A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria.

Microsporidia are small (1-20 micron) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2,3], and are reported to lack mitochondria [2,4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2,4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5-9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.

Review of the sequential development of Loma salmonae (Microsporidia) based on experimental infections of rainbow trout (Oncorhynchus mykiss) and Chinook salmon (O. tshawytscha).

Loma salmonae is a common gill parasite of salmonids, and essentially all species in the genus Oncorhynchus are susceptible. Infections occur in both fresh and salt water. Loma salmonae is directly transmissible by ingestion of spores or infected tissue. The parasite infects the wall of blood vessels of various organs, but the gill is the primary site of infection. Initial infection occurs in the intestine, and xenomas are easily detected in the gills by standard histology at 4-6 wk post-exposure. A few presporogonic stages of the parasite are found in the heart endothelium prior to xenoma formation in the gills. Ultrastructure studies of early infections demonstrated that wandering blood cells transport the meronts to the gills, and that merogony occurs in pillar cells and other cells underlying the gill endothelium. Xenomas develop in these cells, resulting in hypertrophied host cells filled with spores. Xenomas ultimately rupture, and are associated with severe inflammation in which free spores are found in macrophages. The parasites are most pathogenic during this phase of the infection, resulting in severe vasculitis and clinical disease. Both rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus ishawytscha) recover from infections, but free spores persist in kidney and spleen phagocytes for many months after xenomas are absent in Chinook salmon. Fish that have recovered from the infection show strong immunity against the parasite, lasting up to 1 year. Fish are susceptible to infection by other routes of exposure by spores; co-habitation, anal gavage, and intramuscular, intraperitoneal and intravascular injection. Autoinfection probably occurs following release of spores in blood vessels after xenomas rupture. The optimal temperature for L. salmonae infections is 15-17 degrees C, with a permissive range of 11-20 degrees C.

Diagnosis of infections caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis using polymerase chain reaction in stool specimens.

OBJECTIVE: To study the usefulness of polymerase chain reaction (PCR) for the species identification of microsporidia in stool specimens obtained from HIV-infected patients with Enterocytozoon bieneusi or Encephalitozoon intestinalis infections. SETTING: Infectious disease clinic in a university hospital. PATIENTS: Thirty-seven stool specimens from 29 HIV-infected patients with microsporidiosis were tested. The diagnosis of microsporidian infection was made by light microscopy of stool specimens and species identification was made by transmission electron microscopy of duodenal biopsies. Sixty-one stool specimens from 45 HIV-infected patients without microsporidiosis served as controls. METHODS: PCR was performed using DNA extracted from stools with two primers sets, one specific for E. bieneusi and one specific for E. intestinalis. RESULTS: A 1265 base-pair fragment of the small subunit ribosomal RNA (rrs) gene could be amplified from all 31 stool specimens infected with E. bieneusi. In addition, a 930 base-pair fragment of the rrs gene could be amplified from all six stool specimens infected with E. intestinalis. The 61 control stools were negative with both primers. CONCLUSIONS: These results suggest that a PCR based assay using species-specific primers sets can be used successfully for microsporidian species differentiation from stool specimens, thus obviating the need for invasive biopsy procedures.

Microsporidiosis: human diseases and diagnosis.

Microsporidia are considered opportunistic pathogens in humans because they are most likely to cause diseases if the immune status of a host is such that the infection cannot be controlled. A wide spectrum of diseases has been reported among persons infected with microsporidia and different diagnostic techniques have been developed during the last decade.

Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae.

In molecular phylogenies based on ribosomal RNA, three amitochondriate protist lineages, Microsporidia, Metamonada (including diplomonads) and Parabasala (including trichomonads), are the earliest offshoots of the eukaryotic tree. As an explantation for the lack of mitochondria in these organisms, the hypothesis that they have diverged before the mitochondrial endosymbiosis is preferred to the less parsimonious hypothesis of several independent losses of the organelle. Nevertheless, if they had descended from mitochondrion-containing ancestors, it may be possible to find in their nuclear DNA genes that derive from the endosymbiont which gave rise to mitochondria. Based on similar evidence, secondary losses of mitochondria have recently been suggested for Entamoeba histolytica and for Trichomonas vaginalis. In this study, we have isolated a gene encoding a chaperone protein (HSP70, 70 kDa heat shock protein) from the microspordian Nosema locustae. In phylogenetic trees, this HSP70 was located within a group of sequences that in other lineages is targetted to the mitochondrial compartment, itself included in the proteobacterial clade. In addition, the N. locustae protein contained the GDAW(V) motif shared by mitochondrial and proteobacterial sequences, with only one conservative substitution. Moreover, microsporidia, a phylum which was assumed to emerge close to the base of the eukaryotic tree, appears as the sister-group of fungi in the HSP70 phylogeny, in agreement with some ultrastructural characters and phylogenies based on alpha- and beta-tubulins. Loss of mitochondria, now demonstrated for several amitochondriate groups, indicates that the common ancestor of all the extant eukaryotic species could have been a mitochondriate eukaryote.

