Transient transfection of Cryptosporidium baileyi.

Here we demonstrate the transient transfection of C. baileyi. We describe the transfection of this apicomplexan parasite and the cultivation in ovo. The functionality of heterologous sequences in C. baileyi was demonstrated by the expression and detection of the mCherry protein in ovo. The mCherry protein was expressed in parasitic stages up to the oocyst stage under the control of the heterologous promoter region of the C. parvum actin gene.

Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae): the mode of survival in the gut of Enchytraeus albidus (Annelida, Oligochaeta, Enchytraeidae) is close to that of the coccidian genus Cryptosporidium.

A selenid gregarine Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae), harboring the gut lumen of the oligochaete Enchytraeus albidus, was studied by light and electron microscopy. The trophozoite of Ditrypanocystis sp. is attached to the gut wall with its apical end to be anchored eventually between enterocytes in the crypts. Simultaneously, between the surfaces of the parasite and the host cell a peculiar contact is formed made of membranous channels and vesicles of unknown origin, the host cell surface in the contact area lacking cilia. The trophozoite becomes progressively enclosed within a parasitophorous vacuole made of layers of fused ciliar membranes of enterocytes. The fused cilia may be a source of membranes lining channels and vesicles of the contact area. Such a mode of parasitophorous arrangements has never been described before for gregarines, however, it bears a some likeness with that of the coccidian genus Cryptosporidium (similarity and differences being discussed). With regard to some molecular phylogeny constructions, claiming the "sister" relationship between gregarines and the coccidian genus Cryptosporidium (Carreno et al., 1999; Leander et al., 2003), this common feature in host-parasite relationships enabled us to put forward an idea of a possible evolutionary route from extracellularity of gregarines to intracellularity of coccidia, as exemplified by species of Cryptosporidium.

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia).

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.

Microneme secretion in Coccidia: confocal laser scanning and electron microscope study of Sarcocystis muris in cell culture.

A monoclonal antibody (mcab) raised against a subcellular fraction of Sarcocystis muris cystozoites was used to localize microneme antigens before, during and after invasion of cultured cells. The mcab recognized a 20 and 22 kDa protein under reducing and non-reducing conditions on Western blots and localized an antigen in cystozoites in the apical part of the parasites. Confocal laser scanning microscopy of invading cystozoites revealed the secretion of a microneme antigen at the apical tip of the parasite. The secreted microneme antigen was attached to the host cell surface at the invasion site and spread along the surface of the infected cells. Electron microscopy using immunogold labeling showed that the microneme antigen was distributed in patches on the surface of infected cells and present on infected cells more than 60 min post-infection. The function of microneme antigens during parasite-host cell interactions is discussed.

Immunoelectron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sarcocystis muris (Protozoa, Apicomplexa).

Merozoites of the parasitic protozoon Sarcocystis muris (Apicomplexa) possess three types of characteristic organelles with electron dense contents named rhoptries, micronemes, and dense granules, which are supposed to be involved in the parasite-host cell interactions during and after invasion. Dense granules were purified from a merozoite homogenate by centrifugation on a sucrose density gradient. It was shown by SDS polyacrylamide gel electrophoresis that they contain a major protein of 21 kDa. Polyclonal antibodies raised against this protein were applied to ultrathin frozen and Lowicryl-K4M-embedded sections of the parasite before and after host cell invasion. Dense granules were distinctly labeled by immunogold before and after invasion. After host cell invasion the parasite is enclosed in a secondary parasitophorous vacuole which contains an electron-dense material. This deposition was heavily labeled by anti 21 kDa antibodies which clearly demonstrated that the dense granule contents is released into the secondary parasitophorous vacuole.

Characterization of cDNA clones encoding a major microneme antigen of Sarcocystis muris (Apicomplexa) cyst merozoites.

Two monoclonal antibodies directed against a microneme antigen of Sarcocystis muris cyst merozoites (16/17 kDa band doublet) were used to isolate cDNA clones from a lambda ZAP expression library. Restriction analysis revealed that the inserts were highly similar, with sizes ranging between 1.8 and 2.3 kb. In addition, a full-length cDNA insert of 2.6 kb was obtained by hybridization screening. On Northern blots, a single mRNA species of 2.7 kb was detected by a cDNA-derived probe. Southern blot hybridization suggests that the gene is present as a single copy. The nucleotide sequence of the full-length clone contains a single reading frame with a coding capacity of 26.5 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide followed by a hydrophilic domain of unknown function, and the mature protein sequence. After purifying the 16/17 kDa antigen from cyst merozoites, a partial N-terminal amino acid sequence was obtained. Thus, the identity of the cDNA sequence was confirmed. The deduced sequence of the mature protein is predominantly hydrophilic and rich in cysteine (8.7%). Database searching suggested weak homologies of the hypothetical polypeptide to plasma kallikrein, tenascin and blood coagulation factors.

Structure and function of the parasitophorous vacuole in Eimeria species.

