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Govinda S. Visvesvara - One of the best experts on this subject based on the ideXlab platform.

  • Genotyping Encephalitozoon Hellem isolates by analysis of the polar tube protein gene.
    Journal of clinical microbiology, 2001
    Co-Authors: Lihua Xiao, Simonetta Gatti, Massimo Scaglia, Elizabeth S Didier, Hercules Moura, Irshad M. Sulaiman, Altaf A. Lal, Govinda S. Visvesvara
    Abstract:

    To develop an alternative genotyping tool, the genetic diversity of Encephalitozoon Hellem was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24 E. Hellem isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate E. Hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of E. Hellem in clinical samples.

  • Immunologic, Microscopic, and Molecular Evidence of Encephalitozoon intestinalis (Septata intestinalis) Infection in Mammals Other than Humans
    The Journal of infectious diseases, 1998
    Co-Authors: Fernando J. Bornay-llinares, Hercules Moura, Govinda S. Visvesvara, Alexandre J. Da Silva, David A. Schwartz, Norman J. Pieniazek, Antonio Cruz-lópez, Pablo Hernández-jaúregui, Jorge Guerrero, F. Javier Enriquez
    Abstract:

    Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species. This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon Hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using "quick-hot" Gram-chromotrope stain. None of the 10 samples reacted with anti-E. Hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. Hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.

  • Identification of the microsporidian Encephalitozoon Hellem using immunoglobulin G monoclonal antibodies.
    Archives of pathology & laboratory medicine, 1998
    Co-Authors: G P Croppo, Sara Wallace, Govinda S. Visvesvara, D A Schwartz
    Abstract:

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

  • Effects of nifedipine, metronidazole, and nitric oxide donors on spore germination and cell culture infection of the microsporidia Encephalitozoon Hellem and Encephalitozoon intestinalis.
    Antimicrobial agents and chemotherapy, 1996
    Co-Authors: Gordon J. Leitch, Govinda S. Visvesvara, S Wallace
    Abstract:

    Two species of microsporidia, Encephalitozoon Hellem and Encephalitozoon intestinalis, were isolated from AIDS patients and cultured in green monkey kidney cells. A spore germination assay and a cultured-cell infection assay were used to test the efficacy of candidate antiparasitic agents. The calcium channel blocker nifedipine, metronidazole, and two nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, were tested in the two assays. Nifedipine (10(-8) M) significantly inhibited E. Hellem spore germination in three of four germination media. Metronidazole (10(-5) M) inhibited germination weakly and significantly inhibited E. intestinalis germination in a single germination medium. The inhibitory effect of nifedipine and metronidazole used together was greater than the sum of the effects of the drugs used alone in all E. Hellem germination assays. The NO donors also inhibited spore germination. The inhibitory effect of nifedipine and metronidazole could be reversed by washing the spores, while that of the NO donors was not reversible. In early cultured-cell infections, both nifedipine (10(-8) M) and metronidazole (10(-5) M) significantly reduced the number of cells being infected. As the infection spread, these agents were less effective. Some inhibition of the spread of the infection was also demonstrated with the NO donors at a concentration (10(-5) M) not obviously toxic to the cultured cells. These data suggest that combination drug therapy targeting spore germination and intracellular parasite development is promising.

  • Calcium and hydrogen ion concentrations in the parasitophorous vacuoles of epithelial cells infected with the microsporidian Encephalitozoon Hellem.
    The Journal of eukaryotic microbiology, 1995
    Co-Authors: Gordon J. Leitch, Govinda S. Visvesvara, Mary Scanlon, Sara Wallace
    Abstract:

    Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon Hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack ofa pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. Hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.

Michel Drancourt - One of the best experts on this subject based on the ideXlab platform.