Identification of the microsporidian parasite, Enterocytozoon bieneusi in faecal samples and intestinal biopsies from an AIDS patient.

The microsporidian parasite, Enterocytozoon bieneusi, is currently recognized as a potentially important cause of chronic diarrhoea in patients infected with the human immunodeficiency virus (HIV). Faecal concentrates from a 38-year-old, HIV-seropositive patient examined by light and electron microscopy revealed the presence of numerous microsporidian spores. The structural characteristics of the spores were consistent with those previously described for Enterocytozoon bieneusi. Each spore contained a single nucleus, a posterior vacuole and a polar filament with 6-7 overlapping coils which appeared in cross-section as a series of 3 doublets. Mature spores were surrounded by an inner unit membrane, an electron-lucent endospore and a thin, electron-dense exospore. The identity of the parasite was confirmed by the detection of unique endogenous developmental stages in duodenal biopsies. Both proliferative and sporogonial plasmodia (meronts and sporonts) were observed and all stages were monokaryotic (single nucleus) and apansporoblastic (sporophorous vesicle absent). Proliferative and sporogonial plasmodia divided by plasmotomy and spore organelles (polar filament, attachment disc and polaroplast) were well developed prior to fission of the sporogonial plasmodium.

Invasion success of Fibrillanosema crangonycis, n.sp., n.g.: a novel vertically transmitted microsporidian parasite from the invasive amphipod host Crangonyx pseudogracilis.

Parasitism is known to be an important factor in determining the success of biological invasions. Here we examine Crangonyx pseudogracilis, a North American amphipod invasive in the United Kingdom and describe a novel microsporidium, Fibrillanosema crangonycis n.sp., n.g. The primary site of infection is the female gonad and the parasite is transovarially transmitted to the eggs. PCR screening reveals a female bias in the distribution of parasites (96.6% of females, N=29; 22.2% of males, N=27), which is indicative of host sex ratio distortion. The morphological and molecular characterisations of this new microsporidium place it outside all currently established genera. On the basis of these differences, we erect the new genus Fibrillanosema n.g. While F. crangonycis is morphologically identical to uncharacterised microsporidia from populations of North American amphipods, it is distinct from microsporidia found in European populations of amphipods. These data support the hypothesis that vertically transmitted parasites may be selectively retained during invasion events. Furthermore where vertical transmission is combined with host sex ratio distortion these parasites may directly enhance host invasion success through increased rates of population growth.

Detection and identification of gastrointestinal microsporidia using non-invasive techniques.

AIMS: To detect enteric microsporidia in faecal specimens from patients with the acquired immunodeficiency syndrome (AIDS), and to identify the spores to species level without using invasive procedures. METHODS: Formalised faecal preparations were examined using a modification of the strong trichrome staining method to demonstrate microsporidian spores. Six positive specimens were prepared for electron microscopy by emulsification and separation using a 9% Ficoll gradient. RESULTS: The modified staining technique readily identified microsporidian spores. Spores of different species showed variation in size. Identification using electron microscopy was successful for five of the six positive specimens examined. It was unsuccessful for one specimen in which spores were less abundant on initial staining. CONCLUSIONS: The modified strong trichrome staining method is a useful way of detecting spores of intestinal microsporidia in faecal specimens. Variation in spore size may permit provisional identification by light microscopy. Electron microscopic examination of faecal preparations is useful for identifying spores to species level.

Electron microscopic changes in Enterocytozoon bieneusi following treatment with albendazole.

