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PubMed2003

Humoral immune response in adult blue foxes (Alopex lagopus) after oral infection with Encephalitozoon cuniculi spores.

Encephalitozoon cuniculi causes severe diseases in blue fox puppies. When pregnant vixens are infected, parasites are transmitted over the placenta to the unborn that subsequently develop encephalitozoonosis. Adult foxes themselves do not have signs of disease, but show antibody titres to E. cuniculi. The purpose of the present study was to gain information on the immune response in adult foxes after experimental infection. Sixteen foxes were infected orally with E. cuniculi spores, eight of them twice and 28 days apart. The two groups of animals showed elevated serological values in both the carbon immunoassay and in the ELISA. Elevated serological levels were recorded up to 1 year after the infection took place. The control group (n=8) remained serologically negative throughout the trial. The results of the study showed that blue foxes could be seropositive for at least a year after oral infection with E. cuniculi.

Animals↗
PubMed1999

Immune response to Encephalitozoon cuniculi infection in laboratory mice.

The study was performed to determine the immune response to Encephalitozoon cuniculi infection in immunocompetent mice during 120 days of experiment. Mice infected with E. cuniculi had an increased number of CD4+ T cells up to Day 20 post infection (p.i.), but counts of CD8+ T lymphocytes were significantly lower up to Day 90 p.i. in peripheral blood. Blood monocytes were significantly increased on the Day 60 and Day 120 of infection. A lack of significant decrease of CD4+ T cells may be considered as an important event in the immune response to E. cuniculi infection in immunocompetent mice.

Animals↗
PubMed1999

The first finding of antibodies to Encephalitozoon cuniculi in cows in Slovakia.

The presence of antibodies against Encephalitozoon cuniculi in cows was observed by the method of indirect immunofluorescence (IFAT). The animals reacting at the titre 1:64 and more were considered positive. Of the total number of 55 sera examined, 20 were positive at the titre 1:64, from which four were positive at the titre 1:256 also. Our results indicate that it is very important to carry out screening examinations in large-scale breeding of cattle due to the persistent high percentage of bovine abortions and disorders in the reproductive cycle of unknown etiology.

Abortion, Veterinary↗
PubMed1997

Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi.

The molecular karyotype of a murine isolate of Encephalitozoon cuniculi, a microsporidian with a wide range of mammalian hosts, comprises eleven chromosomes ranging in size between 217 and 315 kb. To determine specific chromosomal markers, a partial genomic library was constructed and cloned DNA fragments were hybridized to chromosomal bands separated by pulsed-field gel electrophoresis. Most probes were assigned to single chromosomes, indicating prevalence of low-copy number nucleotide sequences within the very small genome of E. cuniculi (2.9 Mb). A few probes were shown to hybridize to all chromosomes. These repetitive DNA fragments corresponded to either rRNA genes or some non-coding regions whose sequences were characterized by short micro- and minisatellites. The chromosomal locations of beta-tubulin genes and six newly identified protein-encoding genes were determined. Genes encoding dihydrofolate reductase, thymidylate synthase, serine hydroxymethyl transferase, a cdc2 kinase-like protein and helicase ERCC6-like protein were each located on a single chromosome whereas genes for both beta-tubulin and aminopeptidase were on two different chromosomes. The mapping will serve as a reference for further analysis of intraspecific karyotype polymorphism in different isolates from different host species.

Animals↗
PubMed2000

Presentation by scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon.

This paper presents, for the first time, documentation by detailed scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon. Phase 1 is represented by the extracellular phase with mature spores liberated by the rupture of host cells. To infect new cells the spores have to discharge their polar filament. Spores with everted tubes show that these are helically coiled. When the polar tubules have started to penetrate into a host cell they are incomplete in length. The infection of a host cell can also be initiated by a phagocytic process of the extruded polar filament into an invagination channel of the host cell membrane. After the penetration process, the tube length is completed by polar tube protein which passes through the tube in the shape of swellings. A completely discharged polar tube with its tip is also shown. The end of a polar tube is normally hidden in the cytoplasm of the host cell. After completion of the tube length the transfer of the sporoplasm occurs and phase 2 starts. Phase 2 is the proliferative phase, or merogony, with the intracellular development of the parasite that cannot be documented by scanning electron microscopy. The subsequent intracellular phase 3, or sporogony, starts when the meronts transform into sporonts, documented as chain-like structures which subdivide into sporoblasts. The sporoblasts finally transform directly into spores which can be seen in their host cell, forming bubble-like swellings in the cell surface.

