Discovery of three novel coccidian parasites infecting California sea lions (Zalophus californianus), with evidence of sexual replication and interspecies pathogenicity

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites "A," "B," and "C." Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the "C" sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.     

Monogeneans from Pangasiidae (Siluriformes) in Southeast Asia: IV. Five new species of Thaparocleidus Jain, 1952 (Ancylodiscoididae) from Pangasius krempfi,P. kunyit,P. mekongensis and P. sabahensis

The examination of gill parasites from Pangasius krempfi Roberts & Vidthayanon, 1991; P. kunyit Pouyaud et al., 1999; P. mekongensis Gustiano et al., in press and P. sabahensis Gustiano et al., in press (Siluriformes, Pangasiidae) in Southeast Asia revealed the presence of six species of Monogenea, all belonging to the genus Thaparocleidus Jain, 1952 (Monogenea, Ancylodiscoididae) as defined by Lim (1996) and Lim et al. (2001). One has been previously described (T. vietnamensis Pariselle et al., 2002), five are considered as new species: T. humerus n. sp. and T. culter n. sp. on P. kunyit; T. mehurus n. sp. and T. culteroides n. sp. on P. sabahensis; and T. phuongi n. sp. on the four studied host species. The latter one, due to slight morphometric differences linked to geographical origin of hosts, is described as made up of three sub-species: T. phuongi phuongi n. sub-sp., T. phuongi malaysiensis n. sub-sp. and T. phuongi indonesiensis n. sub-sp.     

Electron microscopic research on cryptosporidia. III. Parasite-host relations

A study was made of the host-parasite relationship with Cryptosporidium parvum (Apicomplexa, Sporozoa), which parasitizes the intestine of newborn rats experimentally infected with oocysts isolated from C. parvum-infected calves. The endogenous development of the parasite occurs extracytoplasmically in the microvillar compartment of the enterocytes. The formation of the extracytoplasmic parasitophorous vacuole (PV), like that surrounding the endogenous stages of C. parvum, is regarded as one of the possible and evolutionary established ways for the intracellular parasite to escape from the host cell lysosomal digestion. Special attention is paid to the attachment zone of C. parvum, where a multimembranous organelle is formed serving eventually as a feeder organelle. No other specialized cytostome, similar to the micropore of other coccidia, has been so far revealed in the growing stages of Cryptosporidium. The characteristic ultrastructural organization of the endogenous stages of C. parvum and of other Cryptosporidium species so far investigated, along with the peculiar structure of the cryptosporidia-surrounding PV, to say nothing of some other distinctive features--all this makes it possible to distinguish between the genus Cryptosporidium and other coccidian genera, and warrants the separation of the former into a separate family Cryptosporidiidae Léger, 1911. Unlike, the addition to this family, besides Cryptosporidium Tyzzer, 1910, of another genus, Epieimeria Dykova, 1981, on the ground of the "epicellular" localization of both the genera claimed by Levine (1984), seems hardly correct, due to the totally different patterns of ultrastructural organization and host-parasite relationship recently reported for Epieimeria anguillae by Molnar and Baska (1986).     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Freeze-fracture study of the site of attachment of Cryptosporidium muris in gastric glands.

The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Freeze-Fracture Study of the Site of Attachment of Cryptosporidium muris in Gastric Glands

ABSTRACT The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

The ultrastructure and reproduction of Amphiamblys capitellides (Microspora, Metchnikovellidae), a parasite of the gregarine Ancora sagittata (Apicomplexa, Lecudinidae), with redescription of the species and comments on the taxonomy

The ultrastructural cytology and reproduction of the hyperparasitic microsporidium Amphiamblys capitellides (Caullery and Mesnil, 1897) is described. Merogonial reproduction was not observed. The sporogony comprises two sequences: a sac-bound sporogony in close contact with the cytoplasm of the host and a free sporogony in parasitophorous vacuoles. The free sporogony, which probably precedes the sac-bound, yields a small number of rounded spores. The sac-bound sporogony is polysporoblastic, generating two rows of elongated spores. All stages have isolated nuclei. Both spore types have an extrusion apparatus of the metchnikovellidean type, with a polar sac devoid of anchoring disc, a polar filament with one manubroid and one bulbous part, and a posterior semicircular membrane fold enclosing rounded or tubular structures. Hosts are gregarines of the species Ancora sagittata living in the intestine of polychaetes of the genus Capitella, probably the species Capitella giardi. The cytology, life cycle and classification are discussed. The species is redescribed and the diagnosis of the genus Amphiamblys Caullery and Mesnil, 1914 is emended.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.     