AIM: To identify and describe electron microscopic changes occurring in Enterocytozoon bieneusi in patients treated with albendazole. METHODS: Eighteen HIV seropositive patients with E bieneusi infection of the small intestine were treated with albendazole 400 mg twice a day for one month. Duodenal biopsy specimens obtained before and at the end of treatment were examined electron microscopically by a pathologist who was unaware of the clinical response. A semiquantitative assessment of the parasite load and description of the parasite morphology was made. RESULTS: A complete resolution of diarrhoea occurred in nine patients and a further three had a greater than 50% reduction in baseline stool frequency or volume. Three of the non-responders were also infected with cryptosporidium. There was a reduction in parasite load in those responding to treatment and an increase in abnormal forms in both responders and non-responders. CONCLUSION: The clinical response to albendazole treatment seen in some patients with small intestine microsporidiosis may be due to damage to the developmental stages, causing a partial inhibition of parasite reproduction.

Persistent damage to Enterocytozoon bieneusi, with persistent symptomatic relief, after combined furazolidone and albendazole in AIDS patients.

AIM: To investigate morphological changes in Enterocytozoon bieneusi and the duration of symptomatic relief after combination treatment with furazolidone and albendazole in AIDS patients. METHODS: Four severely immunocompromised AIDS patients with symptomatic E bieneusi infection of the gut received an 18 day course of combined furazolidone and albendazole (500 + 800 mg daily). All patients were monitored for parasite shedding in stool by light microscopy at the end of treatment and monthly during follow up. At the end of treatment, duodenal biopsy specimens obtained from three patients were studied by transmission electron microscopy by two pathologists blind to the patients' treatment or clinical outcome. Duodenal biopsy specimens obtained from one of the patients two months after completion of treatment were also studied electronmicroscopically. RESULTS: All patients had long lasting symptomatic relief, with a major decrease--or transient absence--of spore shedding in stools from completion of treatment. After treatment, changes in faecal spores were persistently found by light microscopy in all cases, and there was evidence of both a substantial decrease in the parasite load and ultrastructural damage in the parasite in all biopsy specimens. The treatment was well tolerated, and no patient had clinical or parasitological relapse during follow up (up to 15 months). CONCLUSIONS: The long lasting symptomatic relief observed in all four treated patients correlated with the persistent decrease in parasite load both in tissue and in stool, and with the morphological changes observed in the life cycle of the protozoan. These data suggest that combined treatment with furazolidone and albendazole is active against E bieneusi and may result in lasting remission even in severely immunocompromised patients.

Microsporidial keratoconjunctivitis caused by Septata intestinalis in a patient with acquired immunodeficiency syndrome.

PURPOSE: To examine and treat a patient with acquired immunodeficiency syndrome (AIDS) who had mildly hyperemic conjunctiva and epithelial keratopathy in both eyes. METHODS: The patient underwent conjunctival biopsy. The specimen was examined by transmission electron microscopy. RESULTS: Septata intestinalis was demonstrated to be the cause of keratoconjunctivitis in the patient. The keratoconjunctivitis resolved after three weeks of therapy with topical fumagillin. No organisms were seen on repeat conjunctival biopsy. CONCLUSIONS: Microsporidial keratoconjunctivitis in patients with AIDS can be caused by S. intestinalis. This condition appears to respond to topical fumagillin.

Ultrastructural qualitative and quantitative data on the sporogenesis of the protozoan Abelspora portucalensis (Microspora, Abelsporidae): a different approach to the study of microsporidia.

The sporogenesis of the microsporidium Abelspora portucalensis was studied with electron microscopy. In qualitative terms, new aspects of the cytoplasmic ultrastructure of the schizont, sporont, and sporoblast are described: the presence of microtubules, of aggregates of small opaque vesicles, and of dispersed larger vesicles with clear matrix. The hypothesis that the opaque vesicles may represent the Golgi apparatus and the clear vesicles may correspond to the smooth endoplasmic reticulum is discussed. The use of standard stereological and statistical techniques gives us a new perspective on the development of this microsporidium. The most relevant quantitative data display that the amount of rough endoplasmic reticulum (either in relative or absolute terms) presents significant differences among the three stages, with the sporont showing the highest values; that the absolute (but not the relative) volume of the large vesicles significantly changes during sporogenesis with the highest values presented by the sporont; that the surface-to-volume ratio of the schizont and sporont cells is similar and significantly greater than that of the sporoblast cell; that the surface density of the nucleus in relation to soma remains constant in the three stages (on the contrary, the surface-to-volume ratio of the nucleus increases and its volumetric density diminishes); and finally, that the nucleolus decreases its relative and absolute volumes. The functional significance of these results is analyzed and the application of similar methodology in quantifying the effects of drugs upon microsporidia is suggested.

