Description Of The Organism Taxonomy

The classification of Blastocystis remains controversial, it has been described as a yeast, a fungus, an alga, the cyst of other organisms (including Trichomonas spp.) and as a morphological form of Dietamoeba fragilis (for reviews, see Zierdt, 1991; Stenzel and Boreham, 1996).

Ultrastructural and physiological studies, first performed by Zierdt et al. (1967), have demonstrated that Blastocystis has protozoan characteristics. Such studies have not, however, allowed definitive taxonomic placement of the organism.

The first molecular study (Johnson et al., 1989), utilising small subunit rRNA sequencing

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Edited by Stephen Gillespie and Richard D. Pearson © 2001 John Wiley & Sons Ltd techniques, indicated that Blastocysts is not closely related to the yeasts, fungi, amoebae, sarcodines or sporozoans, as had been suggested in earlier reports (Stenzel and Boreham, 1996). Blastocystis also did not appear to be closely related to the ciliates, dinoflagellates or amoebae examined in the study. A more recent study (Silberman et al., 1996), using similar molecular techniques and citing previously reported ultrastructural data, has placed Blastocystis with the stramenopiles (a grouping including brown algae and diatoms). However, this proposed taxonomic position needs confirmation, as some ultrastructural characteristics and, particularly, the reproductive modes cited in this work (Silberman et al., 1996) are not in accordance with accepted data (Stenzel and Boreham, 1996).

At the present time, it is considered that only one species of Blastocystis, viz. B. hominis, is present in humans. However, immunological assays, polypeptide patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and DNA hybridisation with random probes, indicate that two or more distinctly different groups or 'demes' of B. hominis can be identified (Kukoschke and Muller, 1991; Boreham et al., 1992; Muller, 1994). Similarly, at least two zymodemes of B. hominis have been distinguished by isoenzyme patterns (Mansour et al., 1995; Gericke et al., 1997). These results warrant further study in order to clarify the speciation of Blastocystis in human hosts and to assess any relevance to pathogenicity.

A number of species of Blastocystis have been proposed for non-human hosts (Boreham and Stenzel, 1993, 1998), although these must be regarded cautiously until further confirmatory evidence is obtained. It also must be recognised that differing phenotypic characteristics do not necessarily indicate genotypic differences and, thus, may not be suitable for differentiating species.

Morphological criteria (predominantly size of the organism) have been used in several reports to differentiate the proposed species B. galli from chickens, B. anatis from domestic ducks and B. anseri from domestic geese (Boreham and Stenzel, 1993). The dimensions given for these proposed species overlap to a considerable degree and also overlap with the reported size range for B. hominis. Hence, size cannot be considered a valid criterion to delineate these species. Differences in nuclear ultrastructure, as compared to B. hominis, appear to be a possible means of distinguishing B. galli, the proposed species from chickens (Boreham and Stenzel, 1993). However, this needs further clarification by more extensive morphological studies and needs to be supported by molecular data. B. lapemi, from a sea-snake, has been differentiated from B. hominis by electro-phoretic karyotyping and by different culture requirements, although morphological differences were not found (Boreham and Stenzel, 1993).

Morphology

A number of morphological forms of Blastocystis have been reported from culture and from faecal material. Considerable morphological variability occurs, and it is not known if this relates to speciation and/or pathogenicity. The forms most commonly reported in faeces include the vacuo-lar, granular, multivacuolar and cyst forms (Figure 15.1, see Plate VI, and Figures 15.2-15.5). An amoeboid form has been reported only rarely, and there are a number of conflicting reports on its morphology. An avacuolar form is thought to be present in the intestine of humans. In culture, the vacuolar or granular forms predominate, and by changing culture conditions it is possible to alter which form is seen.

