Recent advances in Blastocystis hominis research: hot spots in terra incognita

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1 International Journal for Parasitology 32 (2002) Invited review Recent advances in Blastocystis hominis research: hot spots in terra incognita Kevin S.W. Tan*, Mulkit Singh, Eu Hian Yap Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, Singapore Received 12 September 2001; received in revised form 14 December 2001; accepted 18 December 2001 Abstract Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Blastocystis; Taxonomy; Genetic diversity; Morphology; Culture; Immunobiology; Pathogenesis; Programmed cell death; Apoptosis 1. Introduction Blastocystis hominis is a fascinating intestinal protozoan parasite first described in the early 1900s (Alexeieff, 1911; Brumpt, 1912). However, it was Charles Zierdt s numerous studies of this organism (spanning predominantly the 1970s and 1980s) that first attracted the attention of biologists and clinicians. Early workers were unable to classify B. hominis and erroneously described it as the cyst of a flagellate, vegetable material, yeast and fungus (Zierdt, 1991). Its protistan features were subsequently described by Zierdt et al. (1967), based on morphological and physiological criteria. Blastocystis hominis is probably the most common human gut protozoan in the world, with.50% prevalence in developing countries (Stenzel and Boreham, 1996). Its clinical relevance is currently uncertain and shrouded in controversy with numerous conflicting reports on its ability to cause disease. This is one of the major impetuses for the recent surge of interest in this parasite. * Corresponding author. Tel.: ; fax: address: mictank@nus.edu.sg (K.S.W. Tan). Despite the efforts of a handful of laboratories to study B. hominis, until recently, only its morphology had been extensively described (Stenzel and Boreham, 1996). We now have more detailed information on the various morphological forms from numerous light and electron microscopic (EM) studies. Many other aspects of Blastocystis biology remain virtually unexplored territories. Very little is known about its mode of transmission, pathogenicity, culture characteristics, taxonomy, life-cycle, biochemistry and molecular biology. Substantial progress in B. hominis research in recent years has provided us with, or brought us closer to, answers to some of the fundamental questions regarding Blastocystis biology. Blastocystis research has been challenging for a variety of reasons. (1) There is a lack of critical research partly because of the relatively small number of laboratories currently studying the parasite. (2) The existence of genetic and antigenic diversity among morphologically identical parasites complicates the comparison and verification of data. (3) The lack of a suitable animal model hinders investigations into its immuno- and patho-biology. (4) The scarcity of well-characterised genes and proteins in this parasite /02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S (02)00005-X

2 790 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) discourages study of its cell and molecular biology. This review focuses on recent work that has contributed to a better understanding of B. hominis biology, describes current research findings and discusses future research prospects. For an extensive account of the parasite s history, the reader is referred to Zierdt s review (1991) while prior information on the morphology and biology of B. hominis may be found in Stenzel and Boreham s (1996) review. 2. Taxonomy 2.1. Taxonomy and phylogenetic affinities Even though discovered more than 80 years ago, Blastocystis still remains a mysterious parasite with little-understood taxonomic links. The protistan features of this organism were revealed by the observation that it possessed one or more nuclei, had smooth and rough endoplasmic reticulum (ER), Golgi complex and mitochondrion-like organelles; it failed to grow on fungal media and was not killed by antifungal drugs; and some antiprotozoan drugs showed activity against this parasite (Zierdt, 1988, 1991). The latter author classified the organism initially as a sporozoan based on morphology, cultural characteristics and modes of division and later reclassified it as a sarcodine, albeit with insufficient evidence. Stenzel and Boreham (1996), in the most up-to-date review of Blastocystis, lamented, that recent debates on the systematics of the protists have not included the genus Blastocystis in any suggested classifications. As if in response to this, there has been a surge of interest in this area in the last few years. The taxonomy and phylogenetic affinities of Blastocystis have been analysed by comparison of the parasite s ssrrna gene sequences with those from different eukaryotes. By analysis of partial ssrrna sequences, Johnson et al. (1989) showed that B. hominis is not monophylectic with the yeasts (Saccharomyces), fungi (Neurospora), sarcodines (Naegleria, Acanthamoeba and Dictyostelium) or sporozoans (Sarcocystis and Toxoplasma). Based on this data, Johnson et al. (1989) concluded that B. hominis is unrelated to the yeasts and could be placed in an outer group of the clade that links ciliates and Apicomplexa. In a separate study (Silberman et al., 1996) the complete Blastocystis ssrrna gene was sequenced and it showed that Blastocystis could be placed within the Stramenopiles. The Stramenopiles, synonymous with Cavalier-Smith s Heterokonta (Cavalier-Smith, 1997), are defined as a complex and heterogenous evolutionary assemblage that includes unicellular and multicellular protists, with both heterotrophic and photosynthetic representatives. This diverse group includes slime nets, water moulds and brown algae. The study indicated that Blastocystis was, surprisingly, phylogenetically related to the flagellate Proteromonas. These two organisms share certain life-cycle traits. They are gut endosymbionts of vertebrates and encyst to an environmentally resistant form that appears to allow transmission between hosts. However, unlike Proteromonas, Blastocystis does not possess flagella and tubular hairs. According to a revised classification of the six-kingdom system of life (Cavalier-Smith, 1998), Blastocystis is not a fungus or protozoan as previously assumed and because it does not possess cilia, it is placed in a newly created Class Blastocystea in the Subphylum Opalinata, Infrakingdom Heterokonta, Subkingdom Chromobiota, Kingdom Chromista. This