The intracellular life-cycle of Eimeria are located in the host cell within a membrane-bound parasitophorous vacuole. The invasion process and the formation of the parasitophorous vacuole are mediated by characteristic organelles within the apical complex. During invasion, the parasitophorous-vacuole membrane is manipulated by the parasite and functions later in the development cycle as a molecular sieve, allowing the exchange of metabolites between parasite and host cell. Unlike the cyst-forming coccidia, there is little evidence of parasitophorous-vacuole membrane transformation in the later stages of the lifecycle of Eimeria species. Compared with the human pathogens Plasmodium and Toxoplasma, rather little is known about the parasitophorous vacuole and parasitophorous-vacuole membrane of animal pathogens of the genus Eimeria.

A freeze-fracture study of the host cell-parasite interface during and after invasion of cultured cells by cystozoites of Sarcocystis muris.

The parasite-host-cell interface of Sarcocystis muris in cell culture was studied by freeze-fracture electron microscopy. Cystozoites of S. muris have an intra membrane particle (IMP) density of 1347 ± 146 IMP/µm(2) on the P face and 638 ± 109 IMP/µm(2) on the E face. After exposure to trypsin during extraction from the cysts a reversal of IMP density to 820 ± 115 µm(2) on the P face and 1277 ± 140 µm(2) on the E face occurred. Invasion of S. muris cystozoites is followed by the enclosure of the parasite in a primary parasitophorous vacuole (PV 1), limited by a membrane which partly originates by invagination and inward expansion of the plasmalemma of the host cell. A decline of local densities of IMP on the P face of the infected host cell from 2557 ± 567 IMP/µm(2) to 417 ± 217 IMP/µm(2) in the membrane of PV 1 represents a significant decrease of protein elements. S. muris gamonts became translocated to a new vacuole (PV 2) 30 to 45 minutes after invasion. IMP reappeared on both fracture faces of the vacuole membrane. The rapid increase of IMP on the PV 2 membrane coincides with the release of large amounts of dense granule contents.

A scanning electron microscope study of the colon of the mouse (Mus musculus) infected with Eimeria ferrisi (Protozoa, Apicomplexa, Coccidia).

Micromorphological changes in the colon of mice infected with Eimeria ferrisi were studied by scanning electron microscopy. The damage observed consisted of swelling of epithelial cells, profound stretching of the host cell membranes, loss of microvilli and erosion of tissues. Mature meronts were visible at 3 days post-inoculation (PI) in localized areas of cellular rupturing. At 4-6 days PI, the cellular rupturing and epithelial disruption were more widespread, exposing intra- and extracellular meronts, merozoites, and sexual stages. Microgametes of E. ferrisi are equipped with 2 flagella. Even though large portions of the infected area become denuded of surface epithelium, recovery had begun by day 7 PI.

Analysis of antibodies raised against soluble and membrane bound proteins of Nosema grylli (Microspora) spores.

Microsporidia (M), representatives of the phylum Microspora, make a world-wide distributed group of intracellular protists, parasitic in the vast number of hosts, from Protozoa to Primates. In their morpho-functional organization, both very primitive and extremely specialized features are seen definitely combined. Data available on RNA and DNA sequences suggest that M may be the most ancient eukaryotes. By the present, as many as 13 microsporidian species have been recognized as opportunistic pathogens in AIDS and transplant patients. Information about structural, transport and regulatory proteins of M, as well as on their enzymes is scarce, though it could serve as a basis for understanding pathogenicity of M and indicate some possible sites of relevant suppressive therapy. The present study persuaded two main goals: 1) to examine two ways of antigen preparation (from the infected organ and from the purified spores) and to evaluate their relation to the yield of the resulting antibodies; 2) to identify and localize new proteins with the help of the obtained antibodies by means of IFA, IEM and WB. Mice were immunized: 1) with dissolved proteins of heavily loaded with parasites fat bodies isolated from crickets Gryllus bimaculatus infected with Nosema grylli, and 2) with proteins of the purified spores of N. grylli. As a result two antisera were obtained. Antiserum 1 reacted predominantly with spore walls on IFA slides and ultrathin sections (IEM). It also reacted with a broad spectrum of parasite and host cell proteins on WB. Antiserum 2 recognized polar filaments and walls of discharged spores in IFA and IEM tests. It did not react with undischarged spores or fat bodies of uninfected crickets and gave a comparatively weak reaction with those of the infected hosts. Hybridization of spleen cells of immune mice with murine myeloma cells resulted in several hybridoma clones. They produced Mabs, 5 of which were tested by IFA, IEM and WB. Mab 1BF3 recognized 55 kDa protein connected with polar filaments as it was clearly suggested by IEM and IFA. Mab 1BD9 recognized 25, 34, 43 kDa proteins from the fraction of membrane bound proteins of spore walls, the sites of their interaction with antigens being marked with uneven fluorescence (IFA) and by gold precipitates on spore walls (IEM). Mab 1BB9 reacted with 36, 45, 65 and 75 kDa proteins, which belong mainly to the fraction of membrane-bound spore proteins, and gave a weak fluorescence associated with spores. Mab 2AB3 recognized 44 and 60 kDa proteins from the fraction of soluble spore proteins, and Mab 2AD4 acknowledged a single protein of 55 kDa from the same fraction. The obtained antibodies add to the existing microsporidian antibody bank and can be used for further work of isolation, description and sequencing the microsporidian proteins in order to understand eventually their functions.

