Small Subunit Ribosomal DNA Phylogeny of Various Microsporidia with Emphasis on AIDS Related Forms

ABSTRACT. Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans ( Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi ), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi . Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis . Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many of the important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple origins.     

First detection and genotyping of human-associated microsporidia in pigeons from urban parks

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.     

In vitro and in vivo investigations of human microsporidia.

The numerous infections of microsporidia which have been diagnosed in patients with AIDS have revealed the potential of these organisms for establishing themselves when the immune status of the host is compromised. Two species of Encephalitozoon, E. cuniculi and E. hellem, have been diagnosed in man, the former infecting a variety of tissues, the latter restricted to the corneal and conjunctival epithelia. These species are morphologically indistinguishable even at the ultrastructural level but can be separated biochemically. Two human sera were found to react with equal intensity in the ELISA on spores of E. cuniculi and E. hellem purified from in vitro cultures, and gave similar binding patterns in Western blots on SDS-PAGE protein profiles of the two species. This has raised questions about the identity of Encephalitozoon infections diagnosed previously in man. The diagnosis of Enterocytozoon bieneusi, which infects the intestinal enterocytes of AIDS patients and is associated with chronic diarrhoea, requires observation of smears or sections of biopsies or specialist observation of stool preparations. In vitro cultures, which would facilitate the raising of specific antisera, have proved difficult to establish. In vitro and in vivo systems for assaying drugs for microsporidia have revealed that albendazole has a marked effect on parasite numbers and morphology but does not eliminate infection, which resurges when drug pressure is removed.     

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi,Encephalitozoon hellem,and Vittaforma corneae

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.     

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.     

Comparative Analysis of the Proteins with Tandem Repeats from 8 Microsporidia and Characterization of a Novel Endospore Wall Protein Colocalizing with Polar Tube from Nosema bombycis

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal‐infecting microsporidia (Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect‐infecting microsporidia (Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect‐infecting microsporidia was larger than that of mammal‐infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC‐rich codons. The tandem repeat position within proteins of insect‐infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal‐infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite.     

Genital microsporidiosis in women with AIDS: A post-mortem study

BackgroundMicrosporidiosis 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.ObjectiveTo report genital microsporidiosis in female AIDS patients.MethodsTissues 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 Kourí were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified.ResultsWe 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.ConclusionsMicrosporidia 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 microsporidiosis cases so far reported in humans.     

Genetic and phenotypic intraspecific variation in the microsporidian Encephalitozoon hellem.

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.     

A comparison of several serologic tests to detect antibodies to Toxoplasma gondii in naturally exposed bottlenose dolphins (Tursiops truncatus)

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In a previous study, 138 of 141 (97.8%) bottlenose dolphins (Tursiops truncatus) from the coasts of Florida and California had antibodies to T. gondii by the modified agglutination test (MAT). Although the MAT has been found to be highly sensitive and specific for T. gondii antibodies from several species of terrestrial animals, it has not yet been validated for T. gondii infections in marine mammals. Furthermore, T. gondii has yet not been isolated from dolphins. In the present study, sera from 146 (60 from the 2004 samples and 86 from the 2003 samples) T. truncatus from the coastal areas of South Carolina and Florida were tested for antibodies to T. gondii. Sera from 2004 were tested by the MAT, the indirect fluorescent antibody test (IFAT), the Sabin-Feldman dye test (DT), an indirect hemagglutination test (IHAT), an enzyme-linked immunosorbent assay (ELISA), and Western blot. All 60 dolphins were seropositive, with MAT titers of 1:20 in 3, 1:40 in 19, 1:80 in 29, 1:160 in 2, 1:1,280 in 3, 1:2,560 in 2, and 1:5,120 or higher in 2, and these results were confirmed in another laboratory. The DT titers of these dolphins were <1:10 in 53, 1:800 in 3, 1:1,600 in 2, and 1:3,200 in 2. The IHAT titers were <1:64 in 52, 1:128 in 1, 1:512 in 2, and 1:2,048 in 5. The IFAT titers were <1:20 in 3, 1:20 in 11, 1:40 in 36, 1:80 in 2, 1:160 in 1, and 1:320 or higher in 7. All 7 DT-positive dolphins had high MAT titers, but 2 were negative by the IHAT. Western blot results closely followed MAT results; ELISA results matched MAT results, which were 1:40 or higher. In sera from the 2003 samples, MAT antibodies were found in 86 of 86 dolphins with titers of 1:25 in 29, 1:50 in 23, 1:100 in 27, 1:200 in 3, 1:1,600 in 1, and 1:3,200 in 3; these sera were not tested by other means. Overall, MAT antibodies were found in all 146 dolphin sera tested. Because marine mammals are considered sentinel animals indicative of contamination of the coastal and marine waters by T. gondii oocysts, serologically positive infections need to be validated by the detection of T. gondii organisms in the tissues of seropositive animals.     