classification makes Blastocystis the first chromist known to parasitise humans. The elongation factor-1a (EF-1a) gene, a highly conserved gene suitable for use as a phylogenetic marker, has recently been employed for molecular studies in Blastocystis. Nakamura et al. (1996) analysed the amino acid sequence of Blastocystis hominis EF-1a, deduced from a cdna clone, and from a phylogenetic reconstruction analysis using a maximum likelihood method of protein phylogeny, confirmed that B. hominis should not be included within the fungal lineages and suggested that it diverged before Trypanosoma, Euglena, Dictyostelium and other eukaryotes. However, due to low bootstrap probabilities, it was not possible to determine the relationships between Blastocystis, Entamoeba, Plasmodium and Tetrahymena with statistical significance. Ho et al. (2000) examined the nucleotide sequences of the EF-1a gene of Blastocystis and studied the genetic-relatedness of this parasite from humans and animals. A phylogenetic analysis of this gene from 13 isolates of Blastocystis with other eukaryotic EF-1a sequences revealed that the parasite diverged within the same group and the isolates all belonged to the same genus. In contrast to the phylogenetic data based on ssrrna gene sequences, this study showed that Blastocystis branched off together with Entamoeba histolytica. This apparent discrepancy may be explained statistically by the low bootstrap value (58.1) used to group Blastocystis with E. histolytica. Other possibilities exist for the variation in affinities. They may be due to the choice of genes for analysis. The ssrrna genes have been known to possess drastic G 1 C content variation among species (Hasegawa and Hashimoto, 1993), which may give rise to misleading trees and hence other genes (e.g. EF-1a, EF-2 and RNA polymerase III large subunit) with less extreme biases have been suggested to be better candidates for phylogenetic studies. Other possible factors contributing to the discrepancy include inadequate species sampling, relatively few informative positions, mutational saturation and long branch attraction phenomenon (Philippe and Laurent, 1998; Roger et al., 1999). Indeed, it is evident that more studies need to be carried out to clarify the issue of Blastocystis phylogenetic affinities. The analysis of multiple candidate genes and larger numbers of isolates should help place Blastocystis in a more precise taxonomic framework Cryptic genetic diversity Numerous morphologically similar Blastocystis isolates have been described from humans and various animals.

3 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) Various studies utilising sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Kukoschke and Müller, 1991; Boreham et al., 1992; Müller, 1994; Tan et al., 2001b), isoenzyme analysis (Mansour et al., 1995; Gericke et al., 1997), electrophoretic karyotyping (Upcroft et al., 1989; Ho et al., 1994; Carbajal et al., 1997), random amplified polymorphic DNA (RAPD) (Yoshikawa et al., 1998; Init et al., 1999) and restriction fragment length polymorphism (RFLP) analysis (Bohm- Gloning et al., 1997; Clark, 1997; Hoevers et al., 2000; Yoshikawa et al., 2000; Ho et al., 2001) have shown that there is considerable antigenic and genetic heterogeneity among B. hominis isolates within and among geographical regions, suggesting that several strains or species of this parasite exist. Pulsed field gel electrophoresis (PFGE) is a commonly employed technique for chromosome separation and the study of karyotypic diversity. Using this method, significant diversity was revealed among 15 B. hominis isolates (Carbajal et al., 1997). From the study, 11 karyotypic profiles could be clustered into three karyotypes. In contrast, PFGE of five B. hominis isolates from Singapore revealed broadly similar karyotypic patterns (Ho et al., 1994). This discrepancy may be a statistical problem due to the smaller sample size of the Singapore study. RAPD has been used to distinguish isolates of Blastocystis. Yoshikawa et al. (1996) analysed polymerase chain reaction (PCR) banding patterns of a strain (HE87-1) from a patient in Japan, an isolate (CK86-1) from a chicken and the Nand strain (ATCC strain) of B. hominis. The Japanese and the chicken strain shared similar bands, raising the possibility that the chicken isolate may be a zoonotic strain of B. hominis or that certain B. hominis strains are crossinfective. Both strains had common bands with the reference Nand strain. Another human isolate (B) from Singapore did not share any banding patterns with the Nand strain, suggesting that it is an intraspecific variant of B. hominis. The genetic diversity in B. hominis has also been shown by use of RFLP analysis of the ssrrna gene and of the elongation factor-1a. Clark (1997) examined the sequence variation in the ssrrna gene in 30 randomly selected isolates of B. hominis using RFLP analysis of PCR-amplified ssrrna. This method allowed for a rapid comparison of the different isolates. The RFLP patterns obtained with 11 different restriction enzymes were termed riboprints and the different isolates obtained by this method were grouped into ribodemes (genotypes). Seven distinct ribodemes were obtained with varying frequencies. A dendrogram constructed by phylogenetic analysis showed that the seven ribodemes fell into two distinct lineages. Two of the isolates had riboprints identical to that of a guinea pig isolate, indicating that some human Blastocystis infections may be of zoonotic origin, as had been alluded to by other authors (Yoshikawa et al., 1996). Clark (1997) suggested that this genetic diversity in B. hominis might be responsible for the controversial role of Blastocystis in producing disease in humans; isolates vary in their pathogenic role in humans. However, a study on a large number (113) of isolates suggested otherwise (Bohm-Gloning et al., 1997). Their analysis on the RFLP patterns, of PCR-amplified ssrrna gene, using three restriction enzymes showed the existence of five subgroups none of which were significantly associated with intestinal disease. It is difficult to draw conclusions about the association between genotypes and disease based on only three restriction enzymes. Perhaps the use of more discriminatory enzymes in subsequent studies is required before this important issue can be resolved. More recently, Hoevers et al. (2000) examined the genetic diversity in 14 isolates obtained from four different geographical locations by similar RFLP