Microsporidia are eukaryotic, obligate intracellular organisms defined by their small spores containing a single polar tube that coils around the interior of the spore. After appropriate stimuli the germination of spores occurs. Conditions that promote germination vary widely among species, presumably reflecting the organism’s adaptation to their host and external environment as well as preventing accidental discharge in the environment. It appears that calcium may be a key ion in this process. Regardless of the stimuli required for activation, all microsporidia exhibit the same response to the stimuli, that is, increasing the intrasporal osmotic pressure. This results in an influx of water into the spore accompanied by swelling of the polaroplasts and posterior vacuole. The polar tube then discharges from the anterior pole of the spore in an explosive reaction and is thought to form a hollow tube by a process of eversion. If the polar tube is discharged next to a cell, it can pierce the cell and transfer the sporoplasm into this cell. Polar tubes resist dissociation in detergents and acids but dissociate in dithiothreitol. We have developed a method for the purification of polar tube proteins (PTPs) using differential extraction followed by reverse phase high performance liquid chromatography (HPLC). This method was used to purify for subsequent characterization PTPs from Glugea americanus, Encephalitozoon cuniculit E. hellem and E. intestinalis. These proteins appear to be members of a protein family that demonstrate conserved characteristics in solubility, hydrophobicity, mass, proline content and immunologic epitopes. These characteristics are probably important in the function of this protein in its self assembly during the eversion of the polar tube and in providing elasticity and resiliency for sporoplasm passage.
The production of three cytokines, interferon gamma (IFN-y), interleukin 10 (1L-10) and interleukin 12 (IL-12), was measured after intraperitoneal infection of immunocompetent Balb/c mice and immunodeficient SCID mice with the microsporidian, Encephalitozoon cuniculi Levaditi, Nicolau ct Schoen, 1923. High levels of IFN-y were detected in ex vivo cultures of peritoneal exudate cells (PEC) of Balb/c mice, a lower, but earlier IFN-y response was observed in PEC from SCID mice. The early 1L-10 response was detected in ex vivo cultures of splenocytes from Balb/c but not from SCID mice, explaining a delay in the IFN-y response in Balb/c mice. IL-12 was detected in PEC cultures from SCID mice, indicating an alternative pathway of IFN-y production by NK. cells stimulated by IL-12 derived from macrophages.
Two hundred and seventeen captive great apes (150 chimpanzees, Pan troglodytes; 14 bonobos, Pan paniscus; 53 western gorillas, Gorilla gorilla) and 20 personnel from thirteen European zoos and two African sanctuaries were sampled and examined in order to determine the occurrence of Enterocytozoon bieneusi and species of Encephalitozoon in faecal specimens and to compare the epidemiological situation between zoos and sanctuaries. Microsporidia were detected at all sampling sites. Sequence analyses of ITS amplicons generated by using microsporidia-specific primers determined the presence of microsporidia in 87 samples including 13 humans; since two cases of simultaneous occurrence of Encephalitozoon cuniculi and Enterocytozoon bieneusi were identified, 89 full-length ITS sequences were obtained, namely 78 Encephalitozoon cuniculi genotype I, five E. cuniculi genotype II, two E. hellem 1A and four Enterocytozoon bieneusi. No Encephalitozoon intestinalis-positive samples were identified. This is the first report of Encephalitozoon species and Enterocytozoon bieneusi genotypes in captive great apes kept under various conditions and the first record of natural infection with E. hellem in great apes. A comparison of zoos and sanctuaries showed a significantly higher prevalence of microsporidia in sanctuaries (P<0.001), raising a question about the factors affecting the occurrence of microsporidia in epidemiologically and sanitarily comparable types of facilities.
Microsporidia cause opportunistic infections in AIDS patients and commonly infect laboratory animals, as well. Euthymie C57B1/6 mice experimentally infected with intraperitoneal injections of lxlO6 Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923, Encephalitozoon hellem Didier et al., 1991, or Nosema comeum Shadduck et al., 1990 displayed no clinical signs of disease. Athymic mice, however, developed ascites and died 8-16 days after inoculation with N. comeum, 21-25 days after inoculation with E. cuniculi, and 34-37 days after inoculation with E. hellem. All athymic mice displayed hepatomegaly, dilated intestine and accumulation of ascites fluid. Granulomatous lesions were primarily located in the liver, lung, pancreas, spleen, and on serosal surfaces of abdominal organs.
Microsporidia are obligate intracellular eukaryotic parasites that utilize a unique mechanism to infect host cells. One of the main characteristics of all microsporidia is that they produce spores containing an extrusion apparatus that consists of a coiled polar tube ending in an anchoring disc at the apical part of the spore. With appropriate conditions inside a suitable host, the polar tube is discharged through the thin anterior end of the spore, thereby penetrating a new host cell for inoculating the infective sporoplasm into the new host cell. This method of invading new host cells is one of the most sophisticated infection mechanisms in biology and ensures that the microsporidia enter the host cell unrecognized and protected from the host defence reactions. Recent studies have shown that microsporidia gain access to host cells by phagocytosis as well. However, after phagocytosis, the special infection mechanism of the microsporidia is used to escape from the maturing phagosomes and to infect the cytoplasm of the cells. Gaining access to cells by endocytosis, and escaping destruction in the phago-/endo-/lysosome by egressing quickly from the phagocytic vacuole to multiply outside the lysosome, is a common phenomenon in biology that has been evolved several times during evolution. How this is put into execution by the microsporidia is an inimitable principle by which an obligate intracellular organism has managed this problem. The extrusion apparatus of the microsporidia has obviously ensured the success of this phylum during evolution, resulting in a group of obligate intracellular organisms, capable of infecting almost any type of host and cell.