  • Antigenic diversity of Encephalitozoon Hellem demonstrated by subspecies-specific monoclonal antibodies.
    The Journal of eukaryotic microbiology, 2002
    Co-Authors: Michel Drancourt
    Abstract:

    Encephalitozoon Hellem is a unicellular, obligate intracellular microsporidian species detected and isolated in HIV-infected patients presenting with keratoconjunctivitis, sinusitis, tracheobronchitis, nephritis, cystitis, and disseminated infection. A total of 24 monoclonal antibodies were produced against E. Hellem and characterized. The monoclonal antibodies were of the immunoglobulin (Ig) G and Ig M subclasses, and, when incorporated into indirect immunofluorescence and immunoblotting assays, reacted against 13 isolates of E. Hellem originating from three geographic regions. These monoclonal antibodies did not react with one strain each of either Encephalitozoon intestinalis or Encephalitozoon cuniculi, demonstrating their specificity. Two monoclonal antibodies reacted with all karyotype B-E. Hellem isolates but did not react with karyotype A-isolates from North America and the Netherlands, thus demonstrating antigenic diversity among E. Hellem isolates. These results add to the increasing evidence for diversity among E. Hellem, which therefore may be reclassified into subspecies.

  • Genetic Diversity in the Microsporidian Encephalitozoon Hellem Demonstrated by Pulsed-Field Gel Electrophoresis
    The Journal of eukaryotic microbiology, 2001
    Co-Authors: Sebastien Delarbre, Massimo Scaglia, Simonetta Gatti, Michel Drancourt
    Abstract:

    Encephalitozoon Hellem is a microsporidian species responsible for opportunistic infections in AIDS patients. Use of a novel chitinase-based method allowed unsheared chromosomal DNA to be recovered from eleven E. Hellem isolates derived from three geographic regions. All isolates were typed by 18S rDNA sequencing, which showed that they belonged to internal transcribed spacer type 1. After ethidium bromide staining, pulsed-field gel electrophoresis (PFGE) analysis discriminated two new karyotypes comprising 7 and 8 chromosomal bands respectively, ranging in size from 205- to 272-kb pairs. Genomic size was estimated to be 2.39 Mb. Our data indicate PFGE is useful for typing E. Hellem and confirms genetic diversity among E. Hellem genotypes.

  • in vitro susceptibilities of the microsporidia Encephalitozoon cuniculi Encephalitozoon Hellem and Encephalitozoon intestinalis to albendazole and its sulfoxide and sulfone metabolites
    Antimicrobial Agents and Chemotherapy, 1998
    Co-Authors: Olivier Ridoux, Michel Drancourt
    Abstract:

