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Encephalitozoon spp. is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. This study was conducted to determine the prevalence of Encephalitozoon spp. in wild living rodents from Poland, the Czech Republic and Slovakia. Faecal and spleen samples were collected from individuals of Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, and Myodes glareolus (n = 465) and used for DNA extraction. PCR, targeting the ITS region of the rRNA gene was performed. The overall prevalence of microsporidia was 15.1%. The occurrence of Encephalitozoon cuniculi in the abovementioned host species of rodents has been presented for the first time, with the highest infection rate recorded for A. flavicollis. Sequence analysis showed that the most frequent species was E. cuniculi genotype II (92.5%). E. cuniculi genotypes I (1.5%) and III (6.0%) were also identified.

Microsporidia are obligate intracellular parasites that produce spores. The infections caused by these parasites are mostly considered to be opportunistic in immunodeficient patients. Because of the zoonotic nature of microsporidia as well as the increasing prevalence of immunodeficiency diseases, the aim of this study was to evaluate the molecular diagnosis of Enterocytozoon bieneusi (E. bieneusi) and Encephalitozoon spp. in exotic birds in southwestern Iran. Initially, 816 stool specimens were collected and stained by modified trichrome (Weber) staining. The slides were explored using light microscopy. In the next stage, the extracted DNA was amplified using a multiplex/nested PCR method. RFLP with the Mnl1 restriction enzyme was used to differentiate the Encephalitozoon species in the products of the molecular analysis. Out of 816 samples, 138 and 181 cases were found to be positive by the staining and the multiplex/nested-PCR methods, respectively. Of the 181 samples, 103 and 78 samples were positive for E. bieneusi and Encephalitozoon spp., respectively. The Encephalitozoon species were 17 E. cuniculi, 52 E. intestinalis and 9 E. hellem. Of 103 E. bieneusi samples, 57, 39, 2 and 5 cases were detected as genotypes D, M, E and L, respectively. The results showed a relatively high prevalence of microsporidia in exotic birds, and according to the results of the genotyping, these birds can be an important source of microsporidiosis. It is essential that high-risk individuals, including patients with immunodeficiency diseases, receive accurate and valid information about the risk of direct and indirect contact with infected exotic birds. Keywords: Enterocytozoon bieneusi, Encephalitozoon spp., Microsporidia, Exotic bird, PCR, Iran

Intestinal microsporidiosis is known as an opportunistic infection in immunocompromised patients. The current study aimed to investigate intestinal microsporidia infection in human subjects with/without immunodeficiency. Totally, 600 stool samples were collected from immunocompromised (254) and immunocompetent (346) subjects. DNA extraction was performed and the SSU rRNA and the ITS genes were amplified to detect and characterize microsporidia and the relevant genotypes. Phylogenetic trees were drawn using MEGA7 software to illustrate the correlation between isolates. From 600 enrolled subjects, 283 and 317 were male and female, respectively. The average age ± SD of all tested subjects was 28.85 ± 26.92. The results of PCR demonstrated the presence of E. bieneusi and Encephalitozoon sp., among 10/600 (1.67%) and 26/600 (4.33%) of samples, respectively. Accordingly, E. bieneusi was seen among 4/346 (1.15%), 1/53 (1.88%), 3/124 (2.42%), and 2/63 (3.17%), and Encephalitozoon sp., was detected from 17/346 (4.91%), 3/53 (5.36%), 4/124 (3.22%) and 2/63 (3.17%) of healthy subjects, RA patients, cancer patients, and transplantation recipients, respectively. Statistical significant correlation was not seen between the presence of microsporidia and age, gender, stool appearance, and geographical region. Molecular analysis showed that all E. bieneusi were the genotype D. Phylogenetic tree demonstrated no classification according to the presence/absence of immunodeficiency, geographical locations and presence of diarrhea. The high prevalence of Encephalitozoon sp., in comparison to E. bieneusi in this study suggested the importance of this genus alongside with E. bieneusi in Iran. In addition, predominance of the genotype D highlighted the wide distribution of this genotype in Iran.

International audience; Five cases of microsporidioses among leukemic patients, 4 in myeloid-leukemic patients and 1 in a chronic lymphocytic leukemia, have been described until now. We report a case of microsporidiosis and the genomic identification of Encephalitozoon hellem in a patient with CD4(+) T-cell prolymphocytic leukemia.

