Microsporidian species known to infect humans are present in aquatic birds: implications for transmission via water?

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 x 10(5) spores/g) than nonaquatic bird droppings contained (mean, 4.4 x 10(4) spores/g) (P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 x 10(8) microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.     

Primers Designed for Amplification of Echinococcus multilocularis DNA Amplify the DNA of Encephalitozoon-Like Spores in the Polymerase Chain Reaction

ABSTRACT. Microsporidian spores were developed from cells which were grown in vitro from a human liver lesion which was due to larval Echinococcus multilocularis . The microsporidian spores developed in the same fashion as an Encephalitozoon cuniculi . The Encephalitozoon -like spores were completely separated on Percoll gradients. The separated spores contained DNA capable of amplification by two different primer sets designed for the polymerase chain reaction (PCR) of E. multilocularis DNA. However, the cell DNA from which microsporidium developed was thoroughly insensitive to the PCR using the E. multilocularis primer sets. The results strongly suggested that Encephalitozoon should be taken into consideration, when DNA isolated from larval E. multilocularis is analyzed.     

Analysis of four human microsporidian isolates by MALDI-TOF mass spectrometry

Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000-8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.     

Development, Characterization and Future Prospects of Monoclonal Antibodies Against Spores of Glugea atherinae (Protozoa-Microsporidia-Fish Parasites)

ABSTRACT. Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV' patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae . The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV⁺ patients.     

Immunocytochemical Identification of Spore Proteins in Two Microsporidia, with Emphasis on Extrusion Apparatus

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera ( Glugea atherinae and Encephalitozoon cuniculi ) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea , 35, 55 and 150 kDa in Encephalitozoon ). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.     

Microsporidian Species Known To Infect Humans Are Present in Aquatic Birds: Implications for Transmission via Water?

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 × 10⁵ spores/g) than nonaquatic bird droppings contained (mean, 4.4 × 10⁴ spores/g) (P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 × 10⁸ microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.     

Fine Structure of a New Human Microsporidian, Encephalitozoon hellem, in Culture

Encephalitozoon hellem is a new human microsporidian isolated from corneal biopsies and conjunctival scrapings of three AIDS patients and cultured in Madin Darby canine kidney (MDCK) cells. Encephalitozoon hellem and Encephalitozoon cuniculi display different protein profiles with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and unique antibody binding patterns with murine antisera against Western blots of each organism. Developmental stages of E. hellem in culture are similar to E. cuniculi. Meronts are 1.3–2.7 μm in diameter, develop within a parasitophorous vacuole adjacent to the vacuolar membrane, divide by binary fission, and contain one or two discrete nuclei. Sporonts measure 2 × 3 μm, separate from the vacuolar membrane, and have a thickened outer membrane. Sporoblasts display a tri-layered wall and possess the earliest recognized polar filaments. Mature spores measure 1 × 1.5 μm and are more electron-dense than other stages. Each spore contains a single nucleus, a polar tubule with four to nine coils, thin electron-dense exospore and thick, electron-lucent endospore. Although E. hellem and E. cuniculi differ biochemically and immunologically, their fine structure and development are indistinguishable.     

Studies on ocular microsporidia.

Sera from six ocular microsporidiosis patients and eight individuals with no history of microsporidiosis were assayed by enzyme-linked immunosorbent assay (ELISA) and by Western blot immunodetection. Microsporidia used as antigen include Nosema corneum, Encephalitozoon hellem, Encephalitozoon cuniculi, and Nosema algerae. Three AIDS patients with known E. hellem infections displayed ELISA antibody titers to E. hellem ranging from 1:400 to 1:12,800. Two patients with unclassified microsporidial infections displayed highest antibody titers to N. algerae (1:1,600 and 1:3,200), a mosquito microsporidian which, reportedly, cannot infect man. A sixth patient with a known N. corneum infection displayed the same ELISA antibody titer (1:1,600) to all four microsporidia. Western blot patterns also were variable among the patient sera; however, the most intense and complex antibody-binding patterns corresponded with the higher ELISA antibody titers. Sera from eight HIV-seronegative individuals with no history of microsporidiosis reacted variably to the four microsporidia. These results suggest that diagnosis of microsporidiosis may depend upon direct detection of the organisms using species-specific antibodies or molecular probes rather than conventional serology.     

A comparison of two microsporidian parasites in enterocytes of AIDS patients with chronic diarrhea.

Enterocytozoon bieneusi was first described by electron microscopy in 1985 in intestinal biopsies from an AIDS patient. It has subsequently been observed in many AIDS patients with chronic diarrhea from the U.S.A., Africa, and Europe. Morphologically, this parasite meets the criteria for being a microsporidian but has unique features justifying the creation of a a new genus and family. It has organelles not seen in microsporida before, i.e. elongated nuclei, electron-lucent inclusions, electron-dense discs, and development of multiple polar tubules in a single cell prior to the final cytokinetic process producing many sporoblasts. However, it produces typical microsporidian spores. Recently, a second type of microsporidian has been observed in similar biopsies from an AIDS patient which resembles an Encephalitozoon except that it secretes a fine network of material in which the developing organisms become embedded. During sporogony, each cell appears to be in a separate chamber. These two parasites are morphologically and pathologically compared.     