Opportunistic Properties of Nosema algerae (Microspora), a Mosquito Parasite, in Immunocompromised Mice

ABSTRACT. In the last ten years microspordia have been recognized as opportunistic pathogens in AIDS patients. The sources of infection and the mechanisms of transmission of these organisms in humans are mostly uncertain. Transmission of invertebrte microsporidia to mammals is normally considered impossible, temperature being a limiting factor for development. Mice treated with cortisone acetate and with cyclosporin A, respectively, as well as athymic mice were injected intravenously, intranasally, perorally and subcutaneously with spores of Nosema algerae , a microsporidian species of culicine mosquitoes. No infection could be detected in tissue samples of cortisone acetate and cyclosporin A treated mice. However, the experimental inoculation of spores into the tail and foot of athymic mice caused severe infection in skeletal muscles and the connective tissue. In some tails, nerve tissue and bone marrow were also infected. Vegetative stages and spores were seen in direct contact to host cell cytoplasma. For the first time the prolonged and progressive development of an invertebrate microsporidium in a mammalian host is shown. The possibility of invertebrate microsporidia as a source of human microsporidiosis should now be taken into consideration.     

Polar tube proteins of microsporidia of the family encephalitozoonidae

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.     

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.     

Long-Term Storage of Infective Microsporidian Spores in Liquid Nitrogen

Thirty-one species of microsporidia, isolated from insects and stored in liquid nitrogen for up to 25 yr, were infectious when removed from liquid nitrogen. The natural hosts of all of these microsporidia were terrestrial insects, representing six different insect orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera. All microsporidia from terrestrial insects that were tested survived storage in liquid nitrogen, while Nosema algerae , a microsporidium from aquatic mosquito hosts did not survive freezing in liquid nitrogen. A Nosema species from the alfalfa weevil, Hypera postica , lost some infectivity in a water storage medium after 25 yr in liquid nitrogen. Liquid nitrogen storage of microsporidian spores in 50% and 100% glycerol media reduced loss of infectivity and is recommended for extended storage of microsporidia from terrestrial insect hosts.     

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.     

Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia,Spain

In this survey we examined 87 domestic animal stool samples in order to detect the possible presence of microsporidia in animals in close contact with humans in Galicia (NW, Spain). The detection of Enterocytozoon bieneusi spores was confirmed in faecal samples from two dogs and one goat by polymerase chain reaction. None of the positive samples for microsporidia in the staining method were amplified with species-specific primers for Encephalitozoon intestinalis, E. hellem and E. cuniculi. Four rabbits faecal samples reacted with anti-E. cuniculi serum. Our results could indicate the importance of domestic animals as zoonotic reservoirs of microsporidial human infections.     

Microsporida infections in immunocompetent and immunosuppressed subjects]

Parasites of the phylum Microspora are obligatory intracellular protoza with a widespread host range among invertebrates and vertebrates. Species from Nosema, Encephalitozoon, Enterocytozoon and Pleistophora genera can infect immunocompetent and immunosuppressed patients. The emergency of the AIDS epidemic has recently highlighted the role of these parasites in human pathology, microsporidian species being a frequent cause of diarrhoea and ocular infections. Recent acquisitions in the taxonomy and life cycle of this parasite group, as well as pathogenesis, immunopathology, clinical aspects, diagnosis, therapy and epidemiology of human microsporidiosis are reviewed and discussed.     

Intestinal microsporidiosis: a current infection in HIV-seropositive patients in Portugal

Intestinal microsporidiosis is recognised as an important cause of opportunistic infections in immunocompromised patients, especially those with AIDS. Two species are implicated in diarrhoea and other gastrointestinal disease in HIV-infected patients: Enterocytozoon bieneusi and Encephalitozoon intestinalis. Diagnosis of gastrointestinal microsporidiosis was made by detecting spores of the parasite in stool specimens with Weber's modified trichrome stain and with some optical brightening agents such as UVITEX 2B or calcofluor white M2R. The identification of microsporidiosis at the species level was made using appropriate primers with PCR. The diagnosis of intestinal microsporidiosis is currently performed in the parasitology laboratory. In a study of 215 HIV-infected patients, conducted from 1996 to 1999 (approximately n = 60/year), we found a prevalence of spores of microsporidia of 51.5% (n = 31) in 1996, 14.0% (n = 5) in 1997 and 12.5% (n = 8) in 1998 and 42.8% (n = 25) in 1999. Using PCR we found that E. intestinalis was the only species responsible for the gastrointestinal symptoms in 49 patients with microsporidian spores (71%) and E. bieneusi in 29% (n = 20).     