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.

Encephalitozoon is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts. In this study, we used nested PCR to investigate the presence of Encephalitozoon infection in Rodentia and Soricomorpha in Japan. We attempted to amplify and sequence Encephalitozoon-specific DNA from brain and viscera samples of 180 animals collected between 2008 and 2010. Forty-three samples (23.9%) from the orders Rodentia and Soricomorpha were positive for Encephalitozoon. This study is the first report of Encephalitozoon infection in Rodentia and Soricomorpha in Japan, and our findings suggest that these hosts may play a role in the spread of microsporidian spores in the environment.

a b s t r a c t Background: Microsporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare. Objective: To report genital microsporidiosis in female AIDS patients. Methods: Tissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kouri were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified. Results: We report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium. Conclusions: Microsporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract

Microsporidia are considered to be a cause of emerging and opportunistic infections in humans, and the species that infect humans can also infect a wide range of animals, raising concerns for zoonotic transmission. To understand the role of birds in the transmission of diseases caused by microsporidia, we examined 196 fecal specimens from birds, including birds of the families Psittacidae, Emberizidae, Icteridae and Columbidae, using Gram-chromotrope stain and polymerase chain reaction (PCR). Of the 196 fecal samples surveyed, 48 (24.5%) tested positive for microsporidia. The prevalence of microsporidia infection was higher in pigeons (31.1%) than in other birds (18.8%). The species of microsporidia that were detected in the birds surveyed in this study included Encephalitozoon hellem (found in 16.3% of positive samples), Enterocytozoon bieneusi (5.6%), Encephalitozoon intestinalis (1.5%) and Encephalitozoon cuniculi (1%). All the internal transcribed spacer (ITS) sequence of the rRNA from the study samples matched (with 100% identity) their correlate reference genotypes in GenBank, which included E. hellem 1A (AF338367), E. hellem 3 (AF110328), E. cuniculi I (AF338410) and E. bieneusi EpbA (AF076040). No fecal sample contained more than one type of microsporidian species. This study implicates exotic birds and pigeons as potential sources of microsporidia infection for humans living in urban areas.

Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (Co-A test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories.

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).

Being intracellular parasites, microsporidia can only be propagated in cell culture systems. This study evaluated three cell lines to determine the most suitable host-parasite In vitro system. Confluent monolayers of vero, SIRC, and HeLa cell lines, grown in 24-well tissue culture plates, were inoculated with varying concentrations (1 x 10 4 to 1 x 10 8 spores/mL) of Vittaforma corneae, Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis spores. Growth was compared quantitatively at weekly intervals. Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, while there was no significant difference in their growth in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. The analytical sensitivity was found to be 1 x 10 4 spores/mL for vero cell line compared to 1 x 10 5 spores/mL for SIRC cell line and 1 x 10 7 spores/mL for HeLa cell line. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris. This is the first report of the comparison of vero, SIRC, and HeLa for the propagation of microsporidial spores. Vero cell line was found to be more sensitive than SIRC and HeLa cells, and we believe that the inclusion of vero cell line in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield.

Encephalitozoon spp. are the primary microsporidial pathogens of humans and domesticated animals. In this experiment, we test the efficacy of 4 commercial antimicrobials against an Encephalitozoon sp. infecting a grasshopper (Romalea microptera) host. Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts (93% and 88% respectively), whereas spore counts of grasshoppers fed quinine produced a non-significant 53% reduction in spores, and those fed streptomycin a non-significant 29% increase in spores, compared to the control. We observed a moderate dose–response effect for thiabendazole, whereby spore count decreased as drug consumption increased. No thiabendazole-treated animals died, whereas 27% of streptomycin-treated animals died, suggesting that thiabendazole was not toxic at the doses administered. The deaths among streptomycin-treated animals may have been caused by drug toxicity, parasite burden, or both. Although fumagillin and thiabendazole significantly reduced spore counts, in no individual was the pathogen totally eliminated. Our data confirm that microsporidia are difficult to control and that fumagillin and thiabendazole are partially effective antimicrobials against this group. Our study suggests that quinine and related alkaloids should be further examined for antimicrosporidial activity, and streptomycin should be examined as a possible enhancer of microsporidiosis.

