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Purpose: Most eukaryotic cells have a plasma membrane with glycoproteins on the outer leaflet and cytoplasmic proteins on the inner leaflet. However, the microsporidians examined have a peculiar outer membrane with cytoplasmic proteins on the surface. His study was to determine if this is true and identify the presence of key cytoplasmic proteins on the exterior of the sporoplasm and spore stages. Methods: Specific probes including GFP-labeled genes, antibodies, and electron microscopy were applied to studies on the sporoplasm and spore stages of different microsporidian species. Results: Cytoplasmic membrane proteins cadherin, intermediate filaments, dynactin p150Glued and tubulin were identified on the outer leaflet of the exterior membrane of the sporoplasm or spore stages of Spraguea americanus, Anncaliia algerae, Ameson michaelis and Thelohania sp. Conclusion: The invasive sporoplasm stage of microsporidians acquires an outer membrane from a cytoplasmic organelle and it has been shown that the outside surface of this membrane bears cytoplasmic proteins. This membrane retains these cytoplasmic proteins even on the spore stage. For an intracellular parasite that locates directly within the cytoplasm of a host cell, the presence of these cytoplasmic proteins on the surface of the parasite appears to have a significant effect on the host's response. [ABSTRACT FROM AUTHOR]

Parasitological examination of the commercially important pelagic fish Sardinella aurita Valenciennes, 1847 (Clupeidae) from the Eastern coast of Algeria revealed xenomas in the peritoneal cavity, suggesting a microsporidian infection. The prevalence of the disease was approximately 30% on average, higher in smaller individuals and showing significant seasonal variation. The xenomas contained numerous ellipsoidal spores, surrounded by a dense layer of connective tissue. Spore sizes were 6.10 ± 0.38 µm length and 3.54 ± 0.43 µm width. Ultrastructural examination by transmission electron microscopy showed various development stages of the parasite, including meronts, sporonts, sporoblasts and mature spores. The internal organization of the mature spores, with a single nucleus, prominent posterior vacuole, a lamellar polaroplast and an isofilar polar tube arranged in a single row, was typical of the genus Glugea. The DNA sequence of the small subunit ribosomal RNA gene confirmed that this parasite belongs to the genus Glugea. Genetic and morphologic comparison with G. sardinellensis, a species previously described in the same host from Tunisia shows many similarities, although some molecular and morphometric inconsistencies precluded the unambiguous assignment of our samples to G. sardinellensis. At the same time, we do not find sufficient grounds to erect a new taxon for our parasite. We discuss the implications of our findings for the current state of the systematics of Glugea. [ABSTRACT FROM AUTHOR]

A novel microsporidial disease was documented in two ornamental fish species, black tetra Gymnocorymbus ternetzi Boulenger 1895 and cardinal tetra Paracheirodon axelrodi Schultz 1956. The non-xenoma-forming microsporidium occurred diffusely in most internal organs and the gill, thus referring to the condition as tetra disseminated microsporidiosis (TDM). The occurrence of TDM in black tetra was associated with chronic mortality in a domestic farmed population, while the case in cardinal tetra occurred in moribund fish while in quarantine at a public aquarium. Histology showed that coelomic visceral organs were frequently necrotic and severely disrupted by extensive infiltrates of macrophages. Infected macrophages were presumed responsible for the dissemination of spores throughout the body. Ultrastructural characteristics of the parasite developmental cycle included uninucleate meronts directly in the host cell cytoplasm. Sporonts were bi-nucleated as a result of karyokinesis and a parasite-produced sporophorous vesicle (SPV) became apparent at this stage. Cytokinesis resulted in two spores forming within each SPV. Spores were uniform in size, measuring about 3.9 ± 0.33 long by 2.0 ± 0.2 μm wide. Ultrastructure demonstrated two spore types, one with 9–12 polar filament coils and a double-layered exospore and a second type with 4–7 polar filament coils and a homogenously electron-dense exospore, with differences perhaps related to parasite transmission mechanisms. The 16S rDNA sequences showed closest identity to the genus Glugea (≈ 92%), though the developmental cycle, specifically being a non-xenoma-forming species and having two spores forming within a SPV, did not fit within the genus. Based on combined phylogenetic and ultrastructural characteristics, a new genus (Fusasporis) is proposed, with F. stethaprioni n. gen. n. sp. as the type species. [ABSTRACT FROM AUTHOR]

A microsporidium was found in a Mediterranean cricket Gryllus bimaculatus from a pet market in the UK and a lab stock at the Moscow Zoo (originating from London Zoo). The spores were ovoid, uninucleate, 6.3 × 3.7 μm in size (unfixed), in packets by of 8, 16, or 32. The spores were easily discharged upon dessication or slight mechanical pressure. The polar tube was isofilar, with 15-16 coils arranged in 1-2 rows. The polaroplast was composed of thin lamellae and occupied about one third of the spore volume. The endospore was 200 nm thick, thinning over the anchoring disc. The exospore was thin, uniform, and with no ornamentation. Phylogenetics based upon small subunit ribosomal RNA (Genbank accession # MG663123) and RNA polymerase II largest subunit (# MG664544) genes placed the parasite at the base of the Trachipleistophora/Vavraia lineage. The RPB1 locus was polymorphic but similar genetic structure and identical clones were found in both isolates, confirming their common geographic origin. Due to in insufficient ultrastructural data and prominent divergence from described species, the parasite is provisionally placed to the collective taxon Microsporidium. [ABSTRACT FROM AUTHOR]