Animals↗
PubMed2001

Immune response to Encephalitozoon cuniculi infection.

Microsporidia are obligate intracellular parasites, which can cause complications in immunocompromised individuals. Very little is known about the host immune response generated against these infectious agents. Encephalitozoon cuniculi is the best studied microsporidian and the protective immune response against this parasite is mediated by cytotoxic CD8(+) T cells.

Animals↗
PubMed2000

Polymorphism of the gene encoding a major polar tube protein PTP1 in two microsporidia of the genus Encephalitozoon.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78 bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvalI digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.

Amino Acid Sequence↗
PubMed1987

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Encephalitozoon cuniculi and its use in determination of infections in man.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Encephalitozoon cuniculi in man, using spores derived from tissue culture as antigen. Negligible cross-reactions were found with other microsporidia of vertebrate or of invertebrate origin and there was no cross-reaction with Toxoplasma gondii, using ELISA, immunoperoxidase or immunofluorescence staining. A high prevalence of antibodies to E. cuniculi was found in patients suffering from schistosomiasis, malaria and neurological and psychiatric disorders, but not in healthy individuals.

Animals↗
PubMed1991

Evidence for widespread occurrence of antibodies to Encephalitozoon cuniculi (Microspora) in man provided by ELISA and other serological tests.

The enzyme-linked immunosorbent assay (ELISA) was used to survey human sera for antibodies to Encephalitozoon cuniculi using spores obtained from in vitro cultures as antigen. Sera were obtained from patients with tropical diseases, neurological and renal disorders, patients who were HIV positive and those who had been tested for HIV but found to be negative. Sera from inhabitants of the village of Jali, The Gambia and from healthy blood donors were also examined. Numerous sera from all groups except the blood donors gave positive ELISA reactions at dilutions of 1:400. On titration, those with titres of 1:400 were reclassified as negative. Antibody titres of 1:800 and above were considered to be indicative of past or present infections with E. cuniculi. Many of these ELISA seropositives were also positive by IFAT or PAP. When examined by Western blotting of SDS-PAGE protein profiles of E. cuniculi spores, sera from many patients who had a tropical association reacted with the characteristic profiles shown by known positive mouse and rabbit sera. Others in the tropical group showed antibody binding to some but not all of the immunodominant polypeptides and yet others were negative in spite of their reactivity in the ELISA, IFAT or PAP test. Less agreement between ELISA and Western blotting results was obtained with the other groups of patients, although reactivity with one or more of the major polypeptide bands was sometimes seen. Serum from one blood donor, examined by ELISA and Western blotting, was positive. Differences in the methods of antigen preparation and of epitopes recognized by individuals may account for different reactivities in the tests. It is concluded that infections of E. cuniculi are common in the tropics and that reactivations of these infections might be a hazard to AIDS patients.

Adult↗
PubMed2002

Characterization of a cytosolic aminopeptidase from Encephalitozoon intestinalis.

Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70.8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4.8. The enzyme was both heat (> 37 degrees C) and cold (< 0 degrees C) labile with an optimal activity at pH 7.2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.

Animals↗
PubMed2005

Cell invasion and intracellular fate of Encephalitozoon cuniculi (Microsporidia).

Microsporidia are obligate intracellular parasites that utilize a unique mechanism to infect host cells, which is one of the most sophisticated infection mechanisms in biology. Microsporidian spores contain a long coiled polar tube that extrudes from the spores and penetrates the membranes of new host cells. We have initiated a study to investigate the invasive process and intracellular fate of the microsporidium Encephalitozoon cuniculi. Here we show that relatively few cells were infected through the traditional penetration of the polar tube from outside. Rather, phagocytosis of spores occurred at least 10 times more frequently than injection of sporoplasms. Some spores extruded their polar tube inside the cells following phagocytosis. Membranes of the vacuoles surrounding the internalized spores were positive for late endosomal and lysosomal markers. Spores that remained inside these compartments disappeared within 3 days. Thus, our studies demonstrate that in addition to the unique way in which microsporidia infect host cells, E. cuniculi spores can also gain access to host cells by phagocytosis. The presence of intracellular spores that have extruded their polar tube shows that some spores germinate after phagocytosis, thus escaping from the phagosomes that mature into lysosomes.