Microsporidia are obligate intracellular pathogens capable of infecting humans. There is credible evidence to suggest that microsporidial infections may be transmitted through consumption of spores in contaminated water; however, methods to detect this pathogen have not been standardized and microsporidia occurrence studies have not been conducted. Concentration of spores by continuous flow centrifugation (CFC), purification using immunomagnetic separation (IMS), and detection by either microscopy or real-time polymerase chain reaction (PCR) were evaluated for detection of Encephalitozoon intestinalis spores in seeded water samples. Recovery efficiency of CFC using microscopic detection ranged from 38.7–75.5% in filtered tap water. Using an indirect IMS method, 78.8–90.2% of seeded spores were recovered in ultrapure water (18 MΩ); however, the lack of a specific monoclonal antibody and the presence of other particulates interfered with the IMS assay in some turbid samples. Despite low recovery efficiencies and the detectable presence of PCR inhibitors in each of the samples, a combination of CFC concentration, indirect IMS, and real-time PCR produced a positive test result in six of ten natural water samples (turbidity 0.1–28.9 NTU) at a seeding level of 50 spores/L.

Microsporidial infections have been recognized as an increasingly important infection in immunocompromized patients, particularly those infected with HIV/AIDS. This study was designed to study immune responses associated with experimental Encephalitozoon intestinalis infection in immunecompetent rats.Thirty-four rats in 3 groups, A (Control), B (Intraperitoneal) and C (Oral) were given injections of 0.5 ml of 2 x 10(6) of purified spores of Encephalitotozoon intestinalis spores and were observed for serum specific IgG for 21 days using both Direct and Indirect ELISA.In indirect ELISA, specific lgG were detected on days 7, 14 and 21 for the group B rats and on day 21 for group C and in direct ELISA method, specific lgG were detected in-group B rats on days 7 and 21, for group C rats on day 21 only, while in the control rats, specific lgG were not detected. There was no significant difference between the direct and indirect methods (df=1, X(2), P0.05). E. intestinalis was observed in stool samples of rats in 1/12 (08.33%) on days 14 and 21 in group B and in 4/10 (33.33%), 3/10 (25.00%) and 2/10 (16.67%) on days 7, 14 and 21 respectively in group C. In-group, A which is the control rats, no microsporidia were observed on days 0, 7, 14 and 21.There were no changes in the T-lymphocyte counts of rats prior to and after inoculation with spores. Extensive lesions were observed along the intestinal walls especially on the middle and lower sections of group C rats only.

The order Microsporidia contains a number of ubiquitous pathogens that can infect various animals, including humans. Enterocytozoon bieneusi and Encephalitozoon intestinalis have been associated with gastrointestinal illness in humans. The effect of four disinfectants--ammonium hydroxide, hydrogen peroxide, and two commercial disinfectants containing peroxyacetic acid (Tsunami) and N-alkyl dimethyl benzyl ammonium chloride (Timsen)--on E. intestinalis spores was examined using exposure times of 1, 5, and 15 min. Spore viability was determined in vitro with RK-13 cells. Hydrogen peroxide was most efficient at inactivating microsporidial spores at all tested concentrations and treatment times, whereas ammonium hydroxide was effective only at the highest concentration at all exposure times. Tsunami (40 microg/ml) and Timsen (200 and 400 ppm) could inactivate spores when incubated for 5 and 15 min.

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.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

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.

The retention and transport of microsporidium Encephalitozoon intestinales spores in two water-saturated sandy porous media was investigated in this study. The initial breakthrough of the spores in the column effluent occurred essentially simultaneously with that of a non-reactive tracer, indicating no significant velocity enhancement. A large fraction (45-73%) of the spores injected into the columns was not recovered in the effluent, indicating removal from solution through colloid retention processes of attachment and/or straining. The relative significance of attachment and straining to total retention was evaluated in additional experiments. An experiment was conducted with a sieved coarse fraction of porous media for which straining is unlikely to be of significance based on the relative diameters of the spores and porous-medium pores. The spore recovery for this experiment was similar to the recoveries obtained for microsporidia transport in the un-sieved parent porous medium. An additional experiment was conducted with a subsample of the coarse fraction that was acid-washed to reduce potential surface attachment sites. Spore recovery was complete for this experiment. These results suggest surface deposition was the primary removal mechanism in our system. This conclusion is supported by the results of an experiment wherein deionized water was flushed through a column that was previously flushed with electrolyte solution. The effluent spore concentrations were observed to increase upon injection of deionized water, indicating re-mobilization of spores upon a change in water chemistry. The measured data were successfully simulated using a mathematical model incorporating colloid filtration. The results of this study suggest that the transport of microspordia in sandy porous media is governed by established colloid-transport processes.