analysis of ssrrna. The RFLP patterns allowed them to identify 12 genotypes but they did not find any correlation between geographic origins of B. hominis and RFLP banding patterns. In a comparative study with riboprinting (Ho et al., 2001) it was discovered that RFLP analysis of elongation factor-1a (EF-1a) gene could also be useful for determining genetic diversity among Blastocystis isolates. In concurrence with the terminology in RFLP studies with ssrrna, the term elfaprints was designated for the RFLP patterns generated from EF-1a and elfatypes (genotypes) for the isolates with similar elfaprints. Elfaprinting was shown to be useful in distinguishing five B. hominis Singapore isolates from an isolate obtained in Pakistan. From the above studies, it is apparent that extensive genetic diversity exists among B. hominis isolates. The genotypes generated from each study can be useful references for studying taxonomic relationships among morphologically identical parasites. Hoevers et al. (2000) had suggested that in order to optimise the usefulness of these observations, such as for effective comparison of data, some form of standardisation is required. Reference strains and standardised panel of restriction enzymes should be used in future studies Speciation Many Blastocystis isolates resembling B. hominis have been described from primates, rodents, birds, reptiles, amphibians and even insects like the cockroach (Boreham and Stenzel, 1993; Chen et al., 1997a; Teow et al., 1992; Yoshikawa et al., 1998). Species from animals have been characterised by morphological criteria and karyotypic differences. Different species in chickens, duck and geese have been described on the basis of morphological differences (studies of Belova and coworkers, cited by Stenzel and Boreham, 1996). Teow et al. (1991) described a species, Blastocystis lapemi, from a sea snake as a result of growth characteristics and a karyotypic profile that differed from B. hominis. Chen et al. (1997b) and Singh et al. (1996) also used karyotypic analysis to describe Blastocystis ratti from rats and new species from reptiles, respectively. The designation of reptilian Blastocystis species was based on broadly

4 792 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) dissimilar karyotypic profiles and growth conditions when compared with human isolates. The rat isolates, when compared with human ones, also revealed distinctly dissimilar profiles and had the unique property of containing numerous cystic forms in axenised cultures (Chen et al., 1999). Thus, karyotypic differences, when used in conjunction with other discriminatory methods (growth conditions, cell types, gene sequences), can be useful for species designation. Opinions vary amongst researchers as to the validity of these techniques in describing new species. There is a great deal of morphological variation in organisms grown in culture from those seen in faeces. Furthermore, ultrastructural studies on the morphological stages have not been helpful in designating species. Yoshikawa et al. (1998) questioned the use of karyotypic patterns to describe new species from reptilian hosts (Teow et al., 1991; Singh et al., 1996). They cited the extensive karyotypic heterogeneity amongst clinical isolates of Giardia lamblia and Trypanosoma cruzi indicating that karyotypic differences may not be enough for speciation in Blastocystis. They advocated the use of PCR-based RAPD to distinguish species and strains of Blastocystis. The use of karyotypic patterns for speciation of Blastocystis was somewhat supported by studies (Ho et al., 2000, 2001) on the EF-1a gene of Blastocystis. A comparative study on the deduced amino acid sequences of a PCR product, amplified from the coding region of the EF-1a gene, of seven isolates of Blastocystis from rats and reptiles showed them to be different from one another and from B. hominis. Furthermore, when the ssrrna and EF-1a genes were used in a PCR-based RFLP analysis of genetic heterogeneity in Blastocystis, it was shown that the banding patterns (ribodemes and elfatypes) of the animal isolates were distinctly different from those of B. hominis (Ho et al., 2001). The banding patterns of the rat Blastocystis, B. ratti, were similar among the rat isolates and different from that of B. hominis. All four reptilian isolates studied had different banding patterns from one another, indicating that they belonged to different species. Although these observations appear to validate the earlier use of PFGE as a tool in speciation of Blastocystis, there are a number of interesting observations that need to be accounted for. The B. ratti ribodeme (three isolates) was identical to B. hominis ribodeme 3 in Clark s (1997) study, which also included a guinea pig isolate. Also, one of the B. hominis isolates (S) in Ho et al. s (2001) study had a similar riboprint with Clark s ribodeme 1 and a Japanese B. hominis isolate (HE87-1). HE87-1 was observed to share very similar RAPD patterns with a chicken isolate (Yoshikawa et al., 1996). These observations suggest that some genotypes of Blastocystis are not host-specific and therefore some caution is warranted when deciding if a particular Blastocystis isolate is truly of human or animal origin. Also, the abovementioned ribodemes (1 and 3) are distant from the bulk of the other human Blastocystis isolates and may therefore suggest that other mammals represent a huge reservoir for infection of humans (Clark, 1997). In a separate study, Snowden et al. (2000) analysed the RFLP of ssrrna genes belonging to eight Blastocystis isolates that were isolated from various animals (cow, goat, sheep, guinea pig and rhea). Five genotypes were identified in the isolates; multiple genotypes were found in isolates from a single animal host and multiple host species shared a single genotype. Thus, their data further indicate that Blastocystis may not be host specific or may be cross-infective among certain host types. Studies involving more isolates and other conserved genes should help clarify taxonomic relationships among isolates Conclusions In summary, research over the last few years has shown that Blastocystis is a ubiquitous parasite of the animal kingdom. Its taxonomy is still not fully understood although recent studies have linked it closely with either the Stramenopiles or the amoebae. In Cavalier-Smith s treatise it currently stands as a member of the Heterokont group of organisms under the Kingdom Chromista. The species in humans, B. hominis, exhibits a great deal of genetic heterogeneity. Based on limited current data, there does not seem to be any association between distinct genotypes and geographical origin or pathogenicity. In light of the extensive genetic variation observed in B. hominis (Silberman et al., 1996; Clark, 2000), the establishment of distinct specific names for the various genotypes should be considered. Several studies have indicated human-to-human, animalto-human and animal-to-animal transmissions. Molecular techniques such as karyotypic typing, RAPD and RFLP analyses have helped to characterise the different genotypes of Blastocystis. The analyses of ssrrna and elongation factor-1a genes have been found useful in such studies. 