Animals↗
PubMed2006

Expression of two cell wall proteins during the intracellular development of Encephalitozoon cuniculi: an immunocytochemical and in situ hybridization study with ultrathin frozen sections.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

Animals↗
PubMed1997

Two Encephalitozoon cuniculi strains of human origin are infectious to rabbits.

The microsporidian Encephalitozoon cuniculi can infect a wide variety of mammals including man. In this study, E. cuniculi isolates of animal origin were compared with 6 isolates obtained from HIV-infected patients. Based on results of Western blot analysis, random amplified polymorphic DNA (RAPD) and the sequence of the rDNA internal transcribed spacer (ITS) the isolates were classified into 3 groups with the repeated element 5'GTTT-3' in the ITS being a reliable genetic marker. Five isolates from Swiss patients were found to be homologous to isolates from Swiss rabbits (strain I). The sixth isolate from a patient from Mexico differed by all methods and could be attributed to E. cuniculi strain III that has been described from 2 dogs from the USA. All of these isolates were distinguished from isolates from blue foxes from Norway (strain II). Intraspecific nucleotide divergence of the SSU rRNA gene of E. cuniculi belonging to the 3 strains was in the same low range (0.00-0.15%) as was found for the corresponding sequence of 2 E. hellem isolates. Groups of 2 rabbits were infected by oral inoculation of 10(7) E. cuniculi spores (2 isolates from strain I of human and rabbit origin, 1 from strain III) as shown by antibody responses and the re-isolation of the parasites from brain material. The results provide further evidence that per oral transmission of the parasite between various hosts is feasible.

Animals↗
PubMed1999

Molecular karyotype diversity in the microsporidian Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi can infect numerous mammals, including man. Three strains of E. cuniculi have been identified so far, the major marker being the number of a tetranucleotide repeats in the rDNA internal transcribed spacer. We investigated diversity at the chromosomal level through the electrophoretic karyotypes obtained from 15 E. cuniculi isolates from 5 different host species. All preparations provided patterns with 9-12 bands within a narrow molecular size range. Six karyotype forms were distinguished, involving subdivision of strain I into 3 types (A, B, C) and strain II into 2 types (D, E). The types A, B and C were mainly associated with isolates from rabbits of different geographical origins. The types D, E and F were characterized by a reduced chromosome size range, 2 of these appearing specific to a carnivorous host species (D in dog and F in blue fox). Hybridization experiments showed that all E. cuniculi isolates possess 11 chromosomes, with a size polymorphism entailing occasional electrophoretic comigration of heterologous chromosomes and differential migration of homologous ones. DNA rearrangements should occur during mitosis and the hypothesis of diploidy for the basic state of E. cuniculi seems likely.

Animals↗
PubMed2000

Isolation and characterization of an avian isolate of Encephalitozoon hellem.

Members of the phylum Microspora are a group of unusual, obligate intracellular eukaryotic parasites that infect a wide range of hosts. However, there are a limited number of microsporidial infections reported in avian hosts, and no parasite species has been defined as an avian pathogen. A microsporidian organism was recovered from the droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) and established in in vitro culture. Intermittent parasite spore shedding was documented over a 2-month period using calcofluor M2R staining of cloacal swabs. The organism was identified as Encephalitozoon hellem based on protein and antigenic profiles and molecular sequencing of the small subunit and internal transcribed spacer regions of the ribosomal RNA gene.

Animals↗
PubMed2004

An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1.

Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes. Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth. Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates. EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro. We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.

Amino Acid Sequence↗
PubMed2001

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.

Animals↗
PubMed2002

Disseminated microsporidiosis caused by Encephalitozoon cuniculi III (dog type) in an Italian AIDS patient: a retrospective study.

We report a case of disseminated microsporidiosis in an Italian woman with AIDS. This study was done retrospectively using formalin-fixed, paraffin-embedded tissue specimens obtained at autopsy. Microsporidia spores were found in the necrotic lesions of the liver, kidney, and adrenal gland and in ovary, brain, heart, spleen, lung, and lymph nodes. The infecting agent was identified as belonging to the genus Encephalitozoon based on transmission electron microscopy and indirect immunofluorescence. Additional molecular studies, including sequence of the rDNA internal transcribed spacer region, identified the agent as E. cuniculi, Genotype III. We believe that this is the first report of a human case of disseminated microsporidial infection involving the ovary.

AIDS-Related Opportunistic Infections↗