    The microsporidia Encephalitozoon cuniculi, Encephalitozoon Hellem, and Encephalitozoon intestinalis are emerging obligate intracellular pathogens causing infections in human immunodeficiency virus-infected patients (24). Albendazole has been successfully used in Encephalitozoonoses (2, 5, 10, 11, 19, 20). However, incomplete response (25) and relapses have also been documented (15, 23, 26). These clinical investigations have been accompanied by a handful of noncomparative laboratory studies; however, all but one of these (14) have not assessed the role of albendazole-sulfoxide and albendazole-sulfone active metabolites (8). We therefore compared the experimental activities of albendazole, albendazole-sulfoxide, and albendazole-sulfone on Encephalitozoon species in MRC5 cell culture. The three benzimidazoles (SmithKline Beecham, Nanterre, France) were dissolved at 1 mg/ml in sterile dimethyl sulfoxide (DMSO), and 10-fold serial dilutions (100 to 10−4 μg/ml) were prepared in minimum essential medium (MEM) (Eurobio, Paris, France). Spores of E. cuniculi, E. Hellem, and E. intestinalis strains (kindly provided by T. van Gool, University of Amsterdam, Amsterdam, The Netherlands) were cocultivated with MRC5 embryonic lung fibroblasts (BioMerieux, Lyon, France) in a mixture of MEM, 1% glutamine, and 10% heat-inactivated fetal calf serum (Flow Laboratories, Paris, France) at 35°C in a 5% CO2 atmosphere. The infection rate was monitored by rapid-heating Gram-chromotrope staining (21) and microscopic counting of spores in a Kova-slide (Hycor Biomedical, Inc., Irvine, Calif.). For antimicrosporidian activity assays, MRC5 cells subcultured at confluence on glass coverslips in a 24-well microplate were incubated with 100 μl of a suspension of 106 spores/ml for 5 h at 37°C. One milliliter of culture medium, with or without benzimidazole, was then added, and the plate was incubated at 37°C in a 5% CO2 incubator for 6 days. For each plate, a benzimidazole-free positive control and benzimidazole dilutions of 1 to 10−4 μg/ml were tested. After incubation, the mean and standard deviation of the number of microsporidia per field were determined by microscopic examination of rapidly heated Gram-chromotrope-stained coverslips (20 fields observed). The percentage of microsporidian growth inhibition was calculated as [1 − (mean number of infected cells in replicate cultures with benzimidazole/mean number of infected cells in control cultures)] × 100 (± standard error). The benzimidazole concentration inhibiting 90% of microsporidian growth in a control culture (IC90) was estimated from plots of spore number versus log benzimidazole concentration. Each benzimidazole was tested in triplicate. The potential toxicities of benzimidazole and DMSO on MRC5-cultured cells were examined by using a microplaque colorimetric assay adaptated from that previously reported (22). Analysis of variance was used to compare the median and standard deviation values of optical density in the toxicity test and to compare the percentages of growth inhibition for each microsporidian species and for each concentration of each benzimidazole. The Kruskall-Wallis test was used when variances were not homogeneous according to Bartlett’s test. DMSO at a final concentration of 10−3 μg/ml had no toxic effect on MRC5 cells, whereas a statistically significant, moderate toxic effect was observed with albendazole at 100 to 10−4 μg/ml, with albendazole-sulfoxide at 100 μg/ml, and with albendazole-sulfone at 100 to 10−1 μg/ml (P < 0.05) (Table ​(Table1).1). Any benzimidazole concentration tested was significantly effective in inhibiting the growth of any of the three Encephalitozoon species (P < 0.0001) (Table ​(Table1).1). The percentage of E. Hellem growth inhibition varied from 97.34 to 90.68% for