3. Morphology 3.1. Vacuolar and granular forms Blastocystis hominis is a polymorphic organism and the four forms commonly described in literature are the vacuolar, granular, amoeboid and cyst forms. The vacuolar form, also referred to as the central vacuole form, varies greatly in size, from 2 to 200 mm in diameter, with average diameters of 4 15 mm (Stenzel and Boreham, 1996). A surface coat of varying thickness is found surrounding most cells. The plasma membrane of the parasite contains coated pits, which appear to have a function in endocytosis (Stenzel et al., 1989). Vacuolar forms are spherical and characterised by a large central vacuole, which may occupy 90% of the cell s volume. The central vacuole usually contains fine granular or flocculent material of varying electron density and appears to have a storage function (Boreham and Stenzel, 1993). The presence of this large structure results in a

5 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) thin band of peripheral cytoplasm. It is within this region that the nucleus and organelles such as mitochondria, Golgi apparatus and ER reside. The larger organelles are usually clustered in thickened portions of the cytoplasm, usually located at opposite ends of the cell. In some instances, portions of the cytoplasm have been observed to invaginate into the central vacuole, resulting in the deposition of cytoplasmic material (Suresh et al., 1995) and organelles such as mitochondria (Stenzel et al., 1991; Pakandl, 1999; Tan et al., 2001a; Nasirudeen et al., 2001a) within membranebound structures. Interestingly, we have recently observed that B. hominis cells undergoing programmed cell death appear to accumulate such membrane-bound structures, reminiscent of apoptotic bodies in higher organisms, in the central vacuole (Tan et al., 2001a; Nasirudeen et al., 2001a) (see Section 7.1). The vacuolar form is predominant in axenised liquid cultures and is also commonly observed, albeit as a smaller form (,5 mm), in fresh faecal samples. The granular form has been suggested to arise from the vacuolar form and the transition induced by a variety of factors (Stenzel and Boreham, 1996). These include increased serum concentrations in the culture medium, transfer of cells to a different culture medium, axenisation and addition of certain antibiotics (Stenzel and Boreham, 1996). The granular form shares many similarities with the vacuolar form except that numerous granules are found within the thin band of peripheral cytoplasm or, more commonly, within the central vacuole. There is considerable morphological variation in the types of granules within the central vacuole. These may be myelin-like inclusions, small vesicles, crystalline granules and lipid droplets (Dunn et al., 1989). There have also been suggestions that the central vacuole functions in schizogony and endodyogeny with certain reproductive granules representing progeny of Blastocystis (Zierdt et al., 1967; Zierdt, 1991; Suresh et al., 1994). However, there has been some debate as to whether these are bona fide progeny of B. hominis as, ultrastructurally, they appear similar to granule types reported previously. One possibility for the confusion is that many of these observations were also made by conventional light microscopy, which can lead to inaccurate or biased interpretation. For example, it had been suggested (Boreham and Stenzel, 1993) that the less commonly seen multivacuolar form may be misconstrued as a cell undergoing schizogony because the multiple vacuoles can resemble progeny. With the advent of improved live imaging systems, such as deconvolution and/or restoration microscopy, subcellular structures can be observed at very high resolving powers (,0.2 mm). Using fluorescent markers and time-course assays, such systems should be helpful in confirming if certain granular structures reported earlier are indeed progeny of B. hominis Amoeboid form The amoeboid form of B. hominis has been reported infrequently and its morphological descriptions have yielded conflicting and confusing reports (McClure et al., 1980; Dunn et al., 1989; Zierdt, 1991; Tan et al., 1996b). It has been observed in older cultures, cultures treated with antibiotics and occasionally in faecal samples (Zierdt, 1973). An early TEM study (Zierdt and Tan, 1976) described the amoeboid form as oval, with one or two large pseudopods but without a cell membrane. It is rather peculiar that a cell can survive without a plasma membrane and it is more likely that this was an artefact of sample preparation, as suggested by Boreham and Stenzel (1993). Dunn et al. (1989) provided detailed ultrastructural descriptions of the amoeboid form by TEM, although it would have been helpful if there were light micrographs to corroborate their observations. The amoeboid forms were rather small ( mm) and possessed extended pseudopodia. Engulfed bacteria were observed in lysosome-like structures within the cell. Curiously, a central vacuole, Golgi complex, surface coat and mitochondria were not seen. We have observed that colony growth of B. hominis cells induced the formation of numerous amoeba-like forms (Tan et al., 1996b). These cells were, by both light microscopy and TEM, shown to be irregular in outline and possessed distinct pseudopod-like extensions (Tan et al., 1996c, 2001a). In contrast to Dunn et al. s study, our TEM study revealed a central vacuole, numerous Golgi bodies, ER and mitochondria within the pseudopod-like cytoplasmic extensions, suggesting that these cells are involved in highly active, energy-requiring processes. The cells also appeared to contain, within lysosome-like organelles, cellular fragments of degenerating neighbouring cells, suggesting a role of this form in endocytosis. It is currently unclear if the amoeboid forms