Nucleotide Sequences of DNA Fragments of Encephalitozoon cuniculi Amplified by Polymerase Chain Reaction with Primers Regarded as Specific for Echinococcus

Encephalitozoon -like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi , which was shown to have En. cunniculi specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus , were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus -specific primer sets could amplify not only EC. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus -specific primers-amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis.     

Fractionation of Sporogonial Stages of the Microsporidian Encephalitozoon cuniculi by Percoll® Gradients

Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK-13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll. Transmission electron microscopy and SDS-polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.     

Identification and characterization of two subpopulations of Encephalitozoon intestinalis

Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% +/- 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included > or = 25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4',6'-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or beta-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.     

Microsporidian polar tube proteins: highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups

The spore polar tube is a unique organelle required for cell invasion by fungi-related microsporidian parasites. Two major polar tube proteins (PTP1 and PTP2) are encoded by two tandemly arranged genes in Encephalitozoon species. A look at Antonospora (Nosema) locustae contigs (http://jbpc.mbl.edu/Nosema/Contigs/) revealed significant conservation in the order and orientation of various genes, despite high sequence divergence features, when comparing with Encephalitozoon cuniculi complete genome. This syntenic relationship between distantly related Encephalitozoon and Antonospora genera has been successfully exploited to identify ptp1 and ptp2 genes in two insect-infecting species assigned to the Antonospora clade (A. locustae and Paranosema grylli). Targeting of respective proteins to the polar tube was demonstrated through immunolocalization experiments with antibodies raised against recombinant proteins. Both PTPs were extracted from spores with 100mM dithiothreitol. Evidence for PTP1 mannosylation was obtained in studied species, supporting a key role of PTP1 in interactions with host cell surface.     

Quantitative evaluation of the impact of bather density on levels of human-virulent microsporidian spores in recreational water

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.     

Fine structure of a new human microsporidian, Encephalitozoon hellem, in culture

Encephalitozoon hellem is a new human microsporidian isolated from corneal biopsies and conjunctival scrapings of three AIDS patients and cultured in Madin Darby canine kidney (MDCK) cells. Encephalitozoon hellem and Encephalitozoon cuniculi display different protein profiles with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and unique antibody binding patterns with murine antisera against Western blots of each organism. Developmental stages of E. hellem in culture are similar to E. cuniculi. Meronts are 1.3-2.7 microns in diameter, develop within a parasitophorous vacuole adjacent to the vacuolar membrane, divide by binary fission, and contain one or two discrete nuclei. Sporonts measure 2 x 3 microns, separate from the vacuolar membrane, and have a thickened outer membrane. Sporoblasts display a tri-layered wall and possess the earliest recognized polar filaments. Mature spores measure 1 x 1.5 microns and are more electron-dense than other stages. Each spore contains a single nucleus, a polar tubule with four to nine coils, thin electron-dense exospore and thick, electron-lucent endospore. Although E. hellem and E. cuniculi differ biochemically and immunologically, their fine structure and development are indistinguishable.     

Antigenic diversity of Encephalitozoon hellem demonstrated by subspecies-specific monoclonal antibodies

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.     

SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis

Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. The microsporidian invasion process involves the extrusion of a unique polar tube into host cells. Both the spore wall and the polar tube play an important role in microsporidian pathogenesis. So far, five spore wall proteins (SWP1, SWP2, Enp1, Enp2, and EcCDA) from Encephalitozoon intestinalis and Encephalitozoon cuniculi and five spore wall proteins (SWP32, SWP30, SWP26, SWP25, and NbSWP5) from the silkworm pathogen Nosema bombycis have been identified. Here we report the identification and characterization of a spore wall protein (SWP5) with a molecular mass of 20.3 kDa in N. bombycis. This protein has low sequence similarity to other eukaryotic proteins. Immunolocalization analysis showed SWP5 localized to the exospore and the region of the polar tube in mature spores. Immunoprecipitation, mass spectrometry, and immunofluorescence analyses revealed that SWP5 interacts with the polar tube proteins PTP2 and PTP3. Anti-SWP5 serum pretreatment of mature spores significantly decreased their polar tube extrusion rate. Taken together, our results show that SWP5 is a spore wall protein localized to the spore wall and that it interacts with the polar tube, may play an important role in supporting the structural integrity of the spore wall, and potentially modulates the course of infection of N. bombycis.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

A simple method for the detection of antibodies to Encephalitozoon cuniculi in rabbits.

Rabbit antibodies against Encephalitozoon cuniculi were detected in an indirect microagglutination test using a bead substrate to which anti-rabbit immunoglobin G light and heavy chain antibodies were coupled. The test was positive using immune whole serum or F(ab)' and F(ab)'2 fragments of immunoglobin G but negative using the F(c) fragment. The reaction was blocked by saturating the beads with rabbit serum or by absorbing positive sera with excess Encephalitozoon cuniculi. The test provided a simple method to detect antibodies to Encephalitozoon cuniculi, did not require elaborate equipment and could be performed using frozen antigen.