Zschokkella helmii n. sp. (Myxozoa: Myxosporea), a new parasite of marbled spinefoot Siganus rivulatus (Forsskal 1775), Red Sea, Egypt: light and transmission electron microscopy.

Zschokkella helmii n. sp., a new parasite of Siganus rivulatus from the Red Sea, Egypt, was described using light and transmission electron microscopy. However, the infection was severe; single "histozoic" plasmodium was encountered in the gallbladder wall. Spores are ellipsoid with 9-11 valvar striations. Spore mean length is 10.8 microm (10.0-11.0), while the spore mean width is 7.5 microm (7.0-8.0). Polar capsules are nearly round with a diameter of 2.2 microm (2.0-3.0) and have five filaments. Ultrastructure of the plasmodial wall and sporogenesis of the present species followed the usual pattern valid for most studied myxosporean species.

Myxobolus lubati n. sp. (Myxosporea: Myxobolidae), a new parasite of haffara seabream Rhabdosargus haffara (Forsskal, 1775), Red Sea, Egypt: a light and transmission electron microscopy.

A new myxosporean parasite, Myxobolus lubati n. sp., was described from the wall of the intestine of haffara seabream Rhabdosargus haffara (Forsskal 1775), Red Sea, Egypt. Macroscopic plasmodia of about 300 mum diameter were located in the circular muscle layer of the intestine. The spores were ovoid and sometimes ellipsoid and measured 9.8 x 7.2 mum. The shell wall of the spore was thickened at the posterior end and marked with 5-7 sutural markings. Polar capsules were equal and pyriform with three polar filament turns situated in the posterior half of the polar capsule. Polar capsules measured 4.2 x 1.6 mum. Histological evaluation of the infection revealed a slight distention of the intestinal layer of muscularis. Ultrastructure of the plasmodial wall and sporogenesis of the present species followed the usual pattern valid for most studied myxosporean species.

Electron microscope study of merogony preceding cyst formation of Sarcocystis sp. in roe deer (Capreolus capreolus).

Precystic merogony of Sarcocystis sp. was studied in roe deer fawns 33, 45, and 49 days postinoculation (pi) with 2 X 10(4)-10(5) sporocysts recovered from dogs. Single merozoites, but no meronts, were found 33 days pi in liver, spleen, and lymph nodes. Transforming merozoites and meronts were found in myofibroblasts, satellite cells, and endothelial cells of muscle tissue on 45 and 49 days pi; they were surrounded by two membranes. Typical coccidian merozoites differentiated simultaneously around an enlarged, lobed nucleus.

Ultrastructure of gamonts and gametes and fertilization of Sarcocystis sp. from the roe deer (capreolus capreolus) in dogs.

Gamonts of Sarcocystis sp. from the roe deer were examined in the intestine of dogs 10 h after inoculation. Early macrogamonts were limited by a three-membranous pellicle, and situated in a parasitophorous vacuole. Female sexual stages during fertilization, the macrogametes, were limited by five membranes, and microgametes were observed in the parasitophorous vacuole. The outer membranes of the microgamete and macrogamete fuse, and the nucleoplasm of the microgamte enters the cytoplasm of the macrogamete. No wall-forming bodies were observed in macrogamonts and macrogametes.

A comparative light and electron microscope study of the cysts of Sarcocystis species of roe deer (Capreolus capreolus).

Sarcocystis muscle cysts of naturally infected roe deer were examined by light and electron microscopy. Two types of thin- and thick-walled cysts could be distinguished by light microscopy, whereas by electron microscopy six types of cyst walls could be differentiated on the basis of the size and shape of protrusions of the primary cyst wall and by the presence or absence of fibrillar elements. The paper also discusses whether the cyst wall types are species related or represent cysts of different ages. The fine structure of merozoites and metrocytes of Sarcocystis of roe deer resembles that of other Sarcocystis species.

Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures.

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30-45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.

Eimeria tenella: localisation of rhoptry antigens during parasite-host cell interactions by a rhoptry-specific monoclonal antibody in PCKC culture.

A rhoptry-specific monoclonal antibody (mab) A4C6 was obtained by fusion of X63-Ag 8.653 plasmacytoma cells with splenocytes of BALB/c mice. Mab A4C6 reacted with three protein bands at about 94, 66 and 45 kDa under non-reducing conditions. Gel electrophoresis under reducing conditions separated these protein complexes into several protein fragments ranging between 20 and 200 kDa. Antigens recognised by mab A4C6 were concentrated in the rhoptry sacs located in the apical region of sporozoites as shown by immunoelectron microscopy. During parasite-host cell interaction and development in PCKC culture, mab A4C6 located rhoptry antigens in the parasitophorous vacuole space between intracellular sporozoites and the vacuole membrane.