This report describes the characterization and phylogenetic analysis of the deduced amino acid sequences of methionine aminopeptidase 2 (MetAP-2) enzymes from microsporidian species and genotypes of the genus Encephalitozoon. Fragments of DNA encoding 318 to 335 amino acid residues of the MetAP-2 genes were isolated from genomic DNA prepared from cultured spores of Encephalitozoon hellem, Encephalitozoon intestinalis, and Encephalitozoon cuniculi genotypes I-III. Sequence comparisons of the deduced amino acid residues indicated that the microsporidian sequences are MetAP-2-like rather than MetAP-1-like. Alignments demonstrated that the new Encephalitozoon sequences included sequences and structures conserved in eukaryotic MetAP-2s, including the five conserved, active site residues, Asp, Asp, His, Glu, and His, considered to be critical for catalysis and for coordinating the cation (e.g., cobalt) co-factor, and included residues known to interact with the antibiotic, fumagillin. The primary structure of the Encephalitozoon MetAP-2s, however, showed some dissimilarity with human and yeast MetAP-2s, including the absence of the NH2-terminal polylysine tract. Phylogenetic comparison of these Encephalitozoon MetAP-2s with orthologues from related species and from other informative taxa confirmed that the MetAP-2s of these Encephalitozoon species and strains are closely related to each other and cluster with MetAP-2s.

Microsporidia are obligate intracellular eukaryotic parasites that have a distinctive mechanism for infecting host cells. Microsporidian spores contain a long coiled polar tube that extrudes from the spores and can penetrate the membranes of new host cells. The contents of the spores are then forced through the tubes into the host cell cytoplasm. Recent studies have shown that microsporidia also gain access to host cells by phagocytosis. However, after phagocytosis, the microsporidian polar tube is used to escape from the maturing phagosomes and to infect the cytoplasm of host cells.

Encephalitozoon hellem is a single-celled, intracellular parasite of the phylum Microspora. E. hellem was first recognized in immunosuppressed humans where it causes keratoconjunctivitis, sinusitis, and, uncommonly, systemic disease. Increasing evidence strongly suggests that birds are the primary host for E. hellem and that most bird infections are self limiting and do not result in disease. To date, commercially raised lovebirds appear to have the highest prevalence of spore shedding of the birds examined. When disease does occur in birds, it is usually associated with another immunosuppressive infection, inadequate husbandry, or immaturity. The kidney, liver, and intestine are the most common targets of infection, but lesion distribution is variable. Disease has been documented in a wide range of companion birds including parrots and finches, in an ostrich, and in wild hummingbirds. Albendazole has been used for systemic treatment of E. hellem and albendazole in combination with fumagillin have been used to treat ocular manifestations of E. hellem .

For the first time, Encephalitozoon (E.) cuniculi genotype III (‘dog strain’) was verified in two cotton-top tamarins (Oedipomidas oedipus) by light microscopy, immunohistochemistry, electron microscopy, PCR and sequencing. The animals had a disseminated lethal infection with this protist. In earlier reports, genotype III had been found only in domestic dogs, man, emperor tamarins (Saguinus imperator) and golden lion tamarins (Leontopithecus rosalia). This investigation establishes now that the ‘dog strain’ can occur in cotton-top tamarins too. This is further evidence for the zoonotic potential of E. cuniculi. Furthermore, free E. cuniculi spores were identified also in blood vessels of several tissues. These findings indicate that during a disseminated infection E. cuniculi spores can occur in peripheral blood, too. We propose that blood should also be included in the investigations for the detection of microsporidia, so that a possible disseminated course of an infection can be detected.

