Elizabeth U. Canning

A triploblast origin for Myxozoa

Hox genes, which play key roles in the development of body plans, have been described from a variety of metazoans. Here we report the presence of Hox class genes that are typical of triploblasts in Myxozoa, formerly a protozoan taxon. This finding confirms Myxozoas phylogenetic affinity with the Bilateria and reveals an extreme example of parasitic degeneracy.

Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique.

AIMS--To assess the value of a new rapid fluorescence method for the diagnosis of microsporidiosis in HIV seropositive patients. METHODS--Microsporidian spores in stools were demonstrated by using the fluorochrome stain Uvitex 2B. The new technique was evaluated in three groups of HIV seropositive patients with diarrhoea. Group 1: 19 patients with biopsy confirmed E bieneusi infection (186 stool samples); group 2: 143 consecutive patients from whom faeces were submitted for routine investigation of diarrhoea (318 samples); group 3: 16 patients with small intestinal biopsy specimens negative for microsporidia (55 samples). The new method was used to monitor spore shedding during experimental treatment with paromomycin and albendazole in four patients. RESULTS--Brightly fluorescent spores were detected in all stool samples of patients in group 1. In group 2 16 (11%) patients had spores in their stool samples. E bieneusi was found in 11 patients; in the other five another genus of microsporidia, Encephalitozoon, was recognised. Encephalitozoon spores were also found in the urine of three of these patients and in the maxillary sinus aspirate of two of them, suggesting disseminated infection. The results were confirmed by electron microscopic examination. In group 3 negative biopsy specimens were confirmed by negative stool samples in all cases. Treatment with albendazole and paromomycin did not affect the spore shedding in three patients with E bieneusi infection. By contrast, in a patient with Encephalitozoon sp infection albendazole treatment resulted in clinical improvement together with complete cessation of spore excretion in the stool. CONCLUSION--The Uvitex 2B fluorescence method combines speed, sensitivity, and specificity for the diagnosis and treatment evaluation of intestinal and disseminated microsporidiosis.

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.

Biodiversity and evolution of the Myxozoa.

Myxozoans (phylum Myxozoa) are metazoan parasites utilizing invertebrate and (mainly) aquatic vertebrate hosts. They have in common with cnidarians the possession of virtually identical, highly complex organelles, namely the polar capsules in myxozoan spores, serving for attachment to new hosts and the nematocysts in surface epithelia of cnidarians, serving for food capture. Although myxozoan spores are multicellular, the simple trophic body forms of almost all species, reduced to syncytial plasmodia or single cells, reveal no clues to myxozoan ancestry or phylogenetic relationships. The myxozoan genus Buddenbrockia is one of only two known genera belonging to a clade which diverged early in the evolution of the Myxozoa. Today the Myxozoa are represented by two classes, the Myxosporea, containing all the better-known genera, which alternate between fish and annelids, and the Malacosporea, containing Buddenbrockia and Tetracapsuloides, parasitising bryozoans. The latter genus also infects salmonid fish, causing proliferative kidney disease (PKD). The enigmatic Buddenbrockia has retained some of its ancestral features in a body wall of two cell layers and a worm-like shape, maintained by four longitudinally-running muscle blocks, similar to a gutless nematode and suggestive of a bilaterian ancestry. Although some analyses of 18S rDNA sequences tend towards a cnidarian (diploblast) affinity for myxozoans, the majority of these studies place them within, or sister to, the Bilateria. The latter view is supported by their possession of central class Hox genes, so far considered to be synapomorphic for Bilateria. The simple body form is, therefore, an extreme example of simplification due to parasitism. Various hypotheses for the occurrence of identical complex organelles (nematocysts and polar capsules) in diploblast and triploblast phyla are evaluated: common ancestry, convergent evolution, gene transfer and, especially, endosymbiosis. A theory of the evolution of their digenetic life cycles is proposed, with the invertebrate as primary host and secondary acquisition of the vertebrate host serving for asexual population increase.

Gametocyte and gamete development in Plasmodium falciparum.

