Intracellular development of Enterocytozoon, a unique microsporidian found in the intestine of AIDS patients

Enterocytozoon was 1st described in 1985, in an AIDS patient with intestinal malabsorption and diarrhea. Since then, additional cases of infection with this organism have been observed, but only in individuals with AIDS and malabsorption. Intestinal tissue biopsies were obtained from a 45-year-old man prior to AIDS diagnosis, again nine months later and then at autopsy two months later. When the biopsies were examined electron microscopically, both sets contained the microsporidian parasite. However, the 2nd intestinal biopsy, when wasting was much more severe, contained infection in almost every small intestinal enterocyte examined. The parasite was actively developing, allowing us to detail its life cycle. The parasite is apansporoblastic, polysporous and has characteristics not previously reported in the Microsporida: (1) an electron lucent inclusion not usually seen in Microsporida is prominent and always present; (2) extremely elongated sausage-shaped nuclei occur in the proliferative phase of parasite development; (3) the polar tube development uniquely involves the production of electron dense discs, yet results in the formation of a typical spore; and (4) polar tube development occurs prior to the final division of the multi-nucleate sporont. On the basis of these characteristics, we are placing this genus in a new family, Enterocytozoonidae, n. fam.     

Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.     

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.     

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi , a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli , purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.     

Brachiola vesicularum, N. G., N. Sp., a New Microsporidium Associated with AIDS and Myositis

Brachiola vesicularum, n. g., n. sp., is a new microsporidium associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 times 2 μm diplokaryotic spores containing 8-10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron-dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae , its development occurred at warm blooded host temperature (37-38° C) and unlike Nosema connori , which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema , this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.     

Microfilum Lutjani N. G. N. Sp. (Protozoa Microsporida), A Gill Parasite of the Golden African Snapper Lutjanus Fulgens (Valenciennes, 1830) (Teleost Lutjanidae): Developmental Cycle and Infrastructure

ABSTRACT Microfilum lutjani n. g., n. sp. (Microsporida) was found on the gill filaments of Lutjanus fulgens (Teleost) inhabiting the coasts of Senegal. This microsporidium forms xenomas distinguished by the microvilli covering the plasma membrane. At all stages of development individuals have isolated nuclei and are in direct contact with the host cytoplasm. Merogony is binary and sporogony is tetrasporoblastic. the spore (4.75 times 2.60 μm)) is characterized by a manubrium inserted on a laterally offset anchoring disc and extending into a very short, noncoiled polar filament (no longer than 500 nm) in the form of a hook. This type of polar filament has not been described previously in the Microsporida.     

Microfilum lutjani N. G. N. Sp. (Protozoa Microsporida), a gill parasite of the golden African snapper Lutjanus fulgens (Valenciennes, 1830) (Teleost Lutjanidae): developmental cycle and ultrastructure

Microfilum lutjani n. g., n. sp. (Microsporida) was found on the gill filaments of Lutjanus fulgens (Teleost) inhabiting the coasts of Senegal. This microsporidium forms xenomas distinguished by the microvilli covering the plasma membrane. At all stages of development individuals have isolated nuclei and are in direct contact with the host cytoplasm. Merogony is binary and sporogony is tetrasporoblastic. The spore (4.75 x 2.60 microns) is characterized by a manubrium inserted on a laterally offset anchoring disc and extending into a very short, noncoiled polar filament (no longer than 500 nm) in the form of a hook. This type of polar filament has not been described previously in the Microsporida.     

A new microsporidium from the oyster Ostrea lutaria in New Zealand

Microsporidium rapuae n. sp. (Microsporida) forms cysts within the connective tissue surrounding the gut epithelium of the oyster Ostrea lutaria. Except for encapsulation of the cysts by fibroblasts no pathogenic effects were observed. Each cyst contained a large number of spores possessing long well-developed polar filaments. This is the third Microspora recorded from Bivalvia (Mollusca), and the first which does not belong to the family Chytridiopsidae (Minisporida).     

A New Species of Perezia (Microsporida: Pereziidae) from the Argentine Grasshopper Dichroplus elongatus (Orthoptera: Acrididae)1

The new microsporidium (Microsporida: Pereziidae), Perezia dichroplusae n. sp., infects the epithelial cells of the Malpighian tubules of the Argentine grasshopper Dichroplus elongatus. Characteristics of the pathogen include the following: development in direct contact with the host cell cytoplasm; bi-, tetra-, and sometimes multinucleate diplokaryotic meronts, rounded or elongate in shape; unikaryotic sporonts and sporogonial plasmodia, elongate in shape; sporoblasts and spores uninucleated; spores highly variable in size (1.6–6.7 by 1.0–2.7 μm, x?= 3.5 ± 0.09 by 1.5 ± 0.02, n = 100) with eight or fewer polar tube coils and showing a posterior electron-dense inclusion body.     