albendazole, from 96.39 to 92.9% for albendazole-sulfoxide, and from 98.78 to 91.73% for albendazole-sulfone. The IC90s were 2.7 × 10−4 μg/ml for albendazole and <10−4 μg/ml for albendazole-sulfoxide and -sulfone. The percentages of E. cuniculi growth inhibition varied from 97.29 to 53% for albendazole, from 96 to 75.98% for albendazole-sulfoxide, and from 100 to 71.63% for albendazole-sulfone. The IC90s were 3.3 × 10−2 μg/ml for albendazole, 6 × 10−3 μg/ml for sulfoxide, and 2 × 10−3 μg/ml for sulfone. The percentages of E. intestinalis growth inhibition varied from 100 to 29.16% for albendazole, from 100 to 53.43% for albendazole-sulfoxide, and from 98.94 to 50.39% for albendazole-sulfone. The IC90s were 7.1 × 10−2 μg/ml for albendazole, 3.8 × 10−2 μg/ml for sulfoxide, and 10−1 μg/ml for sulfone. TABLE 1 Intracellular efficacy of albendazole and its sulfoxide and sulfone metabolites on microsporidial growth and estimation of MRC5 host cell viability Statistical analysis indicated that albendazole-sulfoxide and albendazole-sulfone at concentrations >10−4 μg/ml were significantly (5 to 15 times) more effective than albendazole against E. intestinalis, and albendazole-sulfone was significantly (5 times) more effective than albendazole-sulfoxide (P < 0.05) against any of the three species. E. intestinalis was significantly less susceptible than the two other microsporidian species, regardless of the benzimidazole concentration (P < 0.0001). For benzimidazole concentrations <10−2 μg/ml, E. cuniculi was significantly less susceptible than E. Hellem (P < 0.0001). The previously unreported toxicity for albendazole and, to a lesser extent, its metabolites, we observed could be due to the fact that the microplaque colorimetric assay we used is highly sensitive as a result of the large number of measurements collected for individual benzimidazole concentrations. This moderate toxic effect had a minimal effect on the IC90 determinations. Previous IC determinations of albendazole activity against Encephalitozoon spp. were 2.5 to 0.008 μg/ml for E. cuniculi (1, 4, 9, 16, 27), 0.005 to 0.008 μg/ml for E. intestinalis (3, 14), and 0.008 μg/ml for E. Hellem (4). The higher ICs we observed may have resulted from the experimental model we used, which included simultaneous inoculation and treatment; previously, albendazole had been added to well-established cultures. We found E. intestinalis to be less susceptible to albendazole and its two major derivatives than E. Hellem and E. cuniculi. The new data were obtained as the three Encephalitozoon species were tested in parallel. Previous studies have included only one species, thus preventing the accurate comparison of interspecies susceptibilities. Albendazole acts by disrupting microsporidian microtubules through β-tubulin binding (17). Even if six β-tubulin residues identified as being predictive for benzimidazole susceptibility in parasites (13) exhibited a sequence predictive of susceptibility in E. cuniculi, E. Hellem (7), and E. intestinalis (6), divergences in primary sequences may support differences in susceptibility. Alternative hypotheses include E. intestinalis-related altered intracellular penetration or metabolism of albendazole. We also confirmed that albendazole-sulfone is significantly more effective against the Encephalitozoon species than albendazole-sulfoxide, which in turn is more effective than albendazole. E. intestinalis has previously been found to be 1.7 times more susceptible to albendazole-sulfoxide than to albendazole (14), but no data have been presented for albendazole-sulfone. Indeed, in patients treated with oral albendazole, albendazole and albendazole-sulfone remained undetectable (i.e., <0.02 μg/ml) (18), whereas albendazole-sulfoxide concentrations varied between 0.1 and 0.5 μg/ml (12, 18).

