Historical Introduction

Of three cases of microsporidiosis reported in the first half of this century, only one (Torres, 1927) remains as a possibly correct identification (see Canning and Lom, 1986, for historical summary). Torres (1927) described Encephalitozoon chagasi as the cause of meningocephalomyelitis with myositis and myocarditis in a baby girl who died 2 days after birth. Unfortunately the material has been lost and confirmation of its microsporidial nature cannot be obtained. The first certain case was that of an 11 year-old boy in Japan who suffered a severe convulsive illness, the cause of which was Encephalitozoon sp. (Matsubayashi et al, 1959). After a gap of 25 years a similar illness in a 2 year-old Colombian child resident in Sweden was found to be of microsporidian origin (Encephalitozoon cuniculi) (Bergquist et al., 1984). Unidentified micro-sporidia in two cases of corneal microsporidosis (Ashton and Wirasinha, 1973; Pinnolis et al., 1981), and Nosema connori (Sprague, 1974) (now Brachiola connori; Cali et al., 1998) causing a generalised infection in an athymic infant (Margileth et al., 1973) complete the list of microsporidia found before the AIDS epidemic. The history of human microsporidiosis might have remained at the level of occasional severe cases had it not been for AIDS.

The first microsporidium found in an AIDS patient was Enterocytozoon bieneusi, an entirely new genus and species causing chronic diarrhoea (Desportes et al., 1988) and to this have been added another three new genera and eight new species in HIV-positive or HIV-negative patients, bringing the known total to 14 species. The new species are: Encephalitozoon hellem (Didier et al., 1991); Vittaforma corneae (Nosema corneum) (Shadduck et al., 1990; Silveira and Canning, 1995); Encephalitozoon intestinalis (Septata intestinalis) (Cali et al., 1993; Hartskeerl et al., 1995); Pleistophora sp. (Ledford et al., 1985); Nosema ocularum (Cali et al., 1991);

Principles and Practice of Clinical Parasitology

Edited by Stephen Gillespie and Richard D. Pearson © 2001 John Wiley & Sons Ltd

Trachipleistophora hominis (Hollister et al., 1996b); Trachipleistophora anthropophthera (Vavra et al., 1998b); Brachiola vesicularum (Cali et al., 1998) and Brachiola algerae (Nosema algerae) (Visvesvara et al., 1999; Lowman et al., 2000). It is highly likely that these do not represent the full range of microsporidia capable of infecting man and that with increasing awareness of these organisms, new species will be added to this list.

DESCRIPTION OF THE ORGANISMS Reproduction

Microsporidia are unicellular organisms which produce very small spores, characterised by an extrusible polar tube which is used to convey the infective agent (sporoplasm) out of the spore directly into host cell cytoplasm (Figures 8.2G, p. 175, and 8.5A, Plate IV). They were first recognised as a distinct group of organisms (microsporidies) in 1882 and are now accommodated within their own phylum Microsporidia Balbiani, 1882. They have no functional mitochondria and their ribosomes are unusual, being of a size typical of prokaryotes (70S with 16S and 23S ribosomal RNAs, the 5.8S rRNA gene being incorporated in the 23S rRNA), but in other respects they are true eukaryotes with membrane-bound nuclear material and nuclear division by intranuclear mitosis. Some genera have isolated nuclei (monokaryotic), others have paired (diplo-karyotic) nuclei which divide synchronously.

All microsporidia are obligate parasites with a life-cycle involving repeated proliferation by merogony, followed by sporogony, in which sporonts divide into two or more sporoblasts that mature into spores. Meronts usually have a simple plasma membrane while sporonts have an electron-dense surface coat which later becomes the outer (exospore) layer of the spore wall. However, meronts of Pleistophora, Trachipleistophora and Brachiola have a well developed surface coat and the sporont of Enterocytozoon does not lay down the surface coat until it is actually undergoing division into sporoblasts. The repeated merogonic divisions by binary or multiple fission are responsible for massive infections, resulting in complete destruction of cells and whole tissues.