Microsporidia are obligate intracellular eukaryotic parasites that infect a wide range of hosts, including invertebrates and vertebrates. Microsporidia have emerged as important opportunistic pathogens of humans with the onset of the AIDS pandemic. The potential impact of these infections in human pathology has required the development of antiparasitic strategies, based on the search for molecules having an effect on the development and/or the multiplication of microsporidia. This creates a demand for a simple and reliable in vitro technique for measuring the multiplication of microsporidia. We developed a new monoclonal antibody (MAb) enzyme-linked immunosorbent assay (ELISA) technique and measured the growth of Encephalitozoon intestinalis in an in vitro culturing system using this method. The monoclonal antibody is specific for a coat protein of E. intestinalis sporogonic stages produced in parasitophorous vacuole. An anti-mouse antibody labeled with peroxidase was used as conjugate. This ELISA is a suitable, specific and semiquantitative technique for measuring the spread of E. intestinalis. It is easy to perform and required 5 h from start to end. A good correlation was observed when the ELISA data were compared with the manual microscopic counts of parasitophorous vacuoles obtained after immunofluorescent assay (IFA). Moreover, the ELISA method proved more accurate than the immunofluorescent assay. In summary, the ELISA system described in this study provides a simple reliable assay for measuring the spread of microsporidia in vitro and may prove valuable for the screening of putative interesting antimicrosporidial compounds.

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal–oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log10 or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm2.

Microsporidia in dogs and cats is primarily caused by the obligate, intracellular parasite Encephalitozoon cuniculi, which is a member of the phylum Microsporidia. The aim of the current study is the detection of this parasite in stool samples of small animals of Iran, by polymerase chain reaction. Microsporidia spp. was found in 31% (31/100) of dogs (E. cuniculi (18/100), Encephalitozoon bieneusi (8/100) and Encephalitozoon intestinalis (5/100)), and 7.5% (3/40) of the specimens obtained from cats were infected with E. bieneusi. Sequencing of PCR products confirmed these results. In conclusion, Microsporidia infection seems to be fairly common in pet animals of Iran, especially in dogs. This finding could indicate the importance of pet animals as zoonotic reservoirs of microsporidial human infections.

This study describes fluorogenic 5′ nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi and E.intestinalis . The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10 4 and 10 −1 spores per PCR sample. The coefficients of variation were ≤5·2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10 4 spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.

Newly recognized waterborne pathogens such as microsporidia are being detected in the world's water supplies with increasing frequency. Many of these organisms have been shown to cause negative health impacts for both immunocompetent as well as immunocompromised individuals. It is imperative that these emerging pathogens be investigated for their ability to resist both traditional and novel disinfection technologies that are currently in use or under consideration for drinking water treatment. Low- and medium pressure UV light is at the cutting edge of disinfection technologies for the drinking water industry. While previous UV disinfection studies have focused on the inactivation of Cryptosporidium and Giardia as well as viruses and common bacteria, this research reports the ability of low- and medium pressure UV light to inactivate > 3.6 log10 of microsporidia Encephalitozoon intestinalis spores at a dose of 6 mJ/cm2 or higher as determined using a cell culture approach.

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.

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.

Encephalitozoon hellem is a microsporidian species that causes disseminated infections in HIV-positive patients. Identical genotypes of E. hellem, as assessed by the sequence of the rDNA internal transcribed spacer, have been identified in isolates from humans and from a psittacine bird. However, by analysing the rDNA ITS of four E. hellem isolates from Switzerland (three) and Tanzania (one), two new genotypes were identified. Differences among the E. hellem isolates were also detected by Western blot analysis, but there was no absolute match between ITS genotype and antigen profile. Hence, strain variation exists in E. hellem and the ITS sequence seems a valuable marker in obtaining further insight into the epidemiology of this pathogen.

Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogen's life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.

The microsporidian Encephalitozoon hellem is being reported with increasing frequency in HIV-positive subjects, as an agent of disseminated microsporidiosis without involving the gastrointestinal tract. We describe a case of pulmonary microsporidiosis in a 27-year-old Italian man with AIDS who developed fever, cough, and dyspnea. A chest X-ray showed multiple bilateral pulmonary opacities and mediastinal lymph-node enlargement. Stained smears of bronchoalveolar lavage sediment showed oval structures consistent with microsporidian spores. Viral, bacterial and fungal cultures were repeatedly negative, whereas microsporidia were successfully cultured in human and bovine fibroblast cell lines. Analysis of electron micrographs indicated that the isolate belonged to the genus Encephalitozoon. Based on further immunological, biochemical and molecular studies it was characterized as E. hellem. Even though a temporary improvement with albendazole therapy was noticed, the patient deteriorated clinically and died of severe respiratory distress.