Intestinal microsporidiosis was documented by detecting abundant slightly curved spores (2.9 x 1.2 pm) in the faeces of five of twelve skinks Mabuya perrotetii Duméril et Bibron, 1839 that originated from Ghana. Clinically, the microsporidiosis was characterized by decreased appetite, diarrhea, and weight loss. Histopathological changes consisted of villous atrophy, blunting of mucosa and flattening of individual epithelial cells in the large intestine. The ultrastructure of microsporidian spores was consistent with an Encephalitozoon species. The PCR-RFLP assay and the heleroduplex mobility shift analyses were used to verify that the skink microsporidian is a species of the genus Encephalitozoon Levaditi, Nicolau et Schoen, 1923 and indicate that this microsporidian is not E. hellem, E. intestinalis or a strain of E. cuniculi. The microsporidia in African skink represent an Encephalitozoon species morphologically identical to Encephalitozoon lacerine Canning, 1981.
The Microsporidia have been reported to cause a wide range of clinical diseases particularly in patients that are immunosuppressed. They can infect virtually any organ system and cases of gastrointestinal infection, encephalitis, ocular infection, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles such as albendazole are active against many species of Microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin, ovalicin and their analogues have been demonstrated to have antimicrosporidial activity in vitro and in animal models of microsporidiosis. Fumagillin has also been demonstrated to have efficacy in human infections due to E. bieneusi. Fumagillin is an irreversible inhibitor of methionine aminopeptidase type 2 (MetAP2). Homology cloning employing the polymerase chain reaction was used to identify the MetAP2 gene from the human pathogenic microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Brachiola algerae and E. bieneusi. The full-length MetAP2 coding sequence was obtained for all of the Encephalitozoonidae. Recombinant E. cuniculi MetAP2 was produced in baculovirus and purified using chromatographic techniques. The in vitro activity and effect of the inhibitors bestatin and TNP-470 on this recombinant microsporidian MetAP2 was characterized. An in silico model of E. cuniculi MetAP2 was developed based on crystallographic data on human MetAP2. These reagents provide new tools for the development of in vitro assay systems to screen candidate compounds for use as new therapeutic agents for the treatment of microsporidiosis.
Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We used sequence data of the rRNA gene for the identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi, and E. hellem in different biological samples of HIV-infected patients by PCR, Southern blot hybridization, restriction endonuclease digestion analysis, cloning, and comparative genetic sequencing. One primer pair was used for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E, intestinalis, and E. hellem from samples with electron microscopy confirmed infection. The amplified 1.2 kb SSU-rRNA gene fragments were ligated into a pMOSBlue T-vector, transfected into pMOSS/ме competent cells, and were used as positive controls. Several primer pairs and hybridization probes were used to amplify and identify microsporidian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron microscopy was done on a subset of patient samples. DNA products were obtained from all samples with confirmed microsporidial infections. The identity of the DNA fragments was determined by Southern blot hybridization or by restriction endonuclease digestion analysis or by DNA sequencing. The results show that PCR is a reliable and sensitive indicator for the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differentiation of microsporidia, even between species which cannot be differentiated by light and/or electron microscopy.
The genome sequence of the microsporidian parasite Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 contains about 2,000 genes that are representative of a non-redundant potential proteome composed of 1,909 protein chains. The purpose of this review is to relate some advances in the characterisation of this proteome through bioinformatics and experimental approaches. The reduced diversity of the set of E. cuniculi proteins is perceptible in all the compilations of predicted domains, orthologs, families and superfamilies, available in several public databases. The phyletic patterns of orthologs for seven eukaryotic organisms support an extensive gene loss in the fungal clade, with additional deletions in E. cuniculi. Most microsporidial orthologs are the smallest ones among eukaryotes, justifying an interest in the use of these compacted proteins to better discriminate between essential and non-essential regions. The three components of the E. cuniculi mRNA capping apparatus have been especially well characterized and the three-dimensional structure of the cap methyltransferase has been elucidated following the crystallisation of the microsporidial enzyme Ecm1. So far, our mass spectrometry-based analyses of the E. cuniculi spore proteome has led to the identification of about 170 proteins, one-quarter of these having no clearly predicted function. Immunocytochemical studies are in progress to determine the subcellular localisation of microsporidia-specific proteins. Posttranslational modifications such as phosphorylation and glycosylation are expected to be soon explored.
Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health., Martin Kváč, Lada Hofmannová, Ynes Ortega, Nikola Holubová, Michaela Horčičková, Marta Kicia, Lenka Hlásková, Dana Květoňová, Bohumil Sak, John McEvoy., and Obsahuje bibliografii