isolated from colonies are artefacts of culture or represent forms that would also be found in vivo. It is interesting to note that the colony-induced amoeboid forms of Trichomonas vaginalis had been postulated to represent the same forms that adhere to vaginal epithelial cells (Scott et al., 1995). With our new knowledge of extensive genetic diversity amongst B. hominis isolates, it is conceivable that the conflicting descriptions from the few studies may have arisen from isolate differences. There is scant information on the differentiation of the amoeboid form or its role in the life-cycle of the parasite. It has been suggested that the amoeboid form is an intermediate between the vacuolar and cyst forms and that it ingests bacteria to provide nutrition for the encystment process (Singh et al., 1995). However, Stenzel and Boreham (1996) had postulated that it arose from the morphologically similar avacuolar form (see Section 3.4). None of these have been proven although the presence of amoeboid forms in colonies does suggest that these had arisen from the vacuolar forms used for inoculating the agar plates. The role of the amoeboid form is clearer. The presence of ingested particulate matter, be they bacteria (Boreham and Stenzel, 1993; Stenzel and Boreham, 1996) or cellular debris (of dead neighbouring cells) (Tan, 1999, PhD thesis, National

6 794 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) University of Singapore; Tan et al., 2001a) in this form does point towards a nutritive or regulatory role (see Section 7.1) Cyst form Despite numerous descriptions of the vacuolar and granular forms since the early 1900s, the existence of a morphologically distinct cystic form was confirmed and described relatively recently (Mehlhorn, 1988; Stenzel and Boreham, 1991; Moe et al., 1997, 1999; Zaman et al., 1997a, 1998, 1999b). The delay in these findings was probably attributed to the smaller size (3 5 mm) and distinct appearance of the cyst, which could also be easily confused with faecal debris. Furthermore, these faecal cysts are seldom seen in axenised cultures. Faecal cysts are spherical to ovoid and are protected by a multilayered cyst wall. Internal cellular contents include one to four nuclei, multiple vacuoles and glycogen and lipid deposits (Stenzel and Boreham, 1996; Zaman et al., 1997a; Moe et al., 1999). Such cysts are commonly surrounded by a loose fibrillar layer that appears to shed as they mature (Zaman et al., 1997a). Blastocystis cysts isolated from animal faecal material revealed distinct morphological differences when compared with faecal cysts of B. hominis. Relatively larger cysts, measuring up to 15 mm in diameter, were isolated from Macaca monkeys whilst multiple individual cysts enclosed by a single fibrillar layer were observed from chicken faecal material (Stenzel et al., 1997). The authors suggested that the disparate morphologies of the cyst forms could indicate the presence of different Blastocystis species. Viability analysis of B. hominis cysts revealed that these do not lyse in water (Zaman et al., 1995), are able to survive at room temperature for up to 19 days, but are fragile at extremes of heat and cold, and in common disinfectants (Moe et al., 1996). In contrast to the cystic forms, the vacuolar and granular forms are sensitive to temperature changes, hypertonic and hypotonic environments and exposure to air (Matsumoto et al., 1987; Zierdt, 1991). Thus, the cystic stage is the transmissible form of the parasite. We have observed that alteration of serum concentration resulted in an increase in cystic forms of B. ratti (Chen et al., 1999) when cultured axenically in Iscove s modified Dulbecco s medium (IMDM). However, mass encystation of vacuolar forms with 30% horse serum was achieved for some, but not all, B. ratti isolates suggesting that other factors specific to different isolates were contributing to the encystation process. To date, in vitro encystation of B. hominis, to produce the faecal cyst morphological type, has not been described and we were unable to induce cyst formation with the method employed for B. ratti (unpublished observations). However, taking into account that the genotype of B. ratti was identical to Clark s B. hominis ribodeme 3 (see Section 2.3), it is also possible that different genotypes of B. hominis possess varying susceptibilities to in vitro encystation. We have, however, reported previously a method to induce, in B. hominis, the formation of in vitro cysts that are morphologically distinct from the faecal cyst and resemble granular forms except for the presence of a thick osmiophilic cyst wall-like structure. These were able to withstand hypotonic shock (Villar et al., 1998) and also infect laboratory rats via oral inoculation (Suresh et al., 1994). When vacuolar forms were cultured in encystation medium, the proportion of in vitro cysts to vacuolar forms increased with time (Villar et al., 1998). Whether such cysts exist in vivo has been a matter of debate (Stenzel and Boreham, 1996) and more work needs to be done to resolve this issue. To avoid confusion, subsequent studies involving B. hominis cysts should indicate if the cysts were faecal- or in vitro-derived morphological types. The stepwise differentiation of cysts (faecal-derived morphological type) to vacuolar forms (excystation) has been elegantly described by time-course TEM (Moe et al., 1999; Chen et al., 1999). It showed that the faecal cyst of B. hominis and B. ratti develop, rather similarly and dramatically, into vacuolar forms within 24 h of inoculation into the growth media. At the 1 h time point, the cysts appeared typical (see this section, first paragraph). However, at 3 h, multiple small vacuoles, with many harbouring inclusions, accumulated throughout the cytoplasm. The cysts gradually lose their cyst wall around 6 h, eventually replacing it with a thick surface coat. This disintegration of the cyst wall appeared to allow for the enlargement of the parasite from the initial 5 mm to about 7 9 mm at 9 h. This cell enlargement occurred concurrently with the apparent aggregation of the multiple small vacuoles of the cyst at the central region of the cell, with these vacuoles eventually coalescing to form the central vacuole. This coalescence resulted in the deposition of the abovementioned vacuolar inclusions into the central vacuole, resulting in a granular form-like appearance. By 12 h, typical vacuolar and granular forms were apparent and binary fission was observed. The