Protein analysis of Theileria sergenti/buffeli/orientalis piroplasms by two-dimensional polyacrylamide gel electrophoresis.

Proteins of the piroplasms of Theileria sergenti, T. buffeli and T. orientalis were analysed by two-dimensional polyacrylamide gel electrophoresis. Protein spot patterns of T. buffeli and T. orientalis were identical except for a few minor proteins, whereas spot patterns of two T. sergenti stocks were differentiated from those of T. buffeli and T. orientalis by a characteristic set of proteins including a major protein of molecular weight 33-34 kDa. This result indicates that Japanese T. sergenti can be phenotypically distinguishable from European and Australia Theileria species; T. orientalis and T. buffeli.

Electrophoretic characterization of chromosomal DNA from two microsporidia.

Spores of two microsporidia, Nosema pyrausta (from the European corn borer, Ostrinia nubilalis) and N. furnacalis (from the Asian corn borer, O. furnacalis) were harvested from laboratory-reared O. nubilalis caterpillars and purified by centrifugation through Percoll. Conditions permitting in vitro germination were defined for both species and found to be different. N. pyrausta spores were incubated in 0.1 N KOH for 30 min, recovered by centrifugation, and resuspended in 1 ml of an equal mixture of 1% low melting point (LMP) agarose and L-15B medium at 37 degrees C to induce germination. N. furnacalis spores were first washed in 10 mM Na2EDTA in 1 mM Tris base, pH 7.5, exposed to 0.01 N KOH in 0.17 M KCl for 30 min, centrifuged, and germinated in 1 ml of an equal mixture of 1% LMP agarose and 0.17 M KCl in 10 mM Na2EDTA (pH 8), at 37 degrees C. Eighty to 90% of the spores of each species germinated. Germinated spores were pipetted into a casting mold. Before electrophoresis, agarose blocks were incubated 48 hr at 50 degrees C in 10 mM Tris base/100 mM Na2EDTA, pH 7.8, with 1 mg/ml proteinase K and 1% N-laurylsarcosine to release the chromosomal DNA from sporoplasms. After pulsed-field electrophoresis, ethidium bromide staining revealed 13 chromosomal bands ranging in size from 1390- to 440-kb pairs and 1360- to 440-kb pairs in N. pyrausta and N. furnacalis, respectively. The difference in size estimates of corresponding chromosomes in the two species was not more than 60-kb pairs.

Demonstration of actin in the protozoon--Gregarina.

Actin was identified in the motile trophozoites of Gregarina by indirect immunofluorescence microscopy. Ultrastructurally actin was found by immunogold labelling to be associated with the internal cytomembranes of the ectoplasmic folds and the general ectoplasmic region below the folds. Western blotting with antibodies to actin identified polypeptides with molecular weights around 43kD and 98kD in non-ionic detergent extracted trophozoites. The possibility of two distinct forms of cytoplasmic actin (43kD and 98kD) was considered.

Phenotypic characterization of Theileria parva schizonts by two-dimensional gel electrophoresis.

Biosynthetically radiolabelled Theileria parva schizonts were purified from bovine lymphoblastoid cells and their proteins were analyzed by two-dimensional gel electrophoresis and autoradiography. The protein spot patterns of schizont proteins from three stocks of T. parva parva indicated that the phenotypic diversity among the stocks was minimal, with the Mariakani and Uganda stocks being identical and the Muguga stock showing only a few differences in minor spots. Comparison of the spot patterns of schizonts of three T. parva subspecies showed that T.p. parva and T.p. bovis differed in only one protein and thus could not be reliably distinguished on the basis of their protein differences. However, T.p. lawrencei showed several protein differences and could be distinguished easily from the other subspecies. Differences in schizont-protein spot patterns were also seen when two different cell lines were infected with the same Theileria stabilate, when one cell line was infected with two different stabilates of the same stock and when uncloned and cloned infected cell lines were used. These results suggest the possibility that selection of phenotypically different parasites could occur in vivo or in vitro.

Isolation of Theileria parva schizonts from infected lymphoblastoid cells.

This study set out to develop a rapid method for the isolation of schizonts from Theileria parva-infected bovine and buffalo lymphoblastoid cells. Parasitized lymphoblastoid cells were lysed by treatment with the cytolytic toxins, aerolysin and Ah-1 hemolysin, produced by Aeromonas hydrophila, and the schizonts were separated by Percoll density-gradient centrifugation. Light and electron microscopic examination showed that the isolated schizonts from lymphoblastoid cells infected with T. parva (Muguga) retained their normal morphology and were essentially free from host cell components. The schizonts also retained antigens as recognized by a series of anti-schizont monoclonal antibodies. The concentrations of toxin and Ficoll 400 in the lysis buffer which gave optimal cell lysis varied for 10 different infected cell lines tested.