  • In vitro susceptibilities of the microsporidia Encephalitozoon cuniculi, Encephalitozoon Hellem, and Encephalitozoon intestinalis to albendazole and its sulfoxide and sulfone metabolites.
    Antimicrobial Agents and Chemotherapy, 1998
    Co-Authors: Olivier Ridoux, Michel Drancourt
    Abstract:

    The microsporidia Encephalitozoon cuniculi, Encephalitozoon Hellem, and Encephalitozoon intestinalis are emerging obligate intracellular pathogens causing infections in human immunodeficiency virus-infected patients (24). Albendazole has been successfully used in Encephalitozoonoses (2, 5, 10, 11, 19, 20). However, incomplete response (25) and relapses have also been documented (15, 23, 26). These clinical investigations have been accompanied by a handful of noncomparative laboratory studies; however, all but one of these (14) have not assessed the role of albendazole-sulfoxide and albendazole-sulfone active metabolites (8). We therefore compared the experimental activities of albendazole, albendazole-sulfoxide, and albendazole-sulfone on Encephalitozoon species in MRC5 cell culture. The three benzimidazoles (SmithKline Beecham, Nanterre, France) were dissolved at 1 mg/ml in sterile dimethyl sulfoxide (DMSO), and 10-fold serial dilutions (100 to 10−4 μg/ml) were prepared in minimum essential medium (MEM) (Eurobio, Paris, France). Spores of E. cuniculi, E. Hellem, and E. intestinalis strains (kindly provided by T. van Gool, University of Amsterdam, Amsterdam, The Netherlands) were cocultivated with MRC5 embryonic lung fibroblasts (BioMerieux, Lyon, France) in a mixture of MEM, 1% glutamine, and 10% heat-inactivated fetal calf serum (Flow Laboratories, Paris, France) at 35°C in a 5% CO2 atmosphere. The infection rate was monitored by rapid-heating Gram-chromotrope staining (21) and microscopic counting of spores in a Kova-slide (Hycor Biomedical, Inc., Irvine, Calif.). For antimicrosporidian activity assays, MRC5 cells subcultured at confluence on glass coverslips in a 24-well microplate were incubated with 100 μl of a suspension of 106 spores/ml for 5 h at 37°C. One milliliter of culture medium, with or without benzimidazole, was then added, and the plate was incubated at 37°C in a 5% CO2 incubator for 6 days. For each plate, a benzimidazole-free positive control and benzimidazole dilutions of 1 to 10−4 μg/ml were tested. After incubation, the mean and standard deviation of the number of microsporidia per field were determined by microscopic examination of rapidly heated Gram-chromotrope-stained coverslips (20 fields observed). The percentage of microsporidian growth inhibition was calculated as [1 − (mean number of infected cells in replicate cultures with benzimidazole/mean number of infected cells in control cultures)] × 100 (± standard error). The benzimidazole concentration inhibiting 90% of microsporidian growth in a control culture (IC90) was estimated from plots of spore number versus log benzimidazole concentration. Each benzimidazole was tested in triplicate. The potential toxicities of benzimidazole and DMSO on MRC5-cultured cells were examined by using a microplaque colorimetric assay adaptated from that previously reported (22). Analysis of variance was used to compare the median and standard deviation values of optical density in the toxicity test and to compare the percentages of growth inhibition for each microsporidian species and for each concentration of each benzimidazole. The Kruskall-Wallis test was used when variances were not homogeneous according to Bartlett’s test. DMSO at a final concentration of 10−3 μg/ml had no toxic effect on MRC5 cells, whereas a statistically significant, moderate toxic effect was observed with albendazole at 100 to 10−4 μg/ml, with albendazole-sulfoxide at 100 μg/ml, and with albendazole-sulfone at 100 to 10−1 μg/ml (P < 0.05) (Table ​(Table1).1). Any benzimidazole concentration tested was significantly effective in inhibiting the growth of any of the three Encephalitozoon species (P < 0.0001) (Table ​(Table1).1). The percentage of E. Hellem growth inhibition varied from 97.34 to 90.68% for albendazole, from 96.39 to 92.9% for albendazole-sulfoxide, and from 98.78 to 91.73% for albendazole-sulfone. The IC90s were 2.7 × 10−4 μg/ml for albendazole and 10−4 μg/ml were significantly (5 to 15 times) more effective than albendazole against E. intestinalis, and albendazole-sulfone was significantly (5 times) more effective than albendazole-sulfoxide (P < 0.05) against any of the three species. E. intestinalis was significantly less susceptible than the two other microsporidian species, regardless of the benzimidazole concentration (P < 0.0001). For benzimidazole concentrations

Elizabeth S Didier - One of the best experts on this subject based on the ideXlab platform.

  • Encephalitozoon Hellem infection in aviary passerine and psittacine birds in spain
    Veterinary Parasitology, 2016
    Co-Authors: J Rosell, M Mainez, Elizabeth S Didier, L C Bowers, A Marco, Carles Juansalles
    Abstract:

    A European goldfinch (Carduelis carduelis), a canary (Serinus canaria), and a lovebird (Agapornis roseicollis) captive-bred at three different private aviaries in Spain were submitted for necropsy with a history of weakness and ruffled feathers, weight loss associated with glossitis, and respiratory disease, respectively. Microscopically, enterocytes in the jejunum and ileum contained colonies of gram- and Stamp-positive, oval to elliptical microorganisms within parasitophorous vacuoles in the apical cytoplasm. Nested PCR using MSP primers that target microsporidian RNA genes produced amplicons of expected size for Encephalitozoon species, and analysis of forward and reverse DNA sequences confirmed the presence of Encephalitozoon Hellem in all cases. The main cause of death of all three birds consisted of concurrent infections. However, intestinal Encephalitozoonosis may have contributed to exacerbated catabolism. Encephalitozoonosis (or microsporidiosis) has been rarely described in passerine birds.