In some genera, sporonts produce sporoblasts (the precursors of spores) by binary or multiple fission in direct contact with host cell cytoplasm, so that the resultant spores are freely dispersed in the host cell. In other genera, an envelope separates from the sporont surface and division, again by binary or multiple fission, occurs within this envelope (sporophorous vesicle), resulting in packets of spores rather than free spores. In one medically important genus, all stages of development take place in a host membrane-bound vacuole (=parasitophorous vacuole).

Spores

Microsporidian spores are unique. Within the spore wall, consisting of an electron-dense exospore and a lucent endospore, together having proteinaceous and chitinous components, lie the cytoplasmic structures limited by a plasma membrane (see Figure 8.3G). At the anterior end, lying within a polar sac, shaped like the cap of a mushroom, is an anchoring disc into which the base of the polar tube is inserted. The tube runs a straight course posteriad for about half the length of the spore, then forms a coil in the peripheral cytoplasm. The coiled part of the tube may be of uniform diameter (isofilar) or show a sharp change to a narrower diameter for the posterior coils (anisofilar). Surrounding the straight section is the polaroplast, an organelle composed of tightly packed or loose membranes, and vesicles. The nucleus (or nuclei), together with undifferentiated cytoplasm, occupy the central and most of the posterior regions of the spore. A membrane-bound posterior vacuole, visible even in fresh spores, is a prominent feature of some species. During germination in a new host or spontaneously in the tissues, the polar tube is evaginated and the sporoplasm (cytoplasm and nucleus) pass through it to be injected into the cytoplasm of a host cell, which the tip of the tube may have penetrated by chance during eversion (Figure 8.2G). No other organisms are known to have this type of infection mechanism.

Phylogeny

On the basis of sequences of rRNA and elongation factor EF1a genes in comparison

Fig. 8.1 Diagrammatic representation of the life-cycles of the seven genera of microsporidia parasitising man. (A-D) monokaryotic; (E-G) diplokaryotic. SP, sporoplasms after emergence from spores. Light stippling, merogonic stages; heavy stippling, sporogonic stages. (A) Encephalitozoon: merogonic and sporogonic stages in a host-derived parasitophorous vacuole; spores retained in vacuole until disintegration of host cell. (B) Pleistophora: plurinucleate meronts surrounded by amorphous coat divide into smaller segments; amorphous coat separates from the surface of multinucleate sporont to form a sporophorous vesicle and the sporont divides within it, to give numerous uninucleate spores in a persistent vesicle. (C) Trachipleistophora: meronts, bearing an amorphous coat with branched extensions, divide by binary fission; coat separates from the surface of a uninucleate sporont to form a sporophorous vesicle; sporont divides repeatedly by binary fission to give numerous spores in a persistent vesicle. (D) Enterocytozoon: meronts with irregular nuclei and electron-lucent slits merge into sporonts without a surface coat, by formation of electron-dense discs and change of nuclei to a rounded shape; after merging of the discs into polar tubes, sporoblast formation occurs by invagination of the membrane, simultaneously with deposition of the amorphous surface coat, to isolate each complex of nucleus, polaroplast and polar tube, and form free spores. (E) Brachiola: all stages are diplokaryotic and surrounded by an electron-dense coat; division by binary fission in merogony and sporogony; merogonic stages, often of bizarre shape, bear tubular structures embedded in amorphous surface coat. Spores free. (F) Vittaforma: all stages surrounded by a complete cisterna of host endoplasmic reticulum, the outer membrane of which is ribosome-bearing; merogony by binary fission of stages without surface coat; sporonts with up to eight diplokarya and a surface coat divide into diplokaryotic sporoblasts. Spores free. (G) Nosema: diplokaryotic meronts without a surface coat divide by binary fission; sporonts acquire a surface coat and divide by binary fission to give two diplokaryotic sporoblasts. Spores free. (Figure drawn by Dr L. A. Winchester)