Immunodetection of the microvillous cytoskeleton molecules villin and ezrin in the parasitophorous vacuole wall of Cryptosporidium parvum (Protozoa: Apicomplexa)

Microvilli - actin - villin - ezrin - Cryptosporidium parvum The sporozoites and merozoites of the Apicomplexan protozoan Cryptosporidium parvum (C. parvum) invade the apical side of enterocytes and induce the formation of a parasitophorous vacuole which stays in the brush border area and disturbs the distribution of microvilli. The vacuole is separated from the apical cytoplasm of the cell by an electron-dense layer of undetermined composition. In order to characterize the enterocyte cytoskeleton changes that occur during C. parvum invasion and development, we used both confocal immunofluorescence and immunoelectron microscopy to examine at the C.parvum-enterocyte interface the distribution of three components of the microvillous skeleton, actin, villin and ezrin. In infected cells, rhodamine-phalloidin and anti-villin and anti-ezrin antibodies recognized ring-like structures surrounding the developing parasites. By immunoelectron microscopy, both villin and ezrin were detected in the parasitophorous vacuole wall surrounding the luminal and lateral sides of the intracellular parasite. In contrast, anti-beta and anti-gamma actin antibodies showed no significant labelling of the vacuolar wall. These observations indicate that the parasitophorous vacuole wall contains at least two microvillus-derived components, villin and ezrin, as well as a low amount of F-actin. These data suggest that C.parvum infection induces a rearrangement of cytoskeleton molecules at the apical pole of the host cell that are used to build the parasitophorous vacuole.     

Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species,Sparus aurata L. and Dicentrarchus labrax L

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.     

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.     

Life Cycle of Goussia pannonica (Molnar, 1989) (Apicomplexa, Eimeriorina), an Extracytoplasmic Coccidium from the White Bream Blicca bjoerkna

ABSTRACT Life cycle stages of Goussia pannonica from naturally-infected white bream Blicca bjoerkna were studied by light and electron microscopy. Fourteen of the sixteen fish examined were infected, with developmental stages found in all parts of the intestine. Merogonial, gamogonial, and sporogonial stages were localized intracellularly and extracytoplasmically in the microvillous region of enterocytes. They were separated from the gut lumen by closely apposed enterocyte and parasitophorous vacuole membranes. There were two types of extracytoplasmic attachment: 1) monopodial, with a single zone of attachment, and 2) spider-like, with several isolated zones of attachment to the host cell. First-generation merozoites were formed by ectomerogony. Second- or third-generation merozoites were formed by endodyogeny and endopolygeny. Thirty to 50 biflagellated microgametes developed at the periphery of a microgamont. Macrogamonts contained lipid inclusions, amylopectin and dense granules; however, granules comparable to wall-forming bodies type I and II were absent. At the beginning of sporogony, the sporont cytoplasm detached from two layers which subsequently became constituents of the oocyst wall. After the rupture of enterocyte and parasitophorous vacuole membranes, the sporont was released into the water where exogenous sporulation was completed within 48 h. The thin sporocyst wall contained a small longitudinal suture. Sporocyst and oocyts walls were of similar structure.     

Cryptosporidia: epicellular parasites embraced by the host cell membrane

The ultrastructure of two gastric cryptosporidia, Cryptosporidium muris from experimentally infected rodents (Mastomys natalensis) and Cryptosporidium sp. 'toad' from naturally infected toads (Duttaphrynus melanostictus), was studied using electron microscopy. Observations presented herein allowed us to map ultrastructural aspects of the cryptosporidian invasion process and the origin of a parasitophorous sac. Invading parasites attach to the host cell, followed by gradual envelopment, with the host's cell membrane folds, eventually forming the parasitophorous sac. Cryptosporidian developmental stages remain epicellular during the entire life cycle. The parasite development is illustrated in detail using high resolution field emission scanning electron microscopy. This provides a new insight into the ultrastructural detail of host-parasite interactions and species-specific differences manifested in frequency of detachment of the parasitophorous sac, radial folds of the parasitophorous sac and stem-formation of the parasitised host cell.     