  • Therapeutic strategies for human microsporidia infections.
    Expert review of anti-infective therapy, 2005
    Co-Authors: Elizabeth S Didier, Joseph A. Maddry, Paul J. Brindley, Mary E. Stovall, Peter J. Didier
    Abstract:

    Over the past 20 years, microsporidia have emerged as a cause of infectious diseases in AIDS patients, organ transplant recipients, children, travelers, contact lens wearers and the elderly. Enterocytozoon bieneusi and the Encephalitozoon spp., Encephalitozoon cuniculi, Encephalitozoon Hellem and Encephalitozoon intestinalis, are the most frequently identified microsporidia in humans, and are associated with diarrhea and systemic disease. The microsporidia are small, single-celled, obligately intracellular parasites that have been identified in water sources, as well as in wild, domestic and food-producing farm animals, thereby raising concerns for waterborne, foodborne and zoonotic transmission. Current therapies for microsporidiosis include albendazole, a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon spp., although it is less effective against Encephalitozoon bieneusi. Fumagillin, an antibiotic and antiangiogenic compound p...

  • Genotyping Encephalitozoon Hellem isolates by analysis of the polar tube protein gene.
    Journal of clinical microbiology, 2001
    Co-Authors: Lihua Xiao, Simonetta Gatti, Massimo Scaglia, Elizabeth S Didier, Hercules Moura, Irshad M. Sulaiman, Altaf A. Lal, Govinda S. Visvesvara
    Abstract:

    To develop an alternative genotyping tool, the genetic diversity of Encephalitozoon Hellem was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24 E. Hellem isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate E. Hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of E. Hellem in clinical samples.

  • Encephalitozoon Hellem in Budgerigars (Melopsittacus undulatus)
    Veterinary Pathology, 1997
    Co-Authors: S. S. Black, L. A. Steinohrt, D. C. Bertucci, L B Rogers, Elizabeth S Didier
    Abstract:

    Microsporidiosis with concurrent megabacteriosis in budgerigar (Melopsittacus undulatus) chicks contributed to significant economic loss in a commercial pet bird aviary in Mississippi. Three budgerigar chicks, 1-2 weeks old, from the aviary were necropsied. Microscopic lesions in the chicks consisted of heavy infection of enterocytes with microsporidia (2/3; autolysis precluded critical evaluation of the intestine of chick No. 2), multifocal hepatic necrosis and inflammation with intralesional microsporidia (1/3), spherical clusters of microsporidia in the hepatic sinusoids in the absence of inflammation (1/3), and gastric megabacteriosis (3/3). The ultrastructure of the microsporidian spores was consistent with an Encephalitozoon species. The polymerase chain reaction and Southern blot analysis were used to identify the microsporidian as Encephalitozoon Hellem, an organism that has only been identified in humans. Encephalitozoon Hellem causes keratoconjunctivitis and respiratory infections in humans with...

  • Diagnosis of disseminated microsporidian Encephalitozoon Hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin.
    Journal of clinical microbiology, 1996
    Co-Authors: Elizabeth S Didier, L B Rogers, Alan D. Brush, S Wong, Vicki Traina-dorge, D. C. Bertucci
    Abstract:

    A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon Hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. Hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. Hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. Hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. Hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. Hellem, and E. intestinalis in clinical specimens.

Ralph T. Bryan - One of the best experts on this subject based on the ideXlab platform.