with other protists (Vossbrinck et al., 1987; Kamaishi et al., 1996), microsporidia were thought to be primitively amitochondrial and to have separated from the main evolutionary line of the eukaryotes before the mitochondrial symbiosis event had occurred. However, recent analyses of a- and ß-tubulin (Edlind et al., 1996; Li et al., 1996) and of the largest subunit of RNA polymerase II (Hirt et al., 1999) have suggested that microsporidia are probably related to fungi and that the absence of typical fungal features, such as hyphae and cell walls, is a result of degeneracy due to parasitism. The detection of genes for mitochondrial-derived heat shock protein (HSP70) in microsporidia (Germot et al., 1997; Hirt et al., 1997) strengthens the view that these organisms once had mitochondria. The very close association of host cell mitochondria with the surface of multiplying microsporidia is indicative of microsporidian reliance on the host for chemical energy but does not preclude the possibility that relic mitochondria are retained in some species. The probable affinity of micro-sporidia with fungi has important implications for chemotherapy.

Enterocytozoon bieneusi (Figures 8.5D,E,I on Plate IV; 8.1D; 8.2f,L)

E. bieneusi is the most strikingly different microsporidian species among those infecting humans. All developmental stages are multinucleate plasmodia with unpaired nuclei and development is in direct contact with the host cell cytoplasm (Figure 8.2J). Merogony. early stages have a small number of irregularly-shaped nuclei and electron lucent clefts with dense borders. Sporogony. characterised by polar tube precursors in the form of electron dense discs which become stacked; nuclei compact, rounded, each associated with an anchoring disc, polar tube (formed by coalescence of the precursor discs), and a stack of membranes representing the future polaroplast. Sporoblasts formed by invagination of plasma membrane around each set of spore organelles with simultaneous secretion of surface coat. Spore maturation requires only the secretion of a thin endospore layer. Spores. 1.5 x< 1.0 |m (fresh), broadly ellipsoid with five or six isofilar coils of polar tube in two rows (Figure 8.2L). (For details, see Desportes et al., 1985; Cali and Owen, 1990.)

The Encephalitozoon Group (Figures 8.5C,F on Plate IV; 8.1A; 8.2A-C,H,I,K)

The feature that distinguishes the Encephalitozoon spp. from almost all other microsporidia is that the entire life-cycle evolves within a host cell vacuole (Figure 8.2H,I). Nuclei are unpaired (monokaryotic).

In Encephalitozoon cuniculi, E. hellem and E. intestinalis (formerly Septata intestinalis) after inoculation of sporoplasm into a host cell, a membrane encloses the multiplying stages in a vacuole. Merogony. binary fission of bi- or tetranucleate meronts attached to vacuolar membrane. Sporogony. sporonts detach from vacuolar

Fig. 8.2 (opposite) (A-F) Fresh spores from culture of some of the microsporidia that infect man, for comparison of size and shape. The Encephalitozoon spp. are similar but not identical. Bar on (A)=10 |m and refers also to (B-E). (A) Encephalitozoon cuniculi. (B) Encephalitozoon hellem. (C) Encephalitozoon intestinalis. (D) Vittaforma corneae—note narrow spores of variable length. (E) Free spores of Trachipleistophora hominis. (F) Sporophorous vesicle of Trachipleistophora hominis. Bar=5 |m. (G) Germination of a spore of E. intestinalis showing everted polar tube in two places in the host cell (arrows) and invagination of the host cell plasma membrane (arrowheads) alongside the polar tube. This membrane is probably the origin of the parasitophorous vacuole. Bar=1.0 |m. From Magaud et al. (1997), by permission of the Journal of Eukaryotic Microbiology. (H) Parasitophorous vacuole of E. cuniculi in culture showing meronts (m) in contact with vacuolar membrane and free sporonts (sp) and sporoblasts (sb). Note sparse matrix between parasites and no septa. Bar=1.0 |im. Original photograph of Professor J. Vavra. (I) Parasitophorous vacuole of E. intestinalis in enterocyte showing meronts (m) in contact with vacuolar membrane and free sporonts (sp) and spores (s) separated by septa (arrowheads) formed by compression of the vacuolar matrix. Bar=2.0 |im. From Canning et al. (1994), by permission of the European Journal of Protistology. (J) Enterocytozoon bieneusi. Meront (m) and adjacent sporont (s) in enterocyte showing electron-lucent slits (arrowheads) nuclei (n) and electron dense precursors of the polar tube (pt). Bar=1.0 |im. Original photograph of Dr A. Curry. (K) Spore of E. intestinalis showing six and a half coils of the polar tube in one rank. Bar=0.25 |im. From Van Gool et al. (1994), by permission of Cambridge University Press. (L) Spore of Encephalitozoon bieneusi showing five coils of the polar tube in two ranks (arrowheads) and poorly developed endospore. Bar=0.25 |m. Original photograph of Dr A. Curry