parasite may also undergo division whilst still surrounded by cyst wall material and up to four daughter cells may be observed within a cyst (Chen et al., 1999; Zaman et al., 1999b) Other forms Besides the four abovementioned forms, there have also been descriptions of some other forms isolated from intestines and fresh faecal material. The description of cells isolated from a colonoscopy sample was distinctly different from the vacuolar and granular forms (Stenzel et al., 1991) but was similar to an earlier study of B. hominis obtained from a patient producing large volumes of diarrhoeal fluid (Zierdt and Tan, 1976). These were termed avacuolar forms for their lack of a central vacuole. In contrast to culture forms, these cells were smaller (approximately 5 mm in diameter) and lacked a surface coat. Differences in the morphology of the mitochondria were also noted. Interestingly, these avacuolar cells (Stenzel et al., 1991) could not be established in culture and so it remains to be seen if these were indeed Blastocystis cells to begin with. Multivacuolar

7 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) forms have been reported in fresh human faecal material (Stenzel et al., 1991; Stenzel and Boreham, 1996). These relatively small cells (5 8 mm in diameter) contain multiple small vacuoles varying in size and content and possessed thick surface coats. However, small vacuolar forms were also clearly discernable from the micrographs indicating that the multivacuolar form is not the sole form in faecal material. In a separate study (Lanuza et al., 1997) involving 81 isolates, vacuolar and, in some instances, amoeboid forms were the only Blastocystis cell types seen in faecal samples. The avacuolar and multivacuolar forms have been suggested to represent in vivo stages of the parasite while the larger vacuolar and granular forms predominate in in vitro culture (Boreham and Stenzel, 1993). This needs further confirmation due to the small number of studies that have reported these forms. The multivacuolar forms may well represent intermediate developmental stages of faecal cysts as they undergo differentiation into vacuolar forms, with the coalescence of the described multiple vacuoles forming the central vacuole. The disappearance of the multivacuolar forms in short-term in vitro culture and the subsequent appearance of the vacuolar forms (Stenzel et al., 1991) suggest that these represent transient stages in the B. hominis life-cycle Unusual structures of uncertain function Surface coat A fine fibrillar layer, commonly referred to as a surface coat, usually surrounds the vacuolar and granular forms. It is probably the same structure that surrounds the cyst form (Zaman et al., 1997a) but there have been conflicting reports as to whether the amoeboid form possesses one (Tan and Zierdt, 1973; Dunn et al., 1989; Tan, 1999, PhD thesis). It varies in thickness and morphology and is usually thicker in cells examined from fresh faecal matter as compared with that of cells from in vitro culture (Boreham and Stenzel, 1993). A number of studies have shown that the surface coat of B. hominis does contain carbohydrates (Yoshikawa et al., 1995a; Lanuza et al., 1996, Tan et al., 1996a) although no quantitative analyses were carried out. Lanuza et al. (1996) used a panel of FITC-labelled lectins in their study and observed that the surface coat had components containing alpha-d-mannose, alpha-d-glucose, N-acetyl-d-glucosamine, alpha-l-fucose, chitin and sialic acid. The surface coat appears to be continuously synthesised and shed in the environment (Zaman et al., 1997b). Scanning EM has revealed that the surface coat of B. hominis had a spongy, lace-like appearance and, in some cases, could reach lengths of about 10 mm (Zaman et al., 1999a; Tan et al., 2000). Bacteria may be found in close association with the surface coat (Silard and Burghelea, 1985; Dunn et al., 1989; Zaman et al., 1997b, 1999a). One of these studies (Zaman et al., 1997b) revealed that bacteria attached to the surface coat show loss of electron density, suggesting that it may serve as an entrapment mechanism for nutritive purposes. The surface coat has also been postulated to function as a mechanical and chemical barrier against host innate and acquired immune responses (Boreham and Stenzel, 1993). This hypothesis was supported by our observation that monoclonal antibodies (mab) that bind extensively to B. hominis surface coat did not inhibit growth whereas a particular mab specific for a plasma-membrane protein was cytotoxic to the parasite (Tan et al., 1997) Mitochondria or hydrogenosomes? Blastocystis hominis is an anaerobe (Stenzel and Boreham, 1996) and the presence of mitochondria-like organelles in this parasite is rather unusual. It has been suggested that the mitochondria of B. hominis are, in fact, hydrogenosomes (Boreham and Stenzel, 1993; Stenzel and Boreham, 1996) because biochemical analyses have shown that a number of typical mitochondrial enzymes were absent from B. hominis (Zierdt, 1986, 1988). Hydrogenosomes were first described in trichomonads (Lindmark and Müller, 1973; Lindmark et al., 1975) and have been identified in a broad phylogenetic range of organisms. These organelles have been observed in rumen dwelling ciliates (Yarlett et al., 1981; Lloyd et al., 1989; Paul et al., 1990), free-living ciliates (Fenchel and Finlay, 1991) and rumen fungi (Yarlett et al., 1986; O Fallon et al., 1991). All organisms known to contain hydrogenosomes are either anaerobic or aerotolerant anaerobes, and all appear to lack mitochondria (Johnson et al., 1995). Hydrogenosomes differ from mitochondria in that they lack cytochromes, the tricarboxylic acid cycle and oxidative phosphorylation (Palmer, 1997). TEM of B. hominis cells cultured in the presence of the reducing agent, sodium thioglycollate, revealed that the mitochondria had transformed into numerous structures that, surprisingly, resembled the hydrogenosomes of Trichomonas vaginalis (Tan, 1999, PhD thesis). These hydrogenosome-like structures were bound by a double membrane, appeared as vesicles mm in diameter and contained a fine granular matrix. A possible test to confirm if the hydrogenosome-like structures seem in B. hominis are indeed hydrogenosomes is to detect the presence of its marker enzyme, hydrogenase. This can be achieved by cytochemical tests which detect hydrogenase activity, as has been shown for other protozoa (Zwart et al., 1988; Paul et al., 1990). Considering the growing evidence for the presence of hydrogenosomes in a wide variety of anaerobic organisms, it is not unlikely that the mitochondria of B. hominis are, in reality, hydrogenosomes. 4. In vitro culture Polyxenic or monoxenic B. hominis cultures generally grow well in Jones s (1946) or Boeck and Drbohlav s (1925) inspissated egg medium. Once axenised, B. hominis shows luxuriant growth in enriched monophasic media such as minimum essential medium (MEM) or IMDM that have been supplemented with 10% horse serum and pre-reduced