Encephalitozoon-like microsporidia in the ticks Amblyomma cajennense and Anocentor nitens (Acari: Ixodidae).

A Encephalitozoon-like microsporidia was found in epithelial cells of the midgut and the salivary glands of Amblyomma cajennense (F.) and Anocentor nitens (Neumann) that had fed on rabbits. Ultrastructural studies demonstrated that all stages of the life cycle of the parasite occur in parasitophorous vacuoles and contain only 1 nucleus. The sporonts detach from the limiting membrane of the vacuole and divide by binary fission to produce the sporoblasts, each presenting a thickened electron-dense wall, and a primordium of a polar filament. Each spore contained a single nucleus, an electron-dense and rough exospore, an electron-lucent and thick endospore, and 5 coils of the polar tubule.

Intracellular protozoan infection in small intestinal biopsies of patients with AIDS. Light and electron microscopic evaluation.

Small intestinal biopsies of 21 patients with acquired immunodeficiency syndrome (AIDS) with light microscopic findings diagnostic or suspicious for parasite infection were investigated by transmission electron microscopy (TEM). TEM allowed us to identify and specify the genus and species of involved parasites in 16 out of the 21 cases: 7 Cryptosporidium parvum, 5 Enterocytozoon bieneusi and 4 Isospora belli. Cryptosporidium was easily identified on light microscopy (LM), and only slightly influenced by parasite burden in all the 7 cases; TEM confirmed LM diagnosis and made it possible to characterize the parasites as C. parvum. The identification of Microsporidium on LM in our cases was related to the burden of parasite; its presence was certainty identified in 2 cases and suspected in 3. TEM allowed to identify these parasites as E. bieneusi. Intracytoplasmic coccidia could be detected with certainly in semithin sections in all 4 cases, but TEM was always needed to specify the infectious agent as I. belli. In 5 cases the suspicious of protozoan infection on LM (3 microsporidia, 1 intracytoplasmic coccidia and 1 Cryptosporidium) was not confirmed by TEM. Our data suggest that TEM is an appropriate diagnostic tool in this field of pathology and necessary in most of the cases.

Ultrastructure of atypical (teratoid) sporogonial stages of Enterocytozoon bieneusi (Microsporidia) in naturally infected rhesus monkeys (Macacca mulatta).

OBJECTIVE: To demonstrate the ultrastructural features of normal and atypical (teratoid) developmental stages of Enterocytozoon bieneusi in naturally infected rhesus monkeys (Macacca mulatta). DESIGN AND METHODS: Two rhesus monkeys with chronic simian immunodeficiency virus infection developed naturally acquired microsporidian infections. The gallbladder had a high parasite burden and was evaluated by transmission electron microscopy. The microsporidian agent was confirmed as E bieneusi by polymerase chain reaction. RESULTS: In addition to normal sporogonial plasmodia and spores of E bieneusi, abnormal teratoid structures were noted. These structures were greatly enlarged (up to 10 microm) and were surrounded by an electron-dense exospore and electron-lucent endospore typical of mature spores. Unlike mature spores, the abnormal structures contained multiple nuclei and polar tubes in varying proportions, which were reminiscent of sporogonial plasmodia. CONCLUSIONS: These teratoid structures represent aberrant sporogonial stages, a result of defective maturation in which abnormal cytokinetic replication of organelles occurs, and normal development into uninucleate sporoblasts and spores is inhibited. This leads to the development of teratoid stages having mature spore walls, but containing multiple sets of nuclei and polar tubes, unusual polyribosomal arrays and vacuoles, or persistent cleavage. The biological significance of these atypical spores is unknown, but it is evident that they develop in the absence of antimicrosporidian drugs in extraintestinal tissues from nonhuman primates. Teratoid spores of E bieneusi should not be misinterpreted as another microsporidian species or confused with other pathogenic protozoa, nor should their presence be misconstrued as evidence of antimicrosporidian drug efficacy or toxicity.