Immuno-histochemical and ultrastructural characteristics of a cyst-forming sporozoon associated with encephalomyelitis and myositis in dogs.

Light-microscopy, including immuno-histochemical staining, and transmission and scanning electron microscopy were applied for the characterization of a Toxoplasma-like parasite associated with encephalomyelitis and myositis in dogs of the boxer breed. Most parasites were tachyzoites, occurring in clusters, up to 60 micron in diameter, in parasitophorous vacuoles of host cells. Cyst stages with a definite cyst wall were few, but could be demonstrated in all cases. Immunostaining with antiserum against Toxoplasma gondii was negative, while both tachyzoites and bradyzoites were stained with serum from affected dogs. Ultrastructurally, the parasite was characterized by a great number of rhoptries, few amylopectin granules, lack of micropores in the tachyzoites, and the occurrence of numerous dense bodies in the bradyzoites. Numerous intravacuolar tubules were present in the space of the parasitophorous vacuole. The cyst wall resembled that of T. gondii. Until a definite host is found, the parasite may be regarded as an "orphan", cyst-forming sporozoon that is ultrastructurally different from other known protozoan parasites. It has a light-microscopical similarity to T. gondii but is antigenically different.

Two-dimensional electrophoretic analysis of spore proteins of the Microsporida.

Microsporida are potentially useful as biological control agents for insects of economic and medical importance. Prior to their responsible use, however, an accurate and reliable means of identification to the species and subspecies level is required. Current methods used for identification are not adequate, due to variability of identifiable characters and to the occurrence of dimorphism. Recently, progress has been made in the use of biochemical characteristics to support the more traditional methods of distinguishing between morphologically similar species. We report on an improved method of characterization of microsporidan spore proteins, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). This method increased the number of spore polypeptides resolved from Nosema locustae spore protein extracts 2-3-fold over 1-dimensional PAGE. Also, each of the 2D-PAGE spore protein fingerprints of the species examined, namely Nosema locustae, Nosema bombycis, and Vairimorpha necatrix, were unique and differences in their spore protein composition were easily determined. The major structural proteins of Nosema locustae spores co-electrophoresed with alpha and beta tubulin from calf brain and had similar pI and molecular weight values as reported for tubulin in other species. Each species' 2D-PAGE fingerprint contained a few polypeptides that were present in relatively high concentration and these polypeptides may represent the major proteins of the structural components of the spore.

Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites.

A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria parva, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetically radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/mmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. parva sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens.

Determination of nuclear DNA of five eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella, E. acervulina and Plasmodium berghei, with special reference to gamontogenesis and meiosis in I. (T.) gondii.

DNA contents of individual stages of Isospora (Toxoplasma) gondii and other Eucoccida were measured after Feulgen-pararosaniline (SO2) staining either by direct microfluorometry or by scanning of microphotographic negatives. Frequency distributions were analysed using a computer program based on a mathematical model describing cell division. All stages of I. (T.) gondii, except fertilized macrogametes (2c), contained a haploid amount of DNA (1c), indicating that meiosis in I. (T.) gondii occurs during sporogony. Microgametes possessed 3.3% DNA in excess, presumably mitochondrial DNA. Nuclei of M2- and M3-merozoites differed in two characteristics: a small but distinct nucleolus was observed in almost 50% of the M2-merozoites but in none of the M3-merozoites; all M2 merozoites were strictly haploid, while all M3-merozoites were synthesizing DNA (17% above the haploid value). It may be concluded that all M2- and M3-merozoites are already sexually differentiated, i.e. are macro- and microgamontoblasts, respectively. DNA synthesis necessary for the development of the microgamont starts already in the microgamontoblast stage (M3-merozoite). M2-merozoites macrogametes, synthesize 11% extra DNA before fertilization, (after fertilization an extra amount of 12% of the diploid value was found), probably by amplification of genes for proteins which are needed for rapid maturation and later sporogony. Essentially parallel results have been found in Eimeria tenella and in crescent cystozoites of Sarcocystis cruzi. Absolute DNA values in representatives of the Eucoccida have been estimated as follows (10(-15) g): I. (T.) gondii, 96; E. tenella and E. acervulina, both 75; S. cruzi, 216; Plasmodium berghei, 27. The value of the estimation of total haploid amounts as a tool in taxonomy of Eucoccida is discussed.