  • Disseminated Microsporidiosis Due to Encephalitozoon Hellem: Pulmonary Colonization, Microhematuria, and Mild Conjunctivitis in a Patient with AIDS
    Clinical Infectious Diseases, 1993
    Co-Authors: Rainer Weber, Govinda S. Visvesvara, David A. Schwartz, Herbert Kuster, Ralph T. Bryan, Ruedi Lüthy
    Abstract:

    Four genera of microsporidia have been associated with disease in humans, which predominantly affects immunocompromised persons. Systemic infection with a newly characterized microsporidian species, Encephalitozoon Hellem, was recently reported in a patient with AIDS. This article describes a second patient with AIDS and disseminated E. Hellem infection. In this case the parasite was detected in sputum, urine, and conjunctival swab specimens. Apart from recurrent mild conjunctivitis and asymptomatic microhematuria, the patient had no findings or symptoms that could be related to this parasite. Specifically, no microsporidian-associated pulmonary pathology was documented

  • Treatment of microsporidial keratoconjunctivitis with topical fumagillin
    American journal of ophthalmology, 1993
    Co-Authors: Hans E. Grossnikiaus, Govinda S. Visvesvara, Michael C. Diesenhouse, Louis A. Wilson, George F. Corrent, Ralph T. Bryan
    Abstract:

    Encephalitozoon Hellem is a newly described cause of microsporidial keratoconjunctivitis, occurring chiefly in patients with significantly diminished CD4+ T-lymphocyte levels. This disorder is symptomatically disabling and generally recalcitrant to topical antimicrobial therapy. Two homosexual men with E. Hellem keratoconjunctivitis diagnosed by Gram stain, transmission electron microscopy, and specific indirect immunofluorescent assay were treated with topical fumagillin (Fumidil B). Both patients had marked symptomatic improvement with reduction of clinical findings. Symptoms and signs recurred with temporary discontinuation of the drug. Both patients, however, remained symptom-free on maintenance levels of topical fumagillin with no evidence of toxic side effects.

  • Pathology of symptomatic microsporidial (Encephalitozoon Hellem) bronchiolitis in the acquired immunodeficiency syndrome : a new respiratory pathogen diagnosed from lung biopsy, bronchoalveolar lavage, sputum, and tissue culture
    Human pathology, 1993
    Co-Authors: David A. Schwartz, Gordon J. Leitch, Govinda S. Visvesvara, Louise Tashjian, Mark Pollack, Jeanine Holden, Ralph T. Bryan
    Abstract:

    Abstract 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

  • Successful treatment of microsporidial keratoconjunctivitis with topical fumagillin in a patient with AIDS.
    Cornea, 1993
    Co-Authors: Daniel F. Rosberger, Govinda S. Visvesvara, David A. Schwartz, Ralph T. Bryan, Olivia N. Serdarevic, Robert A. Erlandson, Paul C. Keenan
    Abstract:

    There recently have been several reports of microsporidial keratoconjunctivitis caused by the organism Encephalitozoon Hellem. However, treatment of this infection has been largely ineffective. We report a case of a 35-year-old Hispanic woman with AIDS and E. Hellem keratoconjunctivitis confirmed with light, electron, and immunofluorescence microscopy that resolved promptly with topical fumagillin, a crystalline antibiotic with proven efficacy against Encephalitozoon species. No corneal or systemic toxicities were noted using the dosage and preparation employed (10 mg/ml suspension in balanced salt solution). An easily prepared, topical fumagillin suspension appears to be a safe, effective treatment for E. Hellem keratoconjunctivitis.