8 796 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) for 48 h prior to culture (Boreham and Stenzel, 1993; Ho et al., 1993). There have been some new methods for the culture of B. hominis, particularly in the area of colony growth and axenisation. The ability to grow microorganisms as colonies on solid or semisolid medium has been enormously useful in viability, genetic and biochemical studies because it provides a convenient means of generating and analysing clonal populations. Culture of protozoa as discrete colonies in or on agar/agarose has been established for T. vaginalis (Hollander, 1976), entamoebae (Gillin and Diamond, 1978), Giardia intestinalis (Gillin and Diamond, 1980) and trypanosomatids (Tanuri et al., 1981; Carruthers and Cross, 1992). Colony formation of B. hominis in soft agar was reported relatively recently (Tan et al., 1996b) and was achieved by plating parasites in a mixture of Bacto agar, IMDM and 10% horse serum. Addition of the reducing agent, sodium thioglycollate, improved yields significantly (efficiency of plating ranging from 71 to 98%) and clonal growth was achievable (Tan et al., 1996c). Individual colonies were easily expanded in liquid cultures, providing a method for isolation and expansion of discrete parasite clones. The colony growth method was successfully employed for screening surface-reactive mab for cytotoxic effects (Tan et al., 1997) and for the axenisation of Blastocystis species isolated from a human, a reptile and laboratory rats (Chen et al., 1997b; Ng and Tan, 1999). More recently, we have described an improved, high-efficiency method for clonal growth of B. hominis on solid agar (Tan et al., 2000). This method was better than the previously described ones as the colonies were now exposed on the agar surface, which made it easier for enumeration and manipulation. The buff-coloured colonies grew to 2 mm in diameter by 7 days post-inoculation and clones were easily expanded in liquid cultures. With the aid of fluorescent viability dyes and by flow cytometry, these colony forms were shown to be viable for 14 days. This is in contrast to liquid cultures, which enter death phase around day 5 (Ho et al., 1993; Lanuza et al., 1997). Such colony growth had allowed for subculturing to be carried out once in 2 weeks instead of weekly. Axenisation of Blastocystis spp. is often achieved by antibiotic treatment (Zierdt and Williams, 1974; Kukoschke and Müller, 1991; Ho et al., 1993; Teow et al., 1992; Lanuza et al., 1997; Ng and Tan, 1999) and not by harsher physical methods because of the inherent fragility of the culture forms. There have been reports of unsuccessful axenisation (Boreham and Stenzel, 1993; Lanuza et al., 1997) indicating that in some instances, B. hominis may require bacteria for survival. This is somewhat supported by the observation that once axenised, certain Blastocystis isolates grew slower than when cultured in the presence of accompanying bacteria (Chen et al., 1997b). Another problem encountered by earlier workers was the lengthy periods required to axenise cultures (usually over a month). This can usually be shortened by methods, such as differential centrifugation, that aid in the physical separation of the parasites from the bacterial bulk, when used in concert with conventional antibiotic treatment. The combination of Ficoll-metrizoic acid gradient and addition of antibiotics was successfully employed to axenise 25 out of 81 B. hominis isolates in an average time of 3 weeks (Lanuza et al., 1997). We have also observed that axenisation was made possible by initial reduction of bacterial load using antibiotics followed by colony growth to isolate pure parasite colonies (Chen et al., 1997b; Ng and Tan, 1999). The cryopreservation protocol described previously (Zierdt, 1991) involved the addition of dimethyl sulphoxide and demanded extreme care. In contrast, glycerol supplemented with foetal calf serum (FCS) was shown to improve yields to about 90% (Suresh et al., 1998). However, this study involved only a single isolate of B. hominis and more work therefore needs to be done before concluding that this formulation would be suitable for other Blastocystis spp. or B. hominis isolates. 5. Immunobiology 5.1. Host immune response Very little is known about the host immune response to B. hominis. A lack of humoral immune response was reported by Chen et al. (1987) who had used Western blot to detect only IgG in sera from only four patients. IgG against B. hominis antigens was subsequently shown, by enzyme linked immunosorbent assay (ELISA), to be elevated in 25 of 28 infected humans in a later study (Zierdt et al., 1995), although the threshold dilution was reported as 1/ 50. Hussain et al. (1997) reported significantly increased IgG2 antibody levels to B. hominis in patients with irritable bowel syndrome, suggesting a possible link between B. hominis and irritable bowel syndrome. The authors suggested that the IgG2 subclass antibody was induced in response to surface coat fragments of the parasite that had been transported, via Peyer s patch M cells, from the gut lumen to lymphocytes and macrophages. From these few studies, it is evident that B. hominis can elicit a humoral immune response in humans. However, little else is known about innate and acquired immunity to blastocystosis. To better understand the host immune response to B. hominis infection, a suitable animal model must be found. Mice are generally ideal for such studies as a considerable variety of murine-related reagents and technologies exist. The Giardia muris-mouse model of giardiasis (Roberts-Thomson et al., 1976) has provided a useful tool for the study of the immune response to Giardia infection (Faubert, 2000). Unfortunately, mice, hamsters and rabbits are not naturally infected with Blastocystis (Chen et al., 1997a). Rats, however, have been observed to be especially susceptible to Blastocystis infections (Chen et al., 1997a) and would perhaps be useful animal models.