The three cortical membranes of the gregarines. I. Ultrastructural organization of Gregarina blaberae.

Gregarines, parasitic protozoa of invertebrates, possess a highly differentiated cell surface, with three cortical membranes and associated structures. Transmission electron microscopy and freeze-fracture reveal the presence of two cytomembranes lying uniformly under the plasma membrane. The density and the distribution of the intramembraneous particles (IMPs) in the plasma membrane of Gregarina blaberae are similar to those reported for other eukaryotic cells. The IMP density is lower in the cytomembranes than in the plasma membrane. The distribution of IMPs in the different fracture faces of the two cytomembranes suggests that they are in topological continuity, forming either side of a flattened vesicle or cisterna. The sizes of the cytomembrane IMPs show a high variability. The nature of the IMPs, both for the plasma membrane and the cytomembrane, is discussed with regard to the integral proteins and glycoproteins of the ghost. The cell surface of G. blaberae exhibits numerous longitudinal folds with three types of cortical membrane-associated structures: 12 nm filaments, an internal lamina, and homogeneous structures described as 'rippled dense structures'. The 12 nm filaments, running under the cytomembranes along the longitudinal axis of each fold, exhibit the properties of intermediate filaments. Their distribution in mature cells and during the growth process suggests a participation in cell surface morphogenesis. The internal lamina, also localized under the cytomembranes, would stabilize each fold and assure a scaffolding function between the numerous folds. The rippled dense structures, settled on the external cytomembrane, show a regular distribution at the top of each fold. The membrane-associated structures are discussed with regard to the gliding movement mechanism.

Surface membrane components of Theileria spp piroplasms and associated complement fixation activity.

Theileria parva (parva) piroplasms were examined for complement fixation (CF) activity with sera from cattle immune to T parva (parva) (Muguga). The piroplasms were found to be highly antigenic, and the antigens responsible for this CF reactivity were completely insoluble in aqueous media. The antigens were traced through a variety of fractionation procedures and the fractions examined by electrophoresis in the presence of sodium docedyl sulphate. Antigenically active preparations were found always to contain two polypeptides having molecular weights of 21,500 and 34,000. When intact piroplasms were surface labelled with iodine 125 these same two polypeptides were the most heavily labelled, which suggested that the antigens responsible for the CF reactivity were plasma-membrane bound. Electrophoretic analysis of fractions containing plasma membrane components from piroplasms of three species of Theileria (T parva [parva], T mutans and T taurotragi) enabled interspecific differentiation to be made. Three stocks of T parva (parva) could not be differentiated by this means; such different stocks, from various locations within East Africa, appeared to be identical chemically.

A cyst-forming Eimeriina found in the kangaroo imported from Australia.

One pair of grey kangaroos was imported from Australia in July 1974. One month after arrival they successively died of serious intestinal bleeding. On biopsy, the epithelium was destroyed and replaced with necrotic degeneration with numerous dot-like flecks of bleeding from the stomach till the jejunum. On histopathological examinations, the epithelium was composed of swollen glandular cells which form giant cells with eccentrically displaced nuclei. The cell forms a cyst which develops solitarily and gathers together to form a racemous shape. These cysts are bound to the thick wall and the parasites are found in the vesicular space. Several sequences of the parasite growth are demonstrated. The parasites migrated initially into the cytoplasm and then to the nucleus of the glandular cell in which they grow, then the cyst is formed and finally the parasites flow out to penetrate again into normal cells. The parasite cell changes to crescent- or spindle-shape as a tachyzoite. The relative nucleic acid content of the parasite in the host cell or its nucleus is measured by spectrophotometry and fluorometry. The data suggest that the parasites are reproduced in intestinal glandular cells of the kangaroo.

[Dionisia bunoi n. g. n. sp., Haemoproteidae parasite of the microchiropteran bat Hipposideros cyclops in Gabon (author's transl)]. / Dionisia bunoi n. gen. n. sp. Haemoproteidae parasite du microchiroptère Hipposideros cyclops au Gabon.

Dionisia bunoi ng. g., n. sp. is characterized by:--a) sexual dimorphism of gametocytes, macrogametocyte of the falciparum type, microgametocyte of the type malariae:--b) schizonts developing in the lumen of liver blood vessels inside a greatly hypertrophied host cell; their size remains moderate and their cytoplasm is not intensely basophilic as is usually in the young stages of Haemosporidia of Mammals. The genus has morphological characters in common with each of the 4 other genera of Haemoproteidae of Mammals. The phyletic hypothesis concerning the morphology of gametocytes and schizonts suggests that Bioccala and Dionisia are primitive.

