Effect of tryptic or peptic digestion or mechanical isolation on the extraction of proteins, antigens, and ribonucleic acids from Sarcocystis muris bradyzoites.

Various methods have been used for the isolation of bradyzoites of Sarcocystis species. Using Sarcocystis muris as a model, the effect of 3 methods, trypsin digestion, pepsin digestion, and mechanical isolation, on the subsequent extraction of S. muris proteins, antigens, and RNA was examined. Although some quantitative differences were found among the proteins, antigens, and RNA extracted after the 3 isolation methods, qualitative differences were not evident. The overall isoelectric focusing protein profile showed approximately 30 bands in the pH range of 3-9 and was dominated by 5 bands with pI values of approximately 5.3, 6.3, 6.8, 7.0, and 7.4. In experimentally infected mice, antibodies were recorded from 35 days postinoculation (PI) until the end of examination (130 days PI). The S. muris RNA appeared to consist of at least 10 subunits in the range of 200-4,200 nucleotides. A Toxoplasma gondii DNA fragment specific for small subunit ribosomal RNA (rRNA) hybridized mainly to the S. muris 1,900-nucleotide subunit and a range of smaller subunits, and a probe specific for large subunit rRNA hybridized mainly to the 4,200-nucleotide subunit and a range of smaller subunits. All 3 methods of bradyzoite isolation gave good yields of intact messenger RNAs that showed similar in vitro translation curves.

Electrophoretic separation of the soluble proteins of different fractions of the sarcocyst of Sarcocystis fusiformis of buffalo (Bubalus bubalis).

The electrophoretic fractionation of the various compounds of the soluble proteins in the cyst wall (CW), cyst fluid (CF) and zoites (ZT) of the sarcocyst of Sarcocystis fusiformis from buffalo (Bubalus bubalis) was studied by polyacrylamide disc gel electrophoresis. The CW fraction was found to contain at least six components, and ZT and CF contained at least four components with varied Rf values. Each gel was subjected to the gel scanner, and the corresponding areas of the peaks along with the percentage of the proteins in the bands were calculated.

Biochemistry of the various fractions of sarcocysts of Sarcocystis fusiformis of buffalo (Bubalus bubalis).

A comparative biochemical study on the various fractions (cyst wall, cyst fluid and zoites) of the sarcocysts of Sarcocystis fusiformis from oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis) was carried out. The study included analysis for glycogen, glucose, pyruvic acid, lactic acid, total lipids, phospholipids, cholesterol, triglycerides and fatty acids. The pattern and the distribution of various biochemical constituents varied in the different fractions. The cell wall had the maximum concentration of glucose and phospholipids. Of all the fractions, cell fluid showed the highest contents of pyruvate and lactate, but with a higher level of pyruvate than lactate.

Interactions between Sarcocystis gigantea lectin and toxin-containing fractions in human lymphocyte cultures.

Sarcocystis gigantea extract (SGE) was separated by affinity chromatography into one lectin-containing fraction (SGL) that was mitogenic to mononuclear cells (MNC) and another that lacked this lectin activity. The SGL-depleted Sarcocystis extract (SGTF) contained the so-called Sarcotoxin, inducing only a slight increase in MNC proliferation. Furthermore, preincubation of MNC with SGTF for 60 min suppressed the mitogenic capacity of SGL by 60%-90%. The results presented indicate that SGTF interacts with human MNC differently than SGL, particularly by interfering with the mitogenic lectin. These findings suggest that SGL and SGTF may be involved in different immunomechanisms induced by the parasite.

Characterization and immunolocalization of the protein contents of micronemes of Sarcocystis muris cystozoites (Protozoa, Apicomplexa).

Affinity-purified polyclonal antibodies generated against micronemes obtained by the subcellular fractionation of Sarcocystis muris contain two major proteins of 16 and 17 kDa. Antibodies cross-react with microneme antigen of S. tenella and S. cuniculi but not with the S. sp. of wild boar (Sus scrofa). Microneme antigens could be traced by immunoelectron microscopy in metrocytes as early as 47 days postinfection and in gamonts of S. muris in cell cultures up to 4 h after inoculation.

[Amino acids and their metabolites in Sarcocystis fusiformis and S. gigantea cysts]. / Aminokisloty i ikh metabolity tsist Sarcocystis fusiformis i S. gigantea.

It has been established that cysts S. fusiformis and S. gigantea of parasites isolated from the esophagi of infected buffaloes and sheep have the identical set of free amino acids and their metabolites. These species differ from each other in 7 components of 34 studied that points to their metabolic closeness and to specific differences of sarcocysts from different hosts.

Histochemical study of Sarcocystis sp. intramuscular cysts in gastrocnemius and soleus of the cat.

Histochemical investigations of Sarcocystis microcysts found in two hindleg muscles of cats were carried out. Genus identification was based on the reinforced cyst membrane structure and its dimensions, the structure of the sarcocysts, and an electron microscopic survey of bradyzoite characteristics. The cyst membrane is partly contributed by the host myofiber, the characteristic histochemical features of which it retains. Materials adjacent to the limiting membrane make it appear thicker than it actually is, particularly when the PAS method as well as techniques for the demonstration of alpha-glycerophosphate dehydrogenase and ATPase activities are used. The ground substance occupying the parasitophorous vacuole is not amorphous and metabolically inert, but rather displays a fairly strong and definite ATPase activity, suggesting a trophic role in the support of metrocytes and zoites embedded therein. Cysts tend to adapt their biochemical characteristics to the particular metabolism of the muscle fibers in which they are located. All of these findings are discussed in terms of host-parasite relationships.

Biochemical and histochemical studies of the sarcocyst of Sarcocystis fusiformis of buffalo Bubalus bubalis.

The glycogen content and activities of alkaline and acid phosphatases of sarcocysts of Sarcocystis fusiformis from naturally infected Indian water buffalo (Bubalus bubalis) were determined biochemically and histochemically.

In vitro cultivation of meronts of Sarcocystis cruzi.

Several established cell lines were tested for their ability to support in vitro development of meronts of Sarcocystis cruzi. Sporozoites penetrated bovine monocytes (BM), bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney cells and mouse macrophages, but developed to meronts in BM and CPA only. Sporozoites developed to large meronts that contained approximately 180-350 merozoites, whereas merozoites formed small meronts with 50-100 merozoites. Mature large meronts were present at 18-86 days after inoculation (DAI) in BM and at 16-72 DAI in CPA. Small meronts were present at 23-115 and 23-91 DAI in BM and CPA. Considerably more merozoites developed in CPA than in BM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that merozoites harvested at 36