infected chicken cultivated in vitro showed the highest production of specific IgY and IgA antibodies in jejunum segments. In the further course of infection, the colon produced the highest amount of IgA, and the ileum and colon produced the highest amount of IgY.

Animals↗
PubMed2006

Pure CD4+ T lymphocytes fail to protect perorally infected SCID mice from lethal microsporidiosis caused by Encephalitozoon cuniculi.

The role of CD4+ and CD8+ T lymphocytes in the protection against intraperitoneally (i.p.) or perorally (p.o.) acquired Encephalitozoon cuniculi (Levaditi et al., C R Soc Biol Paris 89:984-986, 1923) infection was studied by means of reconstitution of severe combined immunodeficiency (SCID) mice with well-defined populations of naive CD8+ or CD4+ T lymphocytes. Adoptive transfer of pure CD8+ T lymphocyte subpopulation protects SCID mice against a lethal microsporidiosis caused by E. cuniculi. The protective effect of CD8+ T lymphocytes is manifested in both i.p. and p.o. infection. On the contrary, the host defense against peroral infection does not require CD8+ T cells. The protective role is not mediated by CD4+ T lymphocytes only. SCID mice reconstitution with pure CD4+ T cell subpopulation led to prolonged survival of perorally infected mice. However, these mice died due to lethal encephalitozoonosis caused by i.p. infection.

Adoptive Transfer↗
PubMed2006

An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia).

Intracellular development of microsporidian parasites comprises a proliferative phase (merogony) followed by a differentiation phase (sporogony) leading to the release of resistant spores. Sporogony implies, successively, meront-to-sporont transformation, sporont division into sporoblasts, and sporogenesis. We report a procedure improving the separation of sporogonial stages of Encephalitozoon cuniculi, a species that develops inside parasitophorous vacuoles of mammalian cells. Supernatants of E. cuniculi-infected Madin-Darby canine kidney cell cultures provided a large number of parasites mixed with host-cell debris. This material was gently homogenized in phosphate-buffered saline containing 0.05% saponin and 0.05% Triton X-100 then filtered through glass wool columns. Centrifugation of the filtrate on 70% Percoll-0.23 M sucrose gradient gave a reproducible pattern of bands at different densities. Transmission electron microscopy showed that three of the four collected fractions were free of visible contaminants. Corresponding prominent cell stages were early sporoblasts (fraction B), late sporoblasts plus immature spores (fraction C), and mature spores (fraction D). Further centrifugation of the lightest fraction (A) on 30% Percoll-0.23 M sucrose gradient generated a sporont-rich fraction (A2). First analysis of proteins from fractions A2 and D by two-dimensional gel electrophoresis suggested a potential use of the described method for proteomic profiling.

Animals↗
PubMed1996

Isolates of Encephalitozoon cuniculi from farmed blue foxes (Alopex lagopus) from Norway differ from isolates from Swiss domestic rabbits (Oryctolagus cuniculus).

Encephalitozoon cuniculi has a wide host range among mammals, but whether it represents a homogeneous species is a subject of controversy. We have isolated, cultivated (in human MRC-5 cells) and, for the first time, characterized by immunological and molecular biological methods four isolates of E. cuniculi from Norwegian blue foxes with a history of encephalitozoonosis. The isolates were compared with nine isolates from domestic rabbits from Switzerland. Two E. cuniculi subtypes were identified according to their host species. A 5'-GTTT-3' tetranucleotide repeat was present twice in the rDNA intergenic spacer in all isolates from foxes as opposed to three times in all isolates from rabbits. Furthermore, random amplified polymorphic DNA analysis showed one polymorphic band among the subtypes, and Western-blot analysis using serum from an infected fox discriminated between the two subtypes on the basis of their banding patterns in the ranges of 31-33 and 38-40 kDa. The 5'-GTTT-3' tetranucleotide repeat is a valuable genetic marker for these two subtypes of E. cuniculi and will be of use in continued studies on the molecular epidemiology of this parasite.

AIDS-Related Opportunistic Infections↗
PubMed2001

Dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient.