9 K.S.W. Tan et al. / International Journal for Parasitology 32 (2002) Antigenic diversity Antigenic heterogeneity has been shown to exist among B. hominis isolates (Kukoschke and Müller, 1991; Boreham et al., 1992; Müller, 1994; Lanuza et al., 1999; Tan et al., 2001b) and was facilitated by techniques such as SDS- PAGE, immunoblotting, immunodiffusion and immunofluorescence. Depending on the study, the differences may be discrete (i.e. no cross-reactivity) (Kukoschke and Müller, 1991; Boreham et al., 1992; Müller, 1994; Tan et al., 2001b) or partial (Lanuza et al., 1999). Such diversity, as has also been shown genetically, reinforces the emerging concept that B. hominis is biologically heterogeneous and strongly suggests that there are more than one species of Blastocystis in humans. It has been postulated that serotypes differ in their pathogenic potential, but the data have been contradictory. Support for the hypothesis came from a single study (Lanuza et al., 1999) that showed that 18 isolates of B. hominis could be classified into two related antigenic groups. Patients suffering from chronic diarrhoea clustered within group 1 while those suffering from acute diarrhoea clustered within group 2. Unfortunately, this study lacked details on the statistical significance of the associations. In a larger study involving 61 B. hominis isolates (Müller, 1994), four distinct serogroups were identified using the immunodiffusion assay but none of them were found to be significantly correlated with intestinal disease. This issue would be more readily resolved if and when a suitable animal model is found. Whatever the case, such antigenic diversity should be taken into consideration when developing serological diagnostic tests for B. hominis infections Monoclonal antibodies There have been two reports on the production and characterisation of mab against B. hominis (Yoshikawa et al., 1995b; Tan et al., 1996a). Yoshikawa et al. (1995b) analysed a panel of eight mabs. The majority of the mabs was IgM and, by immunoem, was observed to bind to surface coat epitopes. Only two of the mabs showed cross-reactivity with a Blastocystis spp. isolated from a chicken. The other six B. hominis-reactive mabs were surface coat-specific while the two cross-reactive mabs bound to material within the central vacuole. The authors suggested that antigenic components on the surface coat are strain- or species-specific while those of the central vacuole were not. mabs against B. hominis were also raised and characterised in our laboratory (Tan et al., 1996a). Most of these IgM mab reacted with multiple carbohydrate epitopes, as evidenced by immunoblot, concanavalin A binding and periodate sensitivity assays. These antigens were later shown by immunogold EM to be located on the surface coat (Tan et al., 1997). However, one particular periodate-resistant surface-reactive mab was cytotoxic to the parasite, as shown by a significant decrease in colony yields when compared with cells exposed to the other mabs (Tan et al., 1997, 2001b). This mab (1D5) appeared to bind to a 30 kda plasma membrane-associated protein. Interestingly, the death process that ensued appeared distinctly apoptotic (see Section 7.1). Despite the cytotoxicity of 1D5, surviving clones within a particular isolate could be generated that, after several culture passages in the presence of the mab, showed almost complete resistance to its killing effect. Our recent data suggest that the 1D5 epitope is species- and isolate-specific, as there was cross-reactivity only with local B. hominis isolates but none against a B. hominis isolate from Pakistan and a panel of rat and reptilian Blastocystis spp. (Tan et al., 2001b). Collectively, the data suggest that B. hominis is predisposed to exist in a heterogeneous state (i.e. 1D5-susceptible or -resistant cells) in long-term axenic cultures and the 30 kda surface protein appears to be functionally important to, but is not present on all, Blastocystis isolates. Unlike 1D5, surface coat carbohydrate-reactive mabs were not cytotoxic to the parasite despite extensive binding (Tan et al., 1997). In other studies, (Hussain et al., 1997; Kaneda et al., 2000) it has been observed that the major immune response in infected patients is probably against surface coat carbohydrates. Taken together, the data suggest that the surface coat of B. hominis may serve as an immunological barrier for the parasite. Another interesting observation is that the surface coat of B. hominis appears to be continuously synthesised and shed into the extracellular environment (Zaman et al., 1997b). These phenomena may have a functional role in vivo. The shed surface coat glycoproteins may act as an immunological smokescreen by promoting the generation of ineffective B cells that produce non-cytotoxic or non-neutralising antibodies against surface coat carbohydrate epitopes. This model for the host immune response to B. hominis needs to be proven experimentally. 6. Clinical aspects Blastocystis hominis is commonly found in healthy individuals as well in patients with gastrointestinal symptoms. In general, prevalence of infection is higher in developing than in developed countries (Stenzel and Boreham, 1996). Within communities, groups from lower socio-economic levels who suffer from poor environmental hygiene, due largely to lack of water supply, sewerage and waste removal services are at greater risk of infection (Borda et al., 1996; Wilairatana et al., 1996; Cirioni et al., 1999; Guignard et al., 2000; Taamasri et al., 2000). Increased risk of infection may also be associated with the workplace. In a recent survey conducted in Malaysia, Rajah Salim et al. (1999) showed that people working closely with animals were at higher risk of acquiring Blastocystis infection. About 41% of local animal handlers from two research institutions, a zoo and an abattoir were infected compared with a rate of 17% in a control group of high-rise city dwellers. The authors