Microsporidia are a group of widespread fungi-related obligate intracellular parasites. Direct contact of most microsporidia with the cytoplasm of an infected host cell entails possible secretion of various proteins from the parasite that allows control physiological processes of the host. Earlier, by means of polyclonal antibodies against α/β-hydrolase of microsporidium Paranosema locustae, the secretion of large amounts of the enzyme into the cytoplasm of fat body cells of infected migratory locust Locusta migratoria was demonstrated. However, yeast fungi Pichia pastoris did not recognize this enzyme as a secretory one during its heterologous expression. In the present study, a library of recombinant single-chain antibodies (scFv fragments) against proteins of the infected fat body of locust was constructed. The use of the phage display technology enabled choosing a miniantibody that specifically recognized the studied enzyme. Immunoblotting and immunolabeling of frozen sections of locust fat body with the selected scFv fragment confirmed the fact of secretion of P. locustae α/β-hydrolase (as two forms of different size) into the infected host cell. Prospects of using the selected scFv fragment for further studies of the secretion mechanism of the parasite's protein and its role in host-parasite interactions are discussed. [ABSTRACT FROM AUTHOR]

Spores of Trachipleistophora extenrec, originally isolated from the muscles of the Madagascan insectivore Hemicentetes semispinosus and maintained by serial passage in severe combined immunodeficiency (SCID) mice, were fed to larvae of the Egyptian cotton leafworm Spodoptera littoralis. Extensive infection of larval tissues ensued and caused larval and pupal mortality. The development of T. extenrec in the insect host, studied both by light and electron microscopy, followed generally the same life cycle as in the mammalian host. However, some differences in the fine structure of the parasite grown in both types of hosts were found. Spores isolated from the insect host caused infection of SCID mice when injected intramuscularly. Our results suggest that T. extenrec may be originally an insect microsporidian. This likelihood is corroborated by its structural similarity and phylogenetic relationship to two other microsporidia having insects either as unique hosts ( Vavraia culicis) or being able to infect both mammalian and insect host ( Trachipleistophora hominis). [ABSTRACT FROM AUTHOR]

Lymantria dispar (Lep., Lymantriidae), an important forest defoliator, is host for a variety of entomopathogenic microsporidia. These are obligate intracellular parasites that completely depend on resources provided by the hosts. The microsporidia infecting L. dispar differ in the host tissues that become infected as well as in virulence, with presumably different effects on host physiology. Endoreticulatus schubergi infects the midgut tissue of the host; its virulence is low. We studied the effects of infection with E. schubergi on carbohydrate and lipid levels in the L. dispar host larva. Trehalose titer in the hemolymph increased with larval age. There were no significant differences between infected and uninfected hosts. Glycogen content of the host tissue also increased with age. Levels of this storage carbohydrate were significantly lower in infected larvae at a late stage of infection when insects were either full grown larvae prior to pupation (57.4 ± 5.7 and 36.4 ± 5.2 μg/mg in uninfected controls and infected hosts, respectively) or prepupae (53.2 ± 3.4 vs. 34.7 ± 4.7 μg/mg). Lipid levels in host tissue likewise increased over time and were slightly but not significantly lower in infected L. dispar at a late stage. The results suggest that L. dispar larvae are able to compensate for most of the loss of nutrients to the developing microsporidia. Differences between these findings and the effects of infection with the virulent microsporidium, Vairimorpha disparis, that infects the host’s fat body and causes severe depletion of nutrients in L. dispar larvae are discussed. [ABSTRACT FROM AUTHOR]

The use of animal cell cultures as tools for studying the microsporidia of insects and mammals is briefly reviewed, along with an in depth review of the literature on using fish cell cultures to study the microsporidia of fish. Fish cell cultures have been used less often but have had some success. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable in understanding and treating microsporidia infections in fish, which are increasingly of economic importance. [ABSTRACT FROM AUTHOR]

Microsporidia are fungi-related obligate intracellular parasites that infect numerous animals, including man. Encephalitozoon cuniculi harbours a very small genome (2.9 Mbp) with about 2,000 coding sequences (CDSs). Most repeated CDSs are of unknown function and are distributed in subterminal regions that mark the transitions between subtelomeric rDNA units and chromosome cores. A potential multigenic family ( interB) encoding proteins within a size range of 579–641 aa was investigated by PCR and RT-PCR. Thirty members were finally assigned to the E. cuniculi interB family and a predominant interB transcript was found to originate from a newly identified gene on chromosome III. Microsporidian species from eight different genera infecting insects, fishes or mammals, were tested for a possible intra-phylum conservation of interB genes. Only representatives of the Encephalitozoon, Vittaforma and Brachiola genera, differing in host range but all able to invade humans, were positive. Molecular karyotyping of Brachiola algerae showed a complex set of chromosome bands, providing a haploid genome size estimate of 15–20 Mbp. In spite of this large difference in genome complexity, B. algerae and E. cuniculi shared some similar interB gene copies and a common location of interB genes in near-rDNA subterminal regions. [ABSTRACT FROM AUTHOR]

Nosema michaelis spores possess a biologically unique external arrangement of microtubules. The microtubules were studied ultrastructurally with the use of thin sections, freeze-etched and negatively stained preparations. Tubules were sheared from the spores by repetitive freeze-thaw cycles and harvested by high speed centrifugation. Electron microscopic observations of negatively stained and freeze-etched samples revealed the globular subunit construction of the tubules. Suspensions of isolated microtubules and tubular fragments formed extended linear array at 4°C, apparently by autoaggregation. Tubule preparations were reduced and alkylated under various conditions and examined in several polyacrylamide disc gel systems. Two bands were obtained from completely reduced and alkylated microtubule proteins separated in polyacrylamide gels containing 8 M-urea, detergent-urea or detergent alone. The two subunit proteins (tubulins) had average molecular weights of ∼45000 and ∼53000. Incompletely reduced samples yielded two additional satellite bands of higher molecular weights, suggestive of homodimers. [ABSTRACT FROM AUTHOR]