The microsporidian spore invasion tube. The ultrastructure, isolation, and characterization of the protein comprising the tube.

The extrusion apparatus of the microsporidian parasitic protozoan Nosema michaelis discharges an invasion (or polar) tube with a velocity suitalbe for piercing cells and injecting infective sporoplasm. The tube is composed of a polar tube protein (PTP) which consists of a single, low molecular weight polypeptide slightly smaller than chymotrypsinogen-A. Assembled PTP tubes resist dissociation in sodium dodecyl sulfate and brief exposures in media at extreme ends of the pH range; however, the tubes are reduced by mercaptoethanol and dithiothreitol. When acidified, mercaptoethanol-reduced PTP self-assembles into plastic, two-dimensional monolayers. Dithiothreitol-reduced PTP will not reassemble when acidified. Evidence is presented which indicates that PTP is assembled as a tube within the spore; that the ejected tube has plasticity during sporoplasm passage; and, finally, that the subunits within the tube polymer are bound together, in part, by interprotein disulfide linkages.

[Amino acid makeup of the oocyst proteins of some species of coccidia of hens]. / Aminokislotnyi sostav belkov ootsist nekotorykh vidov koktsidii kur

The composition of amino acids of sporocysts, membranes of oocysts and oocysts of four species of Coccidia from hens is reported. A considerable figgerence was found to exist in the quantitative composition of amino acids in oocysts of different Coccidia species and in different structures of oocysts. It is necessary to carry out a search of preparations breaking the inclusions of some amino acids into the protein molecule of oocysts of Coccidia from hens.

Observations on a feline coccidium with some characteristics of Toxoplasma and Sarcocystis.

Two morphologically different cysts were found in skeletal muscles of mice inoculated with fecal material from a stray cat containing Isopora-type oocysts. The most common cyst contained bradyzoites resembling those of Toxoplasma and resulted from an oocyst measuring 11 times 13 mum which appeared to be identical to that of Toxoplasma. The other cyst, observed in only a few mice, contained bradyzoites resembling those of Sarcocystis, but the oocyst or sporocyst that gave rise to it was overlooked and apparently lost. Two more strains of the parasite resembling Toxoplasma were found in feces of stray cats. When inoculated into mice, the oocyst of this parasite routinely produced chronic infection and formed cysts similar to Toxoplasma in skeletal muscles and occasionally in the central nervous system. The majority of infected mice developed Toxoplasma antibody, but only to low titers. Cats fed carcasses of infected mice remained healthy and shed nonsporulated oocysts following a prepatent period of about 5 days. Cats did not develop Toxoplasma antibody. There was little or no cross immunity between the parasite and T. gondii in cats or mice. Transmission of the parasite between mice by the cyst stage normally was not possible; however, mice inoculated with cortisone acetate did become infected when inoculated with cysts. In other laboratory animals inoculated orally with the oocyst asymptomatic infection was detected in 3 species of rats, in guinea pigs and in dogs, but not in monkeys, pigeons or Japanese qualis. Fluorescent antibody tests on human sera failed to provide evidence of natural human infection with the parasite.

Hammondia hammondi gen. nov., sp.nov., from domestic cats, a new coccidian related to Toxoplasma and Sarcocystis.

Hammondia hammondi gen.nov.,sp.nov (Eimeriorina:Sarcocystidae) is described as an obligate heteroxenous protozoon of domestic cats (final host) and laboratory mice (experimental intermediate host). Oocysts from the final host are infectious only for the intermediate host; and cysts from the intermediate host are infectious only for the final host. Intracellular cysts develop principally in striated muscle of mice that ingest oocysts, with a few cysts in the brain and perhaps elsewhere. Cysts are without septa or radial spines; bradyzoites are slender, there is no evidence of metrocytes. Cysts are not infectious for mice. After the ingestion of cysts by cats, a multiplicative cycle precedes the development of gametocytes in the epithelium of the samll intestine. Oocysts are shed unsporulated, sporogony is outside of the host, resulting in two sporocysts with four sporozoites each. Oocysts of the species average 11 x 13 mum. The prepatent period i 5s 5 to 8 days, and oocyst shedding persists for 10 to 28 days followed by immunity. Cysts in skeletal muscle measured between 100 and 340 mum in length and 40 and 95 mu-m in width. Experimental intermediate hosts are laboratory mice, rats, hamsters, guinea pigs, Peromyscus and Mastomys. Some of the intermediate hosts develop low levels of antibody and some cross-immunity against Toxoplasma; however, this has not been observed in cats.