By transmission electron microscopy, all forms of B. hominis show a characteristic nuclear morphology—a crescentic band of electron-opaque material at one pole (see Figures 15.215.5). This morphology does not appear to depend on cell cycle stage or physiological conditions. Mitochondria-like organelles are present, varying in number and morphology. They usually contain low numbers of sacculate or tubular cristae. Other eukaryotic cell structures, such as Golgi complex, coated pits, endocytic vesicles, rough endoplasmic reticulum and poly-ribosomes are also seen (Zierdt, 1991; Boreham and Stenzel, 1993; Stenzel and Boreham, 1996).

Vacuolar Form

The vacuolar form (synonyms: vacuolated form, central body form) has been considered to be the typical Blastocystis form and it is the form generally sought in routine diagnosis by light microscopy. It predominates in most cultures of B. hominis.

The vacuolar form appears as a spherical or slightly irregularly shaped cell of varying diameter (2-200 pm; Zierdt, 1991; Stenzel and Boreham, 1996). The average size range of vacuolar forms found in human faecal material is approximately 4-15 Larger cells may be found in culture.

By light microscopy (Figure 15.1E), the nuclei and mitochondria are indistinguishable but are seen in the peripheral rim of the cell, surrounding the central vacuole (synonym: central body). By transmission electron microscopy, the cell is seen to consist of a thin band of peripheral cytoplasm, containing organelles, which surrounds a large central vacuole (Figure 15.2). Multiple nuclei (commonly up to four) are present in many vacuolar cells (Zierdt, 1973; Matsumoto et al., 1987; Dunn et al., 1989).

There is considerable variability in the morphology of the vacuolar forms, particularly in the contents and, therefore, staining reactions, within the central vacuole (MacPherson and MacQueen, 1994; Garcia and Bruckner, 1997). The complete

Fig. 15.2 Transmission electron micrograph of the vacuolar form of B. hominis. A thin rim of cytoplasm, containing a nucleus (Nu), mitochondria-like organelles (m) and other organelles, surrounds a large central vacuole (CV). The cell is surrounded by a thin surface coat (SC), with an adherent bacterium (b)

Fig. 15.2 Transmission electron micrograph of the vacuolar form of B. hominis. A thin rim of cytoplasm, containing a nucleus (Nu), mitochondria-like organelles (m) and other organelles, surrounds a large central vacuole (CV). The cell is surrounded by a thin surface coat (SC), with an adherent bacterium (b)

Fig. 15.3 Transmission electron micrograph of the granular form of B. hominis. Granules of various morphologies are present in the central vacuole (CV). Nu, nucleus

function of the central vacuole has not been defined, although it appears to have a function in metabolism and storage (Dunn et al., 1989; Yoshikawa et al., 1996). A role in reproduction has been suggested (Zierdt, 1988, 1991; Singh et al., 1995) but is not supported by ultrastructural data.

A surface coat, also called 'slime layer' or 'capsule' in some reports (Stenzel and Boreham, 1996), of variable thickness and morphology may surround vacuolar forms. It is altered by laboratory culture, becoming thinner or absent after longer periods in culture (Stenzel et al., 1991).

Granular Form

The granular form (Figure 15.3) is morphologically identical to the vacuolar form, apart from the contents of the central vacuole. This form shows numerous small granules within the central vacuole. Cytochemical and ultrastructural studies have indicated that many of the granules are composed of lipid (Zierdt, 1973; Dunn et al., 1989). Granular forms are not commonly seen in faecal material, but are induced by a number of culture conditions (Stenzel and Boreham, 1996).

Multivacuolar Form

Recent ultrastructural studies (Stenzel et al., 1991; Stenzel and Boreham, 1996) have determined that the form of B. hominis present in fresh human faecal material may be multivacuolar, rather than vacuolar, as has previously been assumed. These multivacuolar forms are usually smaller (approximately 5-8 ^ in diameter) than the 'typical' vacuolar forms. Rather than a large single vacuole, numerous smaller vacuoles are seen (Figure 15.1C, 15.4). These vacuoles may be distinct entities or may comprise an interconnected network: sufficient study has not yet been performed to ascertain this. Often, the vacuoles are too small to be resolved by light microscopy, and the multivacuolar form appears as a small, often irregularly shaped, vacuolar form in stained faecal smears (Figure 15.1A,B). The surface coat surrounding all multivacuolar forms found in human faecal material (Figure 15.4) is thicker than that seen on the cultured forms (compare to Figures 15.2, 15.3). After short-term laboratory culture, the multivacuolar form gives rise to the vacuolar or granular forms, with vacuolar forms being the only form present after longer periods in culture (Stenzel et al., 1991).