membrane as the surface coat is secreted; disporoblastic or tetrasporoblastic division in centre of vacuole. Spores: 2.5x1.5 ^ (fresh) (Figure 8.2A,B,C), ellipsoid, five to eight isofilar polar tube coils in a single row (Figure 8.2K); spores retained in the vacuole in an enlarging host cell until cell destroyed. Differentiation of species: spore morphology similar but species show slightly different sizes and shapes (Figure 8.2A,B,C). Parasitophorous vacuoles of E. intes-tinalis have conspicuous septa formed by compression of vacuolar matrix (Figure 8.2I). Otherwise, species are differentiated by protein profiles (SDS-PAGE), Western blotting, PCR amplification of ribosomal DNA with species-specific primers, restriction analysis and double-stranded heteroduplex mobility shift analysis. (For details, see Canning and Lom, 1986; Didier et al, 1991; Cali et al, 1993; Hartskeerl et al, 1995.)

The Anisofilar Polar Tube Group

The polar tube of several of the monokaryotic species infecting man shows a sharp change of diameter from wide anterior coils to narrow posterior coils (anisofilar). It is not clear whether the spores of Pleistophora sp. of Ledford et al.

(1985) are anisofilar but this species is included in this grouping because it produces spores in sporophorous vesicles like Trachipleistophora. The two species of indeterminate genus, placed in Microsporidium are also anisofilar.

In Trachipleistophora hominis (Figures 8.5J on Plate IV; 8.1C; 8.2E,F; 8.3C-J), all stages are surrounded by a thick surface coat which becomes the sporophorous vesicle envelope. Merogony: binary fission of bi- or tetra-nucleate stages. Surface coat, 25-50 nm thick, extends out as complex branches (Figure 8.3D,I) which make contact with 35-40 nm tubules in lysed host cell cytoplasm. Sporogony: surface coat on uninucleate products of merogony detaches to form the envelope of a sporophorous vesicle simultaneously losing the surface coat branches. Division occurs within the enlarging vesicle, by one to several binary fissions, giving two to many sporoblasts (Figure 8.3E,F) in a sparse fibrillar matrix with granules. Envelope persists round mature spores (Figure 8.2F). Spores: elongate pear-shaped 4.0x2.4 ^ (fresh) (Figure 8.2E,F); prominent posterior vacuole; polar tube with 8-11 wide coils and 2-3 narrow coils (Figure 8.3G,H). (For details, see Hollister et al., 1996b; Field et al., 1996.)

Pleistophora sp. of Chupp et al. (1993) resembles Trachipleistophora hominis. Spores of this 'Pleistophora' sp. are 4.0 x 2.0 ^ (fixed),