Human microsporidiosis is a parasitic infection due to species of four different genera: Encephalitozoon; Enterocytozoon; Nosema; and Pleistophora. Although well known as a cause of disease in animals, microsporidiosis was only occasionally reported in humans. Recently, in human immunodeficiency virus (HIV)-infected patients, microsporidia belonging to Encephalitozoon and Enterocytozoon species have proved to be important opportunistic pathogens. Enterocytozoon bieneusi is associated with chronic intermittent diarrhea, cholangiopathy and sinusitis whereas Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi, the three Encephalitozoon species found in humans, are associated with diarrhea, rhinosinusitis, keratoconjunctivitis, nephritis and hepatitis. Diagnosis of microsporidial infections in humans was until recently an invasive, laborious procedure including electron microscopy of small intestine biopsies. However, new simple staining methods using Uvitex 2B or modified trichrome stain for feces and other body fluids have facilitated clinical diagnosis as well as drug evaluation and epidemiological studies. The application of monoclonal antibodies and molecular techniques such as the polymerase chain reaction have further improved microsporidial diagnosis. Treatment of Entero. bieneusi has, until now, been unsuccessful whereas albendazole has proved to be an effective treatment for Encephalitozoon species infection. Identification of effective treatment for Entero. bieneusi infections and further study of the pathogenicity of these microsporidial infections in immunocompetent hosts are important future challenges.

Summary Ciliatosporidium platyophryae nov. gen., nov. spec, is a microsporidian parasite affecting the soil ciliate Cirrophrya terricola Foissner, 1987. It was found in a soil sample from Utah (USA), pure cultures were not available. The developmental stages seen are uninucleate and divide by binary fission (disporoblastic). Each sporont is enveloped by a membranelike exospore precursor and a parasitophorous vacuole deriving from host endoplasmic reticulum and persisting during spore maturation. The spore is rod-shaped with hemispherical ends and measures about 4×2 μm. It has a short, uncoiled polar filament and conspicuous, possibly transient sacs in the wide anterior lamellae of the bipartite polaroplast. The exospore has the appearance of a unit membrane. Maturing spores induce the formation of appendages (“feet”) at the posterior end of the host which was thus formerly assigned to the genus Cirrophrya . However, our study shows that the feet are a pathological phenomenon and the host thus belongs to the genus Platyophrya: Platyophrya terricola (Foissner, 1987) nov. comb. Despite its unusual host, the fine structure of C. platyophryae is very similar to that of microsporidia from other invertebrate and vertebrate hosts. Very likely, C. platyophryae is related to the genera Oligosporidium, Encephalitozoon, Unikaryon and Endoreticulatus , presently assigned to the families Encephalitozoonidae and/or Unikaryonidae. However, for a definite classification of C. platyophryae more detailed data on its life cycle and membranous envelopes are necessary. Thus, we prefer an incerta sedis position.

The amitochondrial (a-mt) protozoa include four groups of organisms that are of interest as important human parasites and as probable descendents of the earliest branches of eukaryotic evolution. These organisms have not been directly compared in terms of structure and function of a specific molecule. We sequenced portions of their rRNA-encoding genes coding for the internal transcribed spacers (ITS1 and 2) and adjoining small subunit (SS), 5.8S and large subunit (LS) rRNAs. Included are sites for RNA processing, mRNA interaction and aminoglycoside binding, as well as potential protein-encoding genes. The ITS of all a-mt protozoa examined are relatively short, but otherwise diverse. They include one or two predominant nucleotides (A in Entamoeba and Trichomonas, T in Encephalitozoon and C in Giardia) and have minimal potential secondary structure, which may form the basis for the preferential processing of ITS sequences. The mechanism employed by a-mt protozoa to bind mRNA may be unique, since Giardia, Trichomonas and Entamoeba mRNAs have unusually short 5′ non-coding regions. In bacteria, the 3′ terminus of the SS rRNA is involved in mRNA binding; analysis of Entamoeba and Trichomonas mRNA 5′ non-coding sequences suggests an analogous mechanism involving potential base pairing to the loop of the terminal SS rRNA hairpin. Giardia sensitivity to paromomycin was previously correlated with the presence of a C:G by near the decoding region of SS rRNA. This bp is also present in Entamoeba and Trichomonas, consistent with their susceptibility. Its absence in Encephalitozoon and other microsporidia predicts paromomycin resistance, and suggests a distinct evolutionary origin for this group.