The ultrastructural organization of the mature gametocytes of Plasmodium falciparum isolated from the peripheral circulation of naturally infected Gambians is examined and compared with immature forms obtained from the peripheral circulation of a chloroquine treated patient. The latter are recognized as the stage 2 and 3 developmental forms (Hawking, Wilson & Gammage 1971 Trans. R. Soc. trop. Med. Hyg. 65, 549-559) observed by light microscopy and are distinguished in the electron microscope by three characters; (i) they do not fill the host cell, (ii) they contain few, if any, osmiophilic bodies, (iii) they possess an extensive subpellicular tubule system. Maturation (capacitation) of these immature parasites takes many days and is followed by an extended period of maturity during which the gametocytes will exflagellate. Mature macro- and microgametocytes have numerous characters in common with the gametocytes of avian and reptilian Plasmodiidae, namely a tripartite pellicle, cristate mitochondria, a comparatively high density of osmiophilic bodies in the macrogametocyte, cytostomal feeding, Golgi body, and persistent nucleolus in the female gametocyte. These similarities together with the unexpected nuclear changes detected in macrogametogenesis suggest that P. falciparum is best considered as pre-dating the ‘malariae’ and ‘vivax’ groups and not as having evolved from them. Light microscopy, scanning and transmission electron microscopy and videotape analyses of gamete formation were undertaken. Nuclei in the mature gametocytes are Feulgen negative but upon activation rapidly become Feulgen positive. The gametes also are Feulgen positive. The crescentic parasites swell to become large spherical cells and escape from the host cell by osmotic or enzymic activity. The microgametocyte undergoes three mitotic divisions during which the chromosomes are sequentially reduced in number such that ca. 7 are incorporated into each gametic nucleus. The microtubule organizing centre (m. t. o. c.), which in the mature gametocyte is associated with the intranuclear body, is attached to the centriolar plaque of the first division spindle. There it differentiates into kinetosomes which act as foci for the polymerization of axonemes. The kinetosomes and axonemes remain attached to the centriolar plaques during division and are segregated synchronously with the genome at each division. Subsequently one axoneme enters each haploid gamete at exflagellation. Exflagellation is accompanied by a significant reduction in microgametocyte volume which is associated with an increase in density of the cytoplasm. The female gametocyte does not decrease in volume but undergoes nuclear changes in which a single pole of an intranuclear spindle is detected. Comparisons are made with macrogametogenesis in avian malarial parasites from which it is suggested that this spindle, if not half of a normal mitotic spindle, is an atavistic trait. The possibility of a meiotic gametic division is discussed but discounted. The activity pattern of the microgamete was found to be similar to that of other malarial parasites, with states of high and low activity or immobility. High activity, which results in rapid movement through the medium, is produced by long wavelength (12 μm), low amplitude (1.1μm) waves generated at ca. 12 waves per second; low activity, which results in contorted gyrating on the spot, is produced by long wavelength (14.1), high amplitude (2.3) waves produced at ca 1 wave per second. Following an initial period of continuous activity the gamete usually alternates between high and low activity states. Subsequent low activity and immobility is in turn followed by death. Microgamete activity was profoundly affected by the plasma of some patients, presumably as a result of the antigametocyte antibodies present. The microgamete contains a single axoneme, at one end of which lies the kinetosome with the juxtakinetosomal sphere and granule. It is this end which emerges first from the parental gametocyte. A single nucleus is centrally located in many microgametes although 23% are anucleate.

Gametogenesis and Fertilization in Plasmodium yoelii nigeriensis: A Transmission Electron Microscope Study

The ultrastructure of the micro- and macro-gametocytes of Plasmodium yoelii nigeriensis and the nuclear and cytoplasmic changes during gametogenesis and fertilization were examined with the electron microscope. Osmiophilic bodies, dispersed in the cytoplasm, served to distinguish the gametocytes from other erythrocytic stages and were thought to play a part in the gametocytes’ escape from the host cells by attachment to the parasite’s plasmalemma causing dissolution of the overlying erythrocyte cytoplasm. Macrogametocytes were distinguished from the microgametocytes by their greater density of ribosomes, more elaborate endoplasmic reticulum, which contained electron dense material, more numerous mitochondria and smaller nucleus. In microgametogenesis nuclear division was endomitotic and the genome was segregated on three successive spindle formations. Microtubule organizing centres developed adjacent to the nuclear envelope and gave rise to orthogonal tetrads of kinetosomes which were found at opposite poles of the first nuclear spindle. Axoneme assembly from the kinetosome followed the usual pattern by the addition of sub-units terminally on to the A subfibres, and, with a slight lag, on to the B subfibres. The kinetosomes were closely linked to centriolar plaques in pores of the nuclear envelope, at the spindle poles. The attachment of the kinetosomes and their axonemes to the spindle poles provided the mechanism by which each haploid set of chromosomes was eventually endowed with a single axoneme. At the time of the final nuclear segregation the kinetosome and a newly formed juxta-kinetosomal sphere and granule became surrounded by a basket work of irregular tubules which lay close to a bud of the nucleus containing a spindle pole and the now highly condensed chromatin. During exflagellation the juxta-kinetosomal sphere and granule, together with the kinetosome and axoneme were forced through the perikinetosomal basket perpendicularly towards the surface and distended the plasmalemma. In the final stages of gamete formation, the gamete slid off tangentially to the surface and the nuclear bud also passed through the perikinetosomal basket, became separated from the main body of nucleoplasm and was incorporated as the nucleus of the gamete. The free microgamete contained a single axoneme with its kinetosome and distal juxta-kinetosomal sphere and granule. The condensed nucleus was intertwined with the axoneme. After emergence from the erythrocyte there were contrastingly few changes in the macrogamete. The absence of intranuclear spindles and maturation bodies provided evidence that meiosis did not occur at this stage. At fertilization one pole of the microgamete was closely applied to the surface of the macrogamete, and amorphous material on the outer surfaces became confluent. This allowed contact and fusion of the plasmalemmas. The naked axoneme and the nucleus of the microgamete passed into the cytoplasm of the macrogamete. Decondensation of the microgamete chromatin may have occurred before fusion of the two nuclei. A chromosome number of about ten was estimated for the microgametes from numbers of kinetochores and microtubules in the intranuclear spindles. This accords with the estimate for the sporogonic stages. The perikinetosomal basket and juxta-kinetosomal sphere and granule have not been described previously in malaria parasites. The former may have a skeletal function in directing the axoneme and nuclear bud towards the surface. The latter, in their position at the distal end of the microgamete may assist in the penetration of the macrogamete by mediating the fusion of the plasmalemmas of the two gametes, though we have not been able to determine that the kinetosomal end is that one which contacts the macrogamete.