Fig. 8.3 (opposite) (A) Magnetic resonance image showing Encephalitozoon hellem-induced hypertrophic epithelium (arrow) blocking the nasal airway. From Lacey et al. (1992), by permission of BMJ Publishing Group. (B) Magnetic resonance image showing multiple ring-enhancing lesions (arrows) in cerebral cortex, representing sites of Trachipleistophora anthropophthera. Unpublished micrograph provided by Dr A. T. Yachnis. (C-J) Trachipleistophora hominis: all stages with isolated nuclei, in skeletal muscle of experimentally-infected mouse (C-F,H,J) or AIDS patient (G,I). (C,D) Meronts with well-developed surface coat extensions into host tissue. Bars=2.0 ^m (C) and 1.0 ^m (D). (E,F) Sporogonic division within sporophorous vesicles derived from surface coat, now almost devoid of branched extensions. Bars=2.0 ^m. (C,E,F) From Hollister et al. (1996b) by permission of Cambridge University Press. (G) Spore showing anchoring disc (ad) polaroplast (p) posterior vacuole (pv) and some polar tube coils (pt). Bar=1.0 ^m. From Field et al. (1996), by permission of American Society for Microbiology. (H) Detail of polar tube coil with eight wide coils and three narrow coils (arrowheads). Bar=0.25 ^m. Unpublished micrograph of Dr E. Weidner. (I) Detail of surface coat branches on meront. Bar=0.5 ^m. From Field et al. (1996), by permission of American Society for Microbiology. (J) Part of a sporophorous vesicle with immature spores showing pale fibrillar matrix with granules. Bar=2.0 ^m. Original micrograph of Dr E. Weidner. (K) Part of a sporophorous vesicle of Pleistophora sp. of Chupp et al. (1993), showing labyrinthine surface coat and spore lying in a dense matrix with tubules. Arrows point to polar tube coils. Bar=0.5 ^m. Original micrograph of Dr J. Alroy. (L) Pleistophora sp. of Ledford et al. (1985). Multinucleate (n) plasmodium in skeletal muscle, showing dense labyrinthine surface coat making contact with adjacent sporophorous vesicle. Bar=1.0 ^m. From Cali and Owen (1988), with permission. (M-O) Trachipleistophora anthropophthera in brain of AIDS patient. (M) Polysporous (large arrow) and disporous (small arrows) sporophorous vesicles. Bar=2.0 ^m. (N) Large spore from polysporous sporophorous vesicle showing wide coils and narrow coils (arrowheads) of the anisofilar polar tube. Bar=0.5 ^m. (O) Small spores from disporous sporophorous vesicle showing four or five coils of the isofilar polar tube (arrowheads) and rows of polyribosomes. Bar=1.0 ^m. Original micrograph of Dr J. Vavra. (M,N) From Vavra et al. (1998b), by permission of the Journal of Eukaryotic Microbiology. (P) Microsporidium ceylonensis: anisofilar polar tube with three narrow coils (arrowheads). Bar=0.23 ^m. From Canning et al. (1998), by permission of Princeps Editions, Paris

with 10 wide and three narrow coils of polar tube. The fibrillar matrix with tubules in the sporophorous vesicles (Figure 8.3K) is denser than the matrix of T. hominis (Figure 8.3J). Pleistophora sp. of Grau et al. (1996) is probably also a Trachipleistophora sp. Few details can be discerned from the original publication but one unpublished micrograph provided by D. S. Ellis shows an anisofilar polar tube (Figure 8.4M).

In Trachipleistophora anthropophthera (Figures 8.5G,H,K,L on Plate IV; 8.3B,M,N,0), merogony is as in T. hominis. Sporogony: dimorphic, one sporogonic sequence resembling T. hominis, forming eight or more large spores. A second sequence produces only two spores in small sporophorous vesicles (Figure 8.3M). Spores: (a) 3.7x2.0 ^ (fixed) with six to eight wide and one to three narrow diameter polar tube coils (Figure 8.3N); (b) 2.2-2.5x1.8-2.0 ^m (fixed) with four or five isofilar coils (Figure 8.30). (For details, see Yachnis et al., 1996; Berg et al., 1996; Vavra et al., 1998a,b.)

In Microsporidium ceylonensis (Figure 8.3P), merogony and sporogony are unknown except for synchronous development of several sporoblasts in a vacuole. Spores: 3.5x1.5 ^ (fixed) in groups of eight or more in vacuoles in macrophages. Polar tube anisofilar, with six to ten wide coils and two to three narrow coils (Figure 8.3P). In the case history given by Ashton and Wirasinha (1973), genus is indeterminate, so the species was placed in the collective genus Microsporidium and named by Canning and Lom (1986); ultrastructural data are given by Canning et al. (1998).

