Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of trachipleistophora hominis infections

The microsporidian Trachipleistophora hominis was isolated in vitro from the skeletal muscle of an AIDS patient. Since its discovery several more cases of myositis due to Trachipleistophora have been diagnosed but the source of infection is unknown. Morphologically, T. hominis most closely resembles Pleistophora and Vavraia, which undergo polysporous sporogony in sporophorous vesicles, but differs from these genera in the mode of formation of sporoblasts and in the morphology of the sporophorous vesicles. Alignment and analyses of the small subunit ribosomal DNA sequences of T. hominis and several other polysporoblastic genera indicated that its closest phylogenetic relationships were with species of the genera Pleistophora and Vavraia, in line with morphological predictions. The type species of the latter two genera are Pleistophora typicalis and Vavraia culicis; these are parasites of fish and mosquitoes, respectively. These results suggest two possible routes and sources of infection to AIDS patients, these being perorally by ingestion of inadequately cooked fish or crustaceans or percutaneously during a bloodmeal taken by a haematophagous insect. Support for an insect source has been provided by recent detection of a microsporidium from mosquitoes in human corneal tissue.     

In vitro cultivation and electron microscopy characterization of Trachipleistophora anthropophthera isolated from the cornea of an AIDS patient

We describe an in vitro culture technique for a microsporidian isolated from the corneal biopsy of an HIV-infected patient. The corneal biopsy was inoculated into a monolayer culture of fibroblasts derived from newborn mouse brain and incubated at 37 degrees C in an atmosphere of 5% CO2. Minimum essential medium supplemented with 2% fetal bovine serum appeared to be an optimum medium for growth and maintenance of the parasite and for production of large numbers of spores. This microsporidian was identified as Trachipleistophora anthropophthera based on ultrastructural features. It forms two types of sporophorous vesicles and two types of spores simultaneously: polysporous vesicle type I with eight or more oval spores, 3.7-4.0 microm by 2.0-2.3 microm, and bisporous vesicle type II with two round spores, 1.7-2.2 microm by 1.6-2.0 microm in size.     

Ultrastructure and development of Pleistophora ronneafiei n. sp., a microsporidium (Protista) in the skeletal muscle of an immune-compromised individual

This report provides a detailed ultrastructural study of the life cycle, including proliferative and sporogonic developmental stages, of the first Pleistophora species (microsporidium) obtained from an immune-incompetent patient. In 1985, the organism obtained from a muscle biopsy was initially identified as belonging to the genus Pleistophora, based on spore morphology and its location in a sporophorous vesicle. Since that initial report, at least two new microsporidial genera, Trachipleistophora and Brachiola, have been reported to infect the muscle tissue of immunologically compromised patients. Because Trachipleistophora development is similar to Pleistophora, and as Pleistophora was only known to occur in cold-blooded hosts, the question of the proper classification of this microsporidium arose. The information acquired in this study makes it possible to compare Pleistophora sp. (Ledford et al. 1985) to the known human infections and properly determine its correct taxonomic position. Our ultrastructural data have revealed the formation of multinucleate sporogonial plasmodia, a developmental characteristic of the genus Pleistophora and not Trachipleistophora. A comparison with other species of the genus supports the establishment of a new species. This parasite is given the name Pleistophora ronneafiei n. sp.     

Fine structure and sporogonic development of a Vavraia sp. (Microsporida: Pleistophoridae) in the biting midge Culicoides edeni (Diptera: Ceratopogonidae)

A microsporidium with ultrastructural characteristics of the genus Vavraia was found in the fat body of an adult specimen of Culicoides edeni (Diptera: Ceratopogonidae) collected in northern Florida. The sporogonial stages developed within sporophorous vesicles, which contained variable numbers of oval spores at maturity. The wall of the sporophorous vesicle was composed of two electron-dense outer layers and an electron-lucent intermediate layer. Sporonts contained haplokaryotic nuclei and divided by rosette formation. Mature spores had anisofilar polar filaments and measured 3.8 +/- 0.28 microns in length and 2.2 +/- 0.16 microns in width in thick sections of resin-embedded material. This is the first report of a Vavraia sp. from a species of Culicodes.     