A New Class and Order of Myxozoans to Accommodate Parasites of Bryozoans with Ultrastructural Observations on Tetracapsula bryosalmonae (PKX Organism)

Abstract Tetracapsula bryosalmonae, formerly PKX organism, is a myxozoan parasite that causes proliferative kidney disease in salmonid fish. Its primary hosts, in which it undergoes a sexual phase, are phylactolaemate bryozoans. It develops in the bryozoan coelomic cavity as freely floating sacs which contain two types of cells, stellate cells and sporoplasmogenic cells, which become organised as spores. Eight stellate cells differentiate as four capsulogenic cells and four valve cells which surround a single sporoplasmogenic cell. The sporoplasmogenic cell undergoes meiosis and cytoplasmic fission to produce two sporoplasms with haploid nuclei. Sporoplasms contain secondary cells. The unusual development supports previously obtained data from 18S rDNA sequences, indicating that species of Tetracapsula form a clade. It diverged early in the evolution of the Myxozoa, before the radiation that gave rise to the better known genera belonging to the two orders in the single class Myxosporea. The genus Tetracapsula as seen in bryozoans shares some of the characters unique to the myxosporean phase and others typical of the actinosporean phase of genera belonging to the class Myxosporea. However, it exhibits other features which are not found in either phase. A new class Malacosporea and order Malacovalvulida are proposed to accommodate the family Saccosporidae and genus Tetracapsula. Special features of the new class are the sac-like proliferative body, valve cells not covering the exit point of the polar filament, lack of a stopper-like structure sealing the exit, maintenance of valve cell integrity even at spore maturity, absence of hardened spore walls and unique structure of sporoplasmosomes in the sporoplasms.

The organization of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei.

Ultramicroscopic features of the ookinete/oocyst transformation in Plasmodium berghei are described. The apical complex of organelles and some pellicular components, believed to be responsible respectively for cell penetration and body form were resorbed into the cytoplasm shortly after the ookinete came to rest under the basal lamina of the midgut wall. Within a single digitate nucleus, spindles at different phases of division were observed with kinetochores in early and late anaphase position. From the number of kinetochores the chromosome complement was estimated at 5–10.

Development and ultrastructure of Trachipleistophora hominis n.g., n.sp. after in vitro isolation from an AIDS patient and inoculation into athymic mice.

Continuous culture was achieved in several cell lines of a microsporidium obtained from the skeletal muscle of an AIDS patient. Development in COS-1 and RK13 cells was prolific. Spores from the original biopsy were also inoculated into athymic mice by i.m. and i.p. routes. Infection was found in several organs as well as in skeletal muscle after a few weeks. All stages were surrounded by an electron-dense surface coat. Meronts had 2-4 nuclei and divided by binary fission. In sporogony the surface coat became separated from the plasma membrane to form a sporophorous vesicle, within which division into sporoblasts was effected by repeated binary fissions. The number of sporoblasts (and later spores) within the sporophorous vesicles varied from 2 to > 32 and the sizes of the vesicles varied, according to the number of spores contained therein, from 5 microns diameter to 14.0 x 11.0 microns. Spores measured 4.0 x 2.4 microns and had a prominent posterior vacuole. The parasite differs from the genus Pleistophora in that it does not form multinucleate sporogonial plasmodia and that the sporophorous vesicle enlarges during sporogony and its wall is not a multilayered structure. It is proposed to place it in a new genus and species Trachipleistophora hominis n.g., n.sp.

Vittaforma corneae N. Comb. for the Human Microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, Based on its Ultrastructure in the Liver of Experimentally Infected Athymic Mice

ABSTRACT. A new genus, Vittaforma n. g. is proposed for the human microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, based on the ultrastructure of developmental stages in the liver of experimentally infected athymic mice. The diplokaryotic arrangement of the nuclei is the only character that conforms with the description of the genus Nosema. Sporogony is polysporoblastic, sporonts are ribbon‐shaped, constricting to give rise to linear arrays of sporoblasts and each parasite is enveloped by a complete cistema of host endoplasmic reticulum. Comparison of N. corneum, with established genera revealed that there were none with the same combination of characters. Consequently it is proposed that Nosema corneum be placed in a new genus as Vittaforma corneae n. comb.