Intestinal microsporidiosis in India: A two year study

Intestinal parasitic pathogens in HIV/AIDS patients include Cryptosporidium sp, Cystoisospora sp, microsporidia and less commonly other parasites. The two most common microsporidia causing intestinal infection are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Most of the Indian studies for intestinal parasitic infections in HIV/AIDS patients have not included microsporidia, due to difficult staining and identification of the parasite. The aim of the present study was to find the prevalence of intestinal microsporidiosis and their species identification along with correlation of CD4 count with parasite positivity and diarrhoea in HIV positive individuals. Stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various out-patient departments and in-patients admitted to a tertiary care hospital from August 2008 to October 2009. The stool samples were subjected to examination by wet mount, modified acid fast stain for coccidian parasites and multiplex nested PCR for microsporidia. The overall prevalence of all intestinal parasites among HIV patients in our study was 26.5%. The prevalence of intestinal parasitic pathogens in HIV positive patients with diarrhoea was 43.2%. Microsporidia were the most common parasites detected (14%) in all patients, while in HIV infected patients 15.9% patients had microsporidia infection. The most common species causing intestinal microsporidiosis in our study was E. intestinalis (10.5%). In HIV seropositive individuals with diarrhoea, E. intestinalis was 20.8% and E. bieneusi 8.0% while in HIV-seropositive individuals without diarrhoea, E. intestinalis was 3.8% and E. bieneusi 1.9%. E. intestinalis was present in 10.9% of HIV negative individuals with diarrhoea in whom E. bieneusi was not found. There was a significant association between CD4 count ≤ 200/μl and intestinal parasite positivity. Thus, it can be concluded that intestinal microsporidiosis is under reported but an important disease in India. The predominant species in our study is E. intestinalis , in contrast to other parts of the world where E. bieneusi is more common.     

HTLV-III neutralizing antibody development in transfusion-dependent seropositive patients with beta-thalassemia

Sera collected in New York in 1984 from 77 patients with homozygous beta-thalassemia were assayed for antibodies to HTLV-III by ELISA and Western blot techniques. Eight (12%) of the 66 hypertransfused thalassemics were seropositive. Retrospective sera of these eight individuals were examined by radioimmune precipitation (RIP), and assays for neutralization of virus infectivity were performed. With seroconversion, antibodies to viral envelope proteins appeared first and were correlated with development of neutralizing antibody. Affinity purified gp120, the major envelope glycoprotein of HTLV-III, blocked viral infectivity and absorbed neutralizing antibody activity from a positive serum. Neutralizing antibody titers mirrored antibody titers to gp120 by RIP. Antibody to gp120 sometimes occurred in the absence of neutralizing antibody, although the reverse was not true. One thalassemia patient who exhibited antibody to gp120 for 3 yr post-seroconversion failed to develop neutralizing antibody, acquired the acquired immunodeficiency syndrome with central nervous system involvement and lymphoma, and subsequently died. In contrast, all other seropositive thalassemics possessed neutralizing antibodies, and were asymptomatic or exhibited only lymphadenopathy. These results indicate that gp120 elicits neutralizing antibodies in the course of natural infection with HTLV-III. The relationship seen here between neutralizing antibody and better clinical outcome needs to be verified by additional studies.     

Diversity of Nosema associated with bumblebees (Bombus spp.) from China

Bumblebees (Bombus spp.) are important pollinators of many economically important crops and microsporidia are among the most important infections of these hosts. Using molecular markers, we screened a large sample (n=1,009 bees) of workers of 27 different Bombus spp. from China (Sichuan, Qinghai, Inner Mongolia, and Gansu provinces). The results showed that 62 individuals representing 12 Bombus spp. were infected by microsporidia with an overall prevalence of 6.1%. Based on the haplotypes (ssrRNA sequences), we confirmed the presence of Nosema bombi, Nosema ceranae and (likely) Nosema thomsoni. In addition, four new putatively novel taxa were identified by phylogenetic reconstruction: Nosema A, Nosema B-complex, Nosema C-complex and Nosema D-complex. In many cases, hosts were infected by more than one Nosema taxon. Possible caveats of sequence analyses are discussed.