Microsporidia of the Genus Trachipleistophora—Causative Agents of Human Microsporidiosis: Description of Trachipleistophora anthropophthera N. Sp. (Protozoa: Microsporidia)

Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field. Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick-walled sporophorous vesicle. In contrast to T. hominis , this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I-round to oval polysporous sporophorous vesicle. 7-10 μm in size, usually with eight spores (3.7 × 2.0 μm), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25-30 nm filaments extending into the host cell cytoplasm. Type II—smaller, bisporous sporophorous vesicle (4-5 times 2.2-2.5 μm) with two, nearly round, thin-walled spores, 2.2-2.5 × 1.8-2.0 μm in size, having 4-5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora -like parasites from human muscle, likely represent a group of closely related human microsporidia.     

Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae) from nymphs of mayflies Cloeon dipterum L. (Insecta: Ephemeroptera) in Western Siberia

The ultrastructure of a new microsporidian, Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae), from the fat body of Cloeon dipterum (L.) (Ephemeroptera: Baetidae) is described. The species is monokaryotic throughout the life cycle, developing in direct contact with the host cell cytoplasm. Sporogonial plasmodium divides into 2-8 sporoblasts. Each sporoblast, then spore, is enclosed in an individual sporophorous vesicle. Fixed and stained spores of the type species P. semitubulata are 3.4 x 1.9microm in size. The polaroplast is bipartite (lamellar and vesicular). The polar filament is isofilar, possessing 6 coils in one row. The following features distinguish the genus Pankovaia from other monokaryotic genera of Tuzetiidae: (a) exospore is composed of multiple irregularly laid tubules with a lengthwise opening, referred to as "semitubules"; (b) episporontal space of sporophorous vesicle (SPV) is devoid of secretory formations; (c) SPV envelope is represented by a thin fragile membrane.     

Ovipleistophora gen. n., a new genus for Pleistophora mirandellae-like microsporidia

Based on ultrastructural study and molecular analysis, a new genus, Ovipleistophora, is established for Pleistophora mirandellae-like microsporidia from roach and ruff oocytes. Unlike Pleistophora, Ovipleistophora has a thick additional envelope around the meront. This envelope breaks open to release the cells into the host cell cytoplasm. The cells, becoming multinuclear sporogonic plasmodia, already have a surface coat that transforms into the sporont wall and eventually into the sporophorous vesicle wall. The surface coat and its transformation differ from those of Pleistophora, but bear some resemblance to those of Trachipleistophora. In Trachipleistophora the sporonts, however, do not form plasmodia, as they do in Ovipleistophora and Pleistophora. Small subunit ribosomal DNA analysis supports the establishment of the new genus and assignment of P. mirandellae from 2 different fish hosts to the same species. The same small subunit ribosomal DNA analysis lends support for transferring P. ovariae into the genus Ovipleistophora.     

Aquatic tetrasporoblastic microsporidia from caddis flies (Insecta, Trichoptera): characterisation, phylogeny and taxonomic reevaluation of the genera Episeptum Larsson, 1986, Pyrotheca Hesse, 1935 and Cougourdella Hesse, 1935

Seven microsporidian species infecting caddis fly larvae, corresponding to conventional genera Episeptum, Pyrotheca and Cougourdella were studied using light and electron microscopy. Parts of their small subunit, ITS and large subunit ribosomal RNA genes were sequenced and compared with sequences of rDNA obtained from syntype slides of Cougourdella polycentropi Weiser 1965 and Pyrotheca sp. from Hydropsyche pellucidula. All studied caddis fly microsporidia form a closely related group. Their developmental stages in trichopteran hosts are restricted to fat body cells and oenocytes and have isolated nuclei. In late merogony, uninucleate meronts and binucleate plasmodia are formed. In sporogony a sporogonial plasmodium with four nuclei gives rise by rosette-like budding to four sporoblasts within a non-persistent sporophorous vesicle. Sporoblasts mature into pyriform to lageniform spores. The shape and size of spores, the number of polar filament coils, the structure of the polaroplast and of the exospore, together with morphometric characters present a set of markers unique for respective species. Four new species are established. The new genus Paraepiseptum is proposed to replace the tetrasporoblastic Pyrotheca and Cougourdella species from caddis flies. The genus Episeptum is redefined. Field and laboratory examinations as well as the phylogenetic position within the aquatic clade of microsporidia suggest that the life cycle of trichopteran microsporidia probably involves an alternate (copepod?) host and (or) transovarial transmission.     

Life cycle,ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae)

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.     

A new microsporidian parasite, Potaspora morhaphis n. gen., n. sp. (Microsporidia) infecting the Teleostean fish, Potamorhaphis guianensis from the River Amazon. Morphological, ultrastructural and molecular characterization

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2.8+/-0.3 x 1.5+/-0.2 microm. In mature spores the polar filament was arranged into 9-10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.     

Ultrastructure of a microsporidian hyperparasite, Unikaryon legeri (Microsporida), of trematode larvae

Unikaryon legeri (Dollfus, 1912) Canning and Nicholas, 1974, has been reexamined by electron microscopy from material collected in Portugal. It parasitises metacercariae of Meigymnophallus sp. in Cardium edule. A sporophorous vesicle forms around the sporonts, arising as a blister that separates from the electron-dense surface coat of the sporont. Sporogony is disporoblastic, giving rise to 2 spores that are retained in pairs within the sporophorous vesicle. Unikaryon piriformis, which is the type species of the genus and is also hyperparasitic in platyhelminth larvae, has not been examined by electron microscopy, and it is not known whether this species also produces sporophorous vesicles. If it does, then all that will be required is a simple addition of this character to the definition; if not, U. legeri will have to be transferred to a new genus and reclassified with other disporoblastic genera that sporulate in sporophorous vesicles.     

Resolution of a taxonomic conundrum: an ultrastructural and molecular description of the life cycle of Pleistophora mulleri (Pfeiffer 1895; Georgevitch 1929)

The classification of a microsporidian parasite observed in the abdominal muscles of amphipod hosts has been repeatedly revised but still remains inconclusive. This parasite has variable spore numbers within a sporophorous vesicle and has been assigned to the genera Glugea, Pleistophora, Stempellia, and Thelohania. We used electron microscopy and molecular evidence to resolve the previous taxonomic confusion and confirm its identification as Pleistophora mulleri. The life cycle of P. mulleri is described from the freshwater amphipod host Gammarus duebeni celticus. Infection appeared as white tubular masses within the abdominal muscle of the host. Light and transmission electron microscope examination revealed the presence of an active microsporidian infection that was diffuse within the muscle block with no evidence of xenoma formation. Paucinucleate merogonial plasmodia were surrounded by an amorphous coat immediately external to the plasmalemma. The amorphous coat developed into a merontogenetic sporophorous vesicle that was present throughout sporulation. Sporogony was polysporous resulting in uninucleate spores, with a bipartite polaroplast, an anisofilar polar filament and a large posterior vacuole. SSU rDNA analysis supported the ultrastructural evidence clearly placing this parasite within the genus Pleistophora. This paper indicates that Pleistophora species are not restricted to vertebrate hosts.     

Pseudoloma neurophilia n. g., n. sp., a new microsporidium from the central nervous system of the zebrafish (Danio rerio)

An unusual xenoma-forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 x 2.7 microm with 13-16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the "Ichthyosporidium group" along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.     

Relationships of microsporidian genera, with emphasis on the polysporous genera, revealed by sequences of the largest subunit of RNA polymerase II (RPB1)

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.     

Ultrastructural Study and Description of Ovavesicula popilliae N. G., N. Sp. (Microsporida: Pleistophoridae) from the Japanese Beetle,Popillia japonica (Coleoptera: Scarabaeidae)1

A new genus and species of microsporidia, Ovavesicula popilliae n. g., n. sp., is described from the Japanese beetle, Popillia japonica, on the basis of studies by light and electron microscopy. Parasite development primarily occurs within the Malpighian tubules of larvae, and spores are formed in a sporophorous vesicle. Meronts have diplokaryotic nuclei, develop in direct contact with the host cell cytoplasm, and divide by binary fission. Sporonts have unpaired nuclei, develop within a thick sporophorous vesicle, and undergo synchronous nuclear divisions producing plasmodia with 2, 4, 8, 16, and 32 nuclei. Cytokinesis of sporogonial plasmodia does not occur until karyokinesis is complete with 32 nuclei. Intact sporophorous vesicles are ovoid, containing numerous secretory products, and are surrounded by a persistent two-layered wall. The uninucleate spores are regularly formed in groups of 32, and the polar tube in each has six coils.     

The Small Subunit Ribosomal RNA Gene Sequence of Pleistophora anguillarum and The Use of PCR Primers for Diagnostic Detection of the Parasite

Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA.) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels ( Anguilla japonica ) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum , enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post-infection. No PCR product was obtained with templates prepared from clinically healthy control animals.     

A new microsporidian species, Vairimorpha ocinarae n. sp., isolated from Ocinara lida Moore (Lepidoptera: Bombycidae) in Taiwan

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.     

Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS

A new microsporidium is reported infesting the enterocytes of a Haitian patients with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 micron X 0.5 micron. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.     

Establishment of Endoreticulatus N. G. for Pleistophora fidelis (Hostounský & Weiser, 1975) (Microsporida: Pleistophoridae) Based on the Ultrastructure of a Microsporidium in the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)1,2

A new genus, Endoreticulatus n. g., is described for the microsporidium Pleistophora fidelis (Hostounský & Weiser, 1975) based on light and electron microscopic studies of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say). This latter microsporidium is considered to be conspecific with P. fidelis because both isolates have been shown to be infectious for L. decemlineata where infection is limited to the epithelial cells of the midgut; both are haplokaryotic and develop as groups of sporoblasts and spores in subpersistent vacuoles in the host cell. In addition, meronts, sporonts, and spores of each isolate often occur simultaneously in a common cell, and by light microscopy they both appear similar. Ultrastructural studies of the isolate from L. decemlineata revealed that all developmental stages occur in parasitophorous vacuoles derived from cisternae of rough endoplasmic reticulum of the host cell cytoplasm. Based on the unique nature of the parasitophorous vacuole, a new genus, Endoreticulatus, is proposed for P. fidelis. The genus is compared with the other genera whose species undergo multisporous sporogony in sporophorous vesicles. In addition, the nature of the parasitophorous vacuole of Endoreticulatus fidelis (Hostounský & Weiser, 1975) n. comb. is compared with the parasitophorous vacuole known to encase various developmental stages of several other microsporidian species.     

Vavraia lutzomyiae n. sp. (Phylum Microspora) infecting the sandfly Lutzomyia longipalpis (Psychodidae, Phlebotominae), a vector of human visceral leishmaniasis

Vavraia lutzomyiae (Microsporida; Pleistophoridae) is a new species parasitic in the tropical phlebotomine sandfly, Lutzomyia longipalpis (Diptera, Psychodidae, Phlebotominae), a major vector of Leishmania chagasi in Latin America where human visceral leishmaniasis is endemic. Infected larvae and pupae were parasitized in the abdomen, and some adults were parasitized in Malpighian tubules and midgut. The sporogonial plasmodium divided by multiple divisions into up to 64 uninucleate sporoblasts. These stages were surrounded outside the plasmalemma by a thick, amorphous dense coat and transformed into a merontogenetic sporophorous vesicle within which the sporonts developed into sporoblasts. The mature microsporidian spores were broadly ellipsoidal and measured 6.1+/-0.43 x 3.1+/-0.15 microm. The spore wall consisted of a transparent endospore (approximately 100 nm) and a thin electron dense exospore (approximately 30 nm) with the outer limit slightly undulated. Spores contained a polar filament arranged peripherally in a single layer of eight to nine wide anterior coils (approximately 125 nm diameter), and three to four narrow posterior coils (approximately 70 nm diameter). Transverse sections revealed a concentric layer organization with the internal layer surrounded by numerous (up to 25) longitudinal microfibrils. The angle of tilt of the polar filament was about 65-68 degrees.