Establishment of Endoreticulatus N. G. for Pleistophora fidelis (Hostounský & Weiser, 1975) (Microsporida: Pleistophoridae) Based on the Ultrastructure of a Microsporidium in the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)1,2

A new genus, Endoreticulatus n. g., is described for the microsporidium Pleistophora fidelis (Hostounský & Weiser, 1975) based on light and electron microscopic studies of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say). This latter microsporidium is considered to be conspecific with P. fidelis because both isolates have been shown to be infectious for L. decemlineata where infection is limited to the epithelial cells of the midgut; both are haplokaryotic and develop as groups of sporoblasts and spores in subpersistent vacuoles in the host cell. In addition, meronts, sporonts, and spores of each isolate often occur simultaneously in a common cell, and by light microscopy they both appear similar. Ultrastructural studies of the isolate from L. decemlineata revealed that all developmental stages occur in parasitophorous vacuoles derived from cisternae of rough endoplasmic reticulum of the host cell cytoplasm. Based on the unique nature of the parasitophorous vacuole, a new genus, Endoreticulatus, is proposed for P. fidelis. The genus is compared with the other genera whose species undergo multisporous sporogony in sporophorous vesicles. In addition, the nature of the parasitophorous vacuole of Endoreticulatus fidelis (Hostounský & Weiser, 1975) n. comb. is compared with the parasitophorous vacuole known to encase various developmental stages of several other microsporidian species.     

Molecular Phylogeny and Surface Morphology of Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp. (Gregarinasina,Apicomplexa) Isolated from a Deep Sea Oradarea sp. (Amphipoda) in the West Pacific

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper‐Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. Thiriotia hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this latter new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans.     

Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae)

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.     

Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem)

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion.The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.     

Further comment on the coccidian nature of cryptosporidia (Sporozoa: Apicomplexa)

The coccidian nature of the genus Cryptosporidium was undoubtedly accepted by Tyzzer who was the first to describe this sporozoan parasite in 1907. Electron microscopic studies made in 70-90s demonstrated the intracellular, although extracytoplasmic localization of Cryptosporidium spp. The pattern of Cryptosporidium life cycle fits well that of other intestinal homogeneous coccidian genera of the suborder Eimeriina: macro- and microgamonts develop independently, a microgamont gives rise to numerous male gametes, oocysts serving for parasite's spreading in the environment. Along with these characters, Cryptosporidium spp. demonstrate some secondary peculiarities (an endogenous phase of development in microvilli of epithelial surfaces, two morphofunctional types of oocysts, the smallest number of sporozoites per oocyst, a multi-membraneous "feeder" organelle etc.), which may be due presumably to their early acquisition of specialization in the course of evolution. The recent studies based on molecular sequence data (18S rRNA) applied to 8 eimeriid and isosporid coccidian genera (Morrison, Ellis, 1997), suggested that the subclass Coccidia (class, according to Morrison and Ellis) be considered monophylic if Cryptosporidium were excluded, and this genus was regarded as the sister group to the rest of the Apicomplexa, or as the sister to the suborder (class) Hematozoa within the Apicomplexa. Either of these placements of Cryptosporidium definitely conflicts with both the generally accepted taxonomic scheme by Levine (1982) and the phenotypically based phylogeny of the phylum Apicomplexa (Barta e. a., 1990). The author's opinion is that the differences between the examined eimeriid and isosporid coccidia, on the one hand, and Cryptosporidium, on the other hand, provided by molecular sequence data, may testify primarily to the well known morphofunctional dissimilarities between the compared organisms, rather than cast doubt on the coccidian nature of Cryptosporidium. Again, these data can hardly prove that Cryptosporidium does not belong to the coccidia. Thus, the modern molecular sequence data, despite their obvious scientific value, would make sense for phylogeny estimation only, if they are critically analysed and considered in combination with results of the relevant basic research.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

Karyolysus sp. (Haemogregarinidae, Adeleida, Apicomplexa): Host-Parasite Relationships of Persisting Stages

ABSTRACT. Two persisting stages in the life cycle of a hemogregarine Karyolysus sp. are described from the liver and blood cells of its intermediate host, the lizard Lacerta raddei nairensis. The tissue cell merozoites lie in a parasitophorous vacuole. Despite the protective role of the vacuolar membrane, the intracellular parasites are progressively destroyed and eliminated during the autumn and winter. Some of the merozoites that normally survive within the host cell even in cold seasons appear to be surrounded by another type of parasitophorous vacuole which is connected to the intercellular space by narrow channels. The intraerythrocytic gamonts that persist in the circulating blood are encapsulated and undergo progressive, obvious structural changes. The two persisting stages are compared with hypnozoites of other Sporozoa.