  • disseminated microsporidiosis Encephalitozoon Hellem and acquired immunodeficiency syndrome autopsy evidence for respiratory acquisition
    Archives of Pathology & Laboratory Medicine, 1992
    Co-Authors: David A. Schwartz, Rainer Weber, Govinda S. Visvesvara, Ralph T. Bryan, Karlene Hewanlowe, A Cali, Peter Angritt
    Abstract:

    Microsporidia are obligate intracellular protozoal parasites that infect a variety of cell types in a broad range of invertebrates and vertebrates. They have recently come to medical attention due to the increased frequency with which members of two microsporidian genera, Enterocytozoon and Encephalitozoon, are being diagnosed in patients with the acquired immunodeficiency syndrome (AIDS). The majority of published reports of human microsporidiosis describe Enterocytozoon infection of small intestinal enterocytes. In addition, a growing number of AIDS patients have been identified with infection due to the two species of Encephalitozoon-Encephalitozoon cuniculi and Encephalitozoon Hellem, observed in conjunctival, corneal, and, recently, sinonasal tissues. However, there are scant data regarding the systemic pathology and epidemiology of these infections. This article describes a patient with AIDS who died with systemic Encephalitozoon infection. The etiologic microsporidian was found to be E Hellem by using antemortem biochemical and antigenic analyses. A complete autopsy, the first to be reported in a patient with this infection, revealed organisms in the eyes, urinary tract, and respiratory tract. A surprising observation was the occurrence of numerous organisms within the lining epithelium of almost the entire length of the tracheobronchial tree, suggestive of respiratory acquisition. Detailed light and electron microscopic findings and the biological and diagnostic features of microsporidiosis are discussed.

Louis M. Weiss - One of the best experts on this subject based on the ideXlab platform.

  • The Structure, Function, and Composition of the Microsporidian Polar Tube
    The Microsporidia and Microsporidiosis, 2014
    Co-Authors: Elaine M. Keohane, Louis M. Weiss
    Abstract:

    The spores of microsporidia possess a unique, highly specialized structure, the polar tube, which is used to inject the parasite from the spore into a new host cell. Several theories have been proposed regarding the method by which the sporoplasm exits the spore and on the function of the polar filament or tube in this process. Electron-dense, particulate material fills the center of the filament. Weidner proposed that this material was unpolymerized polar tube protein (PTP). On the basis of ultrastructural observations, the eversion of the polar tube has been likened to a tube sliding within a tube. This chapter presents details on spore activation and discharge. When sporoblasts form, each one contains five to six coils of the preformed polar filament with the anchoring disk positioned at the anterior end. The major amino acids coded by the Encephalitozoon Hellem and Encephalitozoon cuniculi PTP genes were proline and glycine. Application of the techniques of modern biology has resulted in the identification of several PTPs although the interactions and functional significance of these proteins remains to be determined.

  • Branching Network of Proteinaceous Filaments within the Parasitophorous Vacuole of Encephalitozoon cuniculi and Encephalitozoon Hellem
    Infection and immunity, 2011
    Co-Authors: Kaya Ghosh, Ann Cali, Eddie Nieves, Patrick J. Keeling, Jean-françois Pombert, Philipp P. Henrich, Louis M. Weiss
    Abstract:

    The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon Hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

  • Glycosylation of the Major Polar Tube Protein of Encephalitozoon Hellem, a Microsporidian Parasite That Infects Humans
    Infection and immunity, 2004
    Co-Authors: Peter M. Takvorian, George A. Orr, Ann Cali, Louis M. Weiss
    Abstract:

    The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon Hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. Hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. Hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.

  • mitochondrial type hsp70 genes of the amitochondriate protists giardia intestinalis entamoeba histolytica and two microsporidians
    Parasitology International, 2002
    Co-Authors: Nobuko Arisue, Lidya B. Sánchez, Tetsuo Hashimoto, Louis M. Weiss, Miklós Müller
    Abstract:

    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.

  • mitochondrial type hsp70 genes of the amitochondriate protists giardia intestinalis entamoeba histolytica and two microsporidians
    Parasitology International, 2002
    Co-Authors: Nobuko Arisue, Lidya B. Sánchez, Tetsuo Hashimoto, Louis M. Weiss, Miklós Müller
    Abstract:

    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.