Avacuolar Form

The avacuolar form has been reported only twice in the literature: once from a patient producing copious quantities of diarrhoeal fluid (Zierdt and Tan, 1976) and once from a sample taken at colonoscopy (Stenzel et al., 1991). These organisms were approximately 5 ^ in diameter and lacked a central vacuole and a surface coat.

The avacuolar form has not been found in culture. Zierdt and Tan (1976) suggested that this form gave rise to the vacuolar form after laboratory culture. Further work is needed to determine whether the avacuolar form is the form of B. hominis present within the human intestine, as has been assumed (Stenzel and Boreham, 1996).

Cyst Form

The presence of a cyst form (synonyms: cystic form, cyst-like form, resistant form), although first reported by Alexeieff in 1911, was confirmed only recently by ultrastructural studies (Mehlhorn, 1988; Stenzel and Boreham, 1991; Figure 15.5). The cyst form ranges in diameter (approximately 3-10 It is commonly less than 5 ^ in diameter and, hence, may be very difficult to identify by light microscopy.

The condensed cytoplasm of the cyst contains many vacuoles and often large reserves of glycogen or lipid, which give the cell a very refractile appearance in wet mounts. These materials do not stain with trichrome and may be extracted by some microscopy preparation

Fig. 15.4 Transmission electron micrographs of multivacuolar forms of B. hominis. Numerous small vacuoles (v) are noted. The cells are surrounded by a thick surface coat (SC). Nu, nucleus; m, mitochrondion-like organelle

Fig. 15.5 Transmission electron micrograph of the cyst form of B. hominis, showing a multilayered cyst wall (CW) surrounding the cell. A surface cost (SC) is noted externior to, and separating from, the cyst wall. Lipid (L) and glycogen (G) are seen within the cyst cytoplasm. Nu, nucleus; m, mitochondrion-like organelle

Fig. 15.5 Transmission electron micrograph of the cyst form of B. hominis, showing a multilayered cyst wall (CW) surrounding the cell. A surface cost (SC) is noted externior to, and separating from, the cyst wall. Lipid (L) and glycogen (G) are seen within the cyst cytoplasm. Nu, nucleus; m, mitochondrion-like organelle methods, particularly those used for transmission electron microscopy, giving the appearance of a large vacuole within the cyst (see Figures 15.1d, 15.5).

A thick, multilayered cyst wall is present (Figure 15.5). This wall appears to form beneath the surface coat, and cysts are often seen with the surface coat and cellular debris present external to the cyst wall (Figure 15.5).

Amoeboid Form

The amoeboid forms (synonyms: amoeba form, amoeba-like form, amoebiform) is uncommon but has been described from culture and faeces. A number of conflicting definitions have been presented in the literature (Stenzel and Boreham, 1996). Several studies suggest that this form extends pseudopodia and feeds on bacteria (Zierdt, 1973; Dunn et al., 1989; Suresh et al., 1994; Singh et al., 1995). A central vacuole was not noted, and the cells varied in diameter (approximately 3-8

Other Forms

The existence of other forms of B. hominis, as suggested by Zierdt (1991), remains unverified.

Recent studies have indicated that culture conditions affect the morphology of the organism (Stenzel and Boreham, 1996), although these have not been fully defined. Hence, it is important for future studies to clearly discriminate between morphological differences induced by the microenvironment or physiology of the cell and distinct life-cycle stages.

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