This report describes the first dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient. In view of clinical and epidemiological findings, our E. cuniculi isolate was deduced to be of the dog strain. The patient's occupational involvement with dogs indicates that canines should be considered as a reservoir of human infections for both microsporidial species. Furthermore, our report provides detailed clinical and radiological information on a rare case of a symptomatic pulmonary infection by E. cuniculi and its improvement after treatment with albendazole.

AIDS-Related Opportunistic Infections↗
PubMed1986

Humoral antibody response of rabbits to experimental infection with Encephalitozoon cuniculi.

Six female rabbits (Oryctolagus cuniculus) were experimentally infected intravenously with approximately 1.5 X 10(7) live spores of Encephalitozoon cuniculi. Head tilt was observed as the single clinical sign in only one of the six animals. Antibody response was registered over 68 days postinfection using the indirect immunofluorescence test (IFT) for IgM and IgG, and the carbon immunoassay (CIA). IgG titers reached a level of 160-2560 after a latent phase of 13-28 days, followed by a 2-4 week relatively steep increase. The IgM seroconversion was faster than that of IgG and occurred at the beginning of the antibody response. Thus, simultaneous detection of both IgM and IgG allowed the infection to be identified as recent. Long, short, and episodic antibody responses could be distinguished: the IgG titer continued to increase on Day 68 in one animal (long response) and began to decrease between Days 45 and 63 in three other animals (short response). In two additional animals the seroconversion was very short, occurring between Days 13 and 41, and 28 and 52, respectively (episodic response). The CIA proved to be specific, reliable, and simple to perform; titers were slightly higher than in the IFT. Parasite pseudocysts were detected scattered throughout the brain on Day 68 in four of the six rabbits. The persistence of antigen in the brain did not correlate with antibody response, which in most cases was shorter.

Animals↗
PubMed2006

Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR.

A multiplex real-time polymerase chain reaction (PCR) method was developed for the simultaneous detection of Enterocytozoon bieneusi (n = 30) and Encephalitozoon spp. (n = 3) in stool samples. The multiplex PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n = 22) and a range of well-defined stool samples (n = 140), and it achieved 100% specificity and sensitivity. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique in the routine diagnosis of intestinal microsporidian infections.

Animals↗
PubMed2005

The putative chitin deacetylase of Encephalitozoon cuniculi: a surface protein implicated in microsporidian spore-wall formation.

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.

Amidohydrolases↗
PubMed2005

Intraocular microsporidiosis due to Encephalitozoon cuniculi in a patient with idiopathic CD4+ T-lymphocytopenia.

Encephalitozoon cuniculi was documented to cause disseminated microsporidial infection including an iris tumor and endophthalmitis in an adolescent with idiopathic CD4+ T-lymphocytopenia. The diagnosis was established by microscopic, serologic and molecular methods. E. cuniculi (rabbit strain) was identified from the iris tumor, as well as from urine, conjunctival, corneal, and nasal swabs. Treatment with oral albendazole led to rapid improvement. This case raises the possibility of disseminated microsporidial infection in the context of idiopathic CD4+ T-lymphocytopenia and possibly advanced human immunodeficiency virus (HIV) infection, and above all the possibility of intraocular infection with E. cuniculi in humans.

AIDS-Related Opportunistic Infections↗
PubMed2005

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.

Amino Acid Sequence↗
PubMed2005

Functional characterization of a putative aquaporin from Encephalitozoon cuniculi, a microsporidia pathogenic to humans.

The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl(2) failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections.

Amino Acid Sequence↗
PubMed2006

Disseminated microsporidiosis due to Encephalitozoon hellem in an Egyptian fruit bat (Rousettus aegyptiacus).

Disseminated microsporidiosis was diagnosed in an adult female Egyptian fruit bat that died unexpectedly in a zoo. Gross findings, which were minimal, included poor body condition, bilateral renomegaly, and mottling of the liver. Histopathological lesions, which were particularly pronounced in the urogenital tract and liver, consisted primarily of inflammation associated with intracytoplasmic microsporidian spores. Polymerase chain reaction -based methods were used to establish the identity of the microsporidian as Encephalitozoon hellem. E. hellem is an emerging cause of human and avian disease, manifested mainly as opportunistic infection in immunosuppressed patients. This report describes the first documented case of E. hellem in a non-human mammalian species.

Animals↗