Summary A microsporidium, identified as Septata intestinalis Cali, Kotier and Orenstein, 1993, has been studied by light and electron microscopy from four patients with the acquired immunodeficiency syndrome (AIDS) in Sydney, Australia. The parasite was found in small intestinal and colonic enterocytes and lamina propria macrophages, urine, and in nasal epithelium, the last being a site of infection not previously reported for the species. Ultrastructurally it resembles species of Encephalitozoon in that it develops in host cells within parasitophorous vacuoles, with meronts and sporonts dividing mainly by binary fission, the meronts lying adherent to the vacuolar membrane and sporogonic stages free in the lumen. The microsporidium has additional features which are distinctive: the vacuole is lobed and the sporogonic stages that it contains are separated by septa that condense from a granular matrix with additions from host cell cytoplasmic debris derived by coalescence of vacuoles. The parasitophorous vacuole membrane is present throughout development, even around isolated meronts and the matrix is evenly distributed in vacuoles containing only meronts. Tubular structures apparently arising from the outer coat of sporonts and sporoblasts run through the vacuole. On the basis of these results, it is suggested that the description of the genus Septata is modified to state that the entire development is within parasitophorous vacuoles and that sporogony is mainly disporoblastic. These being characters of the genus Encephalitozoon, gene sequencing will be required further to evaluate the relationship of the two genera.

Encephalitozoon hellem is a recently described microsporidian associated with an expanding spectrum of clinical presentations in patients with the acquired immunodeficiency syndrome (AIDS). It is morphologically similar to Encephalitozoon cuniculi, a microsporidian infection of mammals and some avians, and their differentiation rests on biochemical and antigenic analyses. This report describes a patient previously diagnosed with keratoconjunctivitis due to E hellem who subsequently was found to have respiratory tract microsporidiosis by sputum cytology. He subsequently developed pulmonary symptoms and a left lower lobe interstitial infiltrate. A bronchoalveolar lavage and transbronchial biopsy revealed microsporidial bronchiolitis, and the etiologic agent was identified as E hellem using an immunofluorescent antibody technique. Lavage fluid was successfully cultured in monkey kidney cells, and cultivated E hellem organisms were studied using immunohistochemistry as well as scanning and transmission electron microscopy. The pathologic features of this newly described cause of protozoal bronchiolitis, the role of immunofluorescent antibody examination and in vitro tissue culture for species-specific diagnosis, and the significance of microsporidial pulmonary infections in AIDS patients are discussed.

Summary Contour-clamped homogeneous electric field gel electrophoresis was used to separate chromosome-sized DNA from two strains each of Encephalitozoon hellem and Encephalitozoon intestinalis isolated in vitro from AIDS patients. The two isolates of E. hellem differed significantly in molecular karyotype (EhD with 8 bands from 207 to 281 kb, and EhC with 12 bands from 175 to 315 kb), indicating a marked genetic polymorphism. The two E. intestinalis isolates displayed the same molecular karyotype with 10 bands comprised of between 182 and 279 kb. Of microsporidia so far investigated, species of Encephalitozoon have the smallest nuclear genomes (2.3–2.9 Mb).

Purpose To report a case of microsporidial keratoconjunctivitis in an HIV-seronegative. Methods We report on a 40-year-old woman who presented with acute symptoms of 4 days' duration. Visual acuity was 6/6 in the right eye and 6/6 partial in the left eye. In the left eye, there was diffuse conjunctival congestion with papillary changes. There were multiple raised epithelial lesions in the cornea. Results Corneal scrapings stained with potassium hydroxide and calcofluor white showed Microsporidium spores in large numbers. These spores with polar staining were also confirmed in Gram stain. One month after initiation of itraconazole, the left eye was quiet. She had a visual acuity of 6/6 in that eye, with faint corneal haze. Conclusions We report a rare case of microsporidial keratoconjunctivitis in a HIV-seronegative patient who was treated by debridement and oral itraconazole. Microsporidium should be suspected even in an immunocompetent individual if the corneal examination revealed showed multiple raised epithelial lesions.