In Microsporidium africanum, merogony and sporogony are unknown. Spores: 4.5-5.0 x 2.5-

3.0 pm (fixed) in groups in macrophages. The single published electron micrograph shows at least eight wide coils and three narrow coils of the polar tube (Pinnolis et al., 1981).

Spores of all species in this anisofilar group, measured fresh (T. hominis) or fixed (T. anthropophthera, M. ceylonensis, M. africanum) are generally larger (>4.0 pm long) than those of other microsporidia infecting man. The only species in man with larger spores (5.0 x 3.0 is Nosema ocularum (see below). It also appears to have an anisofilar polar tube and, on the published evidence, there is no certainty that the nuclear complement is diplokaryotic. It may also be one of the anisofilar group.

Pleistophora sp. (Figures 8.1B, 8.3L). Merogony and sporogony: multinucleate plasmodia surrounded by a thick surface coat with branched extensions forming links between adjacent parasites. The surface coat becomes a sporophorous vesicle, within which groups of 12 or more sporoblasts are formed from the plasmodium. Spores: 3.2-3.4 x 2.8 ^ (fixed), with 11 coils of the polar tube. The published micrograph suggests that the polar tube may be anisofilar. However, the multinucleate plasmodia resemble the genus Pleistophora rather than Trachipleistophora. (For details, see Ledford et al., 1985; Cali and Owen, 1988.)

The Diplokaryotic Group

All stages diplokaryotic, lying in direct contact with host cell cytoplasm, no sporophorous vesicles.

Fig. 8.4 (opposite) (A-F) Vittaforma corneae: characteristic ribosome-studded encircling cisternae of endoplasmic reticulum (er), indicated by arrows. (A) Meront with two diplokarya (n). Bar=1.0 ^m. (B) Sporonts with electron dense surface coat (arrowheads). Bar=0.5 ^m. (C) Elongate sporont, progenitor of about eight sporoblasts. Bar=1.0 ^m. (D) Early sporont showing membrane-filled invagination of sporont surface membrane and the encircling er cisterna. Bar=1.0 ^m. (E) Almost complete division of sporont with new cross walls in vicinity of membrane-filled invaginations (arrowheads). Bar=0.5 ^m. (A-E) from Silveira and Canning (1995), by permission of the Journal of Eukaryotic Microbiology. (F) Region of spore showing *polaroplast, close apposition (arrow) of the two nuclei (n) and polar tube coils (arrowheads). Bar=0.5 ^m. From Shadduck et al. (1990), by permission of the University of Chicago Press. (G-I) Brachiola vesicularum: characteristic vesiculotubular structures are indicated by arrows. All stages have an electron-dense surface coat. (G) Diplokaryotic proliferative stage. Bar=1.0 ^m. (H) Region of spore showing only the anterior (wide) polar tube coils. Spore is free of tubules but an adjacent meront (m) has a polar group of tubules (arrowhead). Other bundles of tubules (arrows) lie in lysed host cell cytoplasm. Bar=1.0 ^m. (I) Elongate proliferative stage bearing cytoplasmic extensions (arrowheads) with attached tubules and a cap of tubules at one end (arrow). Bar=1.0 ^m. From Cali et al. (1998), by permission of the Journal of Eukaryotic Microbiology. (J-L) Brachiola connori. (J) Spore showing diplokaryon (n). Bar=1 ^m. (K) Vesiculotubular structures free and attached to cytoplasmic extensions of proliferative stage (p). Bar=1.0 ^m. (L) Part of spore showing seven wide coils and five narrow coils. Bar=0.25 ^m. Original micrographs of Dr J. A. Shadduck. (M) Pleistophora sp. of Grau et al. (1996). Region of spore showing anisofilar polar tube. Bar=1.0 ^m. Original micrograph of Dr D. S. Ellis

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