Coinfection with Giardia lamblia and Enterocytozoon bieneusi in a patient with acquired immunodeficiency syndrome and chronic diarrhea.

Diarrhea is an important clinical problem in immunosuppressed patients with acquired immunodeficiency syndrome (AIDS). There are numerous classical as well as emerging enteric pathogens that can produce diarrhea; however, these agents can be missed when only one method, such as microbiological examination of stool, is used for diagnosis. The endoscopic biopsy is a sensitive method for diagnosis of many viral, fungal, and parasitic infections of the gastrointestinal tract. Although only one agent is often identified in mucosal biopsies from these immunosuppressed patients, coinfection with multiple microbial agents is being increasingly recognized. Giardia infection is not as prevalent as other pathogens in patients with AIDS, but it remains an important diarrheal agent that is potentially curable. However, there have been only rare reports of coinfections with giardiasis and other infectious agents. This report describes a patient with AIDS and chronic diarrhea who had repeated negative stool examinations for ova and parasites. Light and electron microscopic examination of subsequent endoscopically obtained small intestinal biopsies revealed coinfection with two parasites, Enterocytozoon bieneusi and Giardia lamblia. Following treatment with metronidazole for giardiasis, the diarrhea persisted, but was less severe. This report also describes the diagnostic features of Giardia and Enterocytozoon infections in biopsy tissues and emphasizes the importance of identifying enteric coinfections in patients with AIDS to ensure timely and specific modes of therapy.

Microsporidiosis in a young ostrich (Struthio camelus).

Microsporidia are obligate, intracellular, protozoan parasites of a wide variety of vertebrates and invertebrates. Confirmed reports of microsporidial infection of avian species are few (lovebirds, a parrot, and a group of budgerigar chicks). At slaughter, a 14-mo-old ostrich was found to have small intestinal serosal hemorrhages during postmortem inspection. Histologic examination of the small intestine revealed a chronic lymphoplasmacytic to purulent enteritis with mucosal hyperplasia, muscular hypertrophy, and numerous microsporidia that were located within the superficial enterocytes and the lamina propria. Microsporidia have a ubiquitous distribution in nature and are suspected as possible zoonotic agents.

Ultrastructure of the microsporidian Inodosporus octospora (Thelohaniidae), a parasite of the shrimp Palaemon serratus (Crustacea, Decapoda).

A parasite of the muscle of the shrimp Palaemon serratus has been examined by light and electron microscopy. Development occurs among myofibrils and induces ultrastructural alterations of the muscle fibers causing white discoloration. This microsporidian is characterized by uninucleate, later diplokaryotic and di-diplokaryotic meronts. The mother cell develops by rosette-like budding into 8 uninucleate sporoblasts, each containing 3 tape-like filaments attached to the wall that is enclosed in a persistent sporophorous vacuole. Each sporoblast gives rise to a uninucleate spore that possesses 3 elongated tape-like filaments attached to the spore wall, like spore tails. The morphological characters of the spores, redescribed in the present study, suggested that the spores belonged to Inodosporous octospora. The possibility that in the future members of Inodosporus sp. may be considered a new parasite group is discussed.

Kabataia gen. n., a new genus proposed for Microsporidium spp. infecting trunk muscles of fishes.

Based on a fine structural study, a new genus, Kabataia gen. n., is proposed for Microsporidium arthuri Lom, Dyková and Shaharom, 1990. It develops in trunk muscles of a South-East Asian freshwater fish, Pangasius sutchi. The genus has nuclei isolated throughout the cycle, merogony stages are multinucleate, sporogony proceeds in 2 steps: multinucleate sporont segments into sporoblast mother cells which produce 2 sporoblasts. Sporoblasts and early spores are characterized by a dense globule at the site of the posterior vacuole. Mature spores are of a rather variable shape. Their exospore is raised into small, irregular fields. The polaroplast is relatively small and its posterior part consists of flat vesicles with dense contents. The polar tube makes a small number (4 to 6) of turns. A congeneric species is Kabataia seriolae (Egusa, 1982) comb. nov. from cultured marine yellowtails Seriola quinqueradiata. Kabataia inflicts heavy damage on muscle tissue. The sarcoplasm within which Kabataia develops is reduced to an amorphous mass with tubule-like fibrils, microfibrils and small vesicles.