A case of fatal malabsorption syndrome caused by strongyloidiasis complicated with isosporiasis and human cytomegalovirus infection]

This 54-year-old Korean coal miner suffered from continuous watery diarrhea and weight loss after corticosteroid treatment (beta-methasone, 4 mg daily for 1 week) due to hip-bone fracture in January 1991. Except for the short therapy of steroid, no other histories were contributory. The malabsorption syndrome was aggravated while the case was treated under the impression of amebiasis or intestinal tuberculosis. AIDS antibody test by EIA was negative and quantitative analysis of serum immunoglobulins was in normal ranges. Nine months after the onset of symptoms, the case was diagnosed as malabsorption syndrome caused by complexed and aggravated infection by Strongyloides stercoralis, Isospora and cytomegalovirus in the small intestine, which were proved by stool examination and duodenal biopsy. His clinical course became worse even after high-dosed and prolonged albendazole treatment for strongyloidiasis with supportive fluid therapy. The patient was discharged in hopeless status in November, 1991 and died after one week at home.     

Host parasite interactions and pathophysiology in Giardia infections

Giardia is a protozoan parasite of the small intestine, and a leading cause of diarrhoeal disease worldwide in a variety of animals, including humans. The host-parasite interaction and pathophysiological processes of giardiasis remain incompletely understood. Current research suggests that Giardia-induced diarrhoeal disease is mediated by small intestinal malabsorption and maldigestion, chloride hypersecretion and increased rates of small intestinal transit. Small intestinal malabsorption and maldigestion results from the CD8+ lymphocyte-induced diffuse shortening of brush border microvilli. Activation of CD8+ lymphocytes occurs secondary to small intestinal barrier dysfunction, which results from heightened rates of enterocyte apoptosis and disruption of epithelial tight junctions. Both host and parasite factors contribute to the pathogenesis of giardiasis and ongoing research in this field may elucidate genotype/assemblage-specific pathogenic mechanisms. Giardia infections can result in chronic gastrointestinal disorders such as post-infectious Irritable Bowel Syndrome and symptoms may manifest at extra-intestinal sites, even though the parasite does not disseminate beyond the gastrointestinal tract. The infection can cause failure to thrive in children. Furthermore, there is now evidence suggesting that Giardia symptoms may vary between industrialised and developing areas of the world, for reasons that remain obscure. More research is needed to improve our understanding of this parasitic infection which was recently included in the World Health Organisation “Neglected Disease Initiative”.     

Pathophysiology of enteric infections with Giardia duodenalius

Giardia is the most prevalent human intestinal parasitic protist in the world, and one of the most common parasite of companion animals and young livestock. Giardia is a major cause of diarrhea in children and in travelers. The host-microbial interactions that govern the outcome of infection remain incompletely understood. Findings available to date indicate that the infection causes diarrhea via a combination of intestinal malabsorption and hypersecretion. Malabsorption and maldigestion mainly result from a diffuse shortening of epithelial microvilli. This enterocytic injury is mediated by activated host T lymphocytes. Pathophysiological activation of lymphocytes is secondary to Giardia-induced disruption of epithelial tight junctions, which in turn increases intestinal permeability. Loss of epithelial barrier function is a result of Giardia-induced enterocyte apoptosis. Recent findings suggest that these effects may facilitate the development of chronic enteric disorders, including inflammatory bowel disease, irritable bowel syndrome, and allergies, via mechanisms that remain poorly understood. A newly discovered SGLT-1 glucose uptake-mediated host cytoprotective mechanism may represent an effective modulator of the epithelial apoptosis induced by this parasite, and, possibly, by other enteropathogens. A better understanding of the pathogenesis of giardiasis will shed light on new potential therapeutic targets.     

An unusual intraerythrocytic parasite of Parablennius cornutus from South Africa

An intertidal horned blenny, Parablennius cornutus, captured at De Hoop Nature Reserve, South Africa, was found to harbor an unusual blood parasite and the haemogregarine Haemogregarina bigemina. In Giemsa-stained blood films, the enigmatic parasite occurred primarily as intraerythrocytic ringlike stages, with unstained centers and peripheral bands of beaded chromatin, not unlike Haemohormidium spp. Larger forms of the same organism stained pink with Giemsa, with nuclei occurring as 4-8 minute structures around the parasite body or distributed within it. These larger parasites apparently segmented into up to 8 individuals that were rounded or oval with deep-stained, comma-shaped or polar regions surrounding blue cytoplasm. Extracellular, binucleate, sporelike structures in clusters of as many as 16 individuals were also seen in blood films. Praniza larvae of the isopod Gnathia africana were seen in histological sections of gill tissue. Examination of spleen tissue by transmission electron microscopy showed intraerythrocytic organisms with ultrastructural characteristics like those of Haematractidium scombri, namely, a single boundary membrane, sometimes closely apposed nuclei with nucleoli, and profiles of dense material of variable structure. It is concluded that the parasite is probably related to Haemohormidium spp. and H. scombri, but it also shares features with some Microsporida.     

Eimeria meleagrimitis, E. adenoeides, and E. dispersa: Severity of infection and changes in the intestinal mucosa of the Turkey

Glucose and methionine were malabsorbed in some intestinal regions of turkeys infected with Eimeria meleagrimitis, E. adenoeides, or E. dispersa. The decrease in absorption was not always related to the numbers of parasites in the cells or the extent of damage to the mucosa. With E. adenoeides, malabsorption was found in the jejunum even though parasites were not present. Conversely, with E. dispersa, no malabsorption was observed in the duodenum even though light microscopy showed numerous parasites. In many intestinal regions, damage to the mucosal surface visible with scanning electron microscopy (SEM) was slight or absent, although malabsorption was marked. No changes were noted with SEM in the structure and orientation of the brush border in these regions. Villar height was significantly reduced in the regions of heaviest infection when intestinal damage was visible. Conversely, the crypts of Lieberkühn were often two or three times as deep in infected poults as in uninfected poults. In general, no differences were found in the thickness of the circular and longitudinal muscle layers between the infected and uninfected poults. The dry weight of the intestinal tissue was less from infected poults than from uninoculated controls and was related to both region of the intestine and severity of the infection.     

Observations on the Spores of Pleistophora gigantea (Thélohan, 1895) Swellengrebel, 1911, a Microsporidan Parasite of the Fish Crenilabrus melops*

SYNOPSIS. After 1914 protozoologists have generally agreed that Pleistophora gigantea (Thélohan, 1895) Swellengrebel, 1911, Ichthyosporidium giganteum (Thélohan, 1895) Swarczewsky, 1914, and I. phymogenes Caullery and Mesnil, 1905, are identical. Because no polar filament was found in the spores, however, some authors have followed Swarczewsky in considering this species to be a haplosporidan, while others have persisted in thinking it a microsporidan. Using preserved material that Swellengrebel saved from a tumor on which he based his studies, we have found a polar filament in the spores both with the PAS reaction and with the electron microscpe. This new information removes the only basis for the doubt which some authors have entertained, that Thélohan and Sweliengrebel correctly considered the parasite to belong to the Microsporida. Since Pleistophora gigantea is believed to be identical with I. phymogenes, recently selected by Sprague as type species of genus Ichthyosporidium Caullery and Mesnil, 1905, then Ichthyosporidium, originally assigned to the Haplosporida, must be regarded as a microsporidan genus. Whether it is distinct from all other microsporidan genera is a matter needing further consideration.     

Spores of a New Microsporidan Species Parasitizing Molluscan Neurons

Synopsis.
A new species of Microsporida was observed in neurons of the marine mollusc Aplysia californica and the ultrastructure of its spores was investigated. The spores were ˜ 1.3 m long and had a thick 3-layered coat. An anchoring disc and polar aperture were present. The polaroplast occupied most of the anterior half of the spore. The vesicular portion included lengths of loosely packed lamellar structures as well as the usual vesicular profiles embedded in fibrillar material; it was entirely surrounded by tightly packed, electron-dense lamellae. The polar tube had 5 or 6 coils. In this stage of the life cycle (mature spores), the parasites did not appear to be disturbing the host cells. The organism was named Microsporidium aplysiae sp. n.     

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.     

Presentation by scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon

This paper presents, for the first time, documentation by detailed scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon. Phase 1 is represented by the extracellular phase with mature spores liberated by the rupture of host cells. To infect new cells the spores have to discharge their polar filament. Spores with everted tubes show that these are helically coiled. When the polar tubules have started to penetrate into a host cell they are incomplete in length. The infection of a host cell can also be initiated by a phagocytic process of the extruded polar filament into an invagination channel of the host cell membrane. After the penetration process, the tube length is completed by polar tube protein which passes through the tube in the shape of swellings. A completely discharged polar tube with its tip is also shown. The end of a polar tube is normally hidden in the cytoplasm of the host cell. After completion of the tube length the transfer of the sporoplasm occurs and phase 2 starts. Phase 2 is the proliferative phase, or merogony, with the intracellular development of the parasite that cannot be documented by scanning electron microscopy. The subsequent intracellular phase 3, or sporogony, starts when the meronts transform into sporonts, documented as chain-like structures which subdivide into sporoblasts. The sporoblasts finally transform directly into spores which can be seen in their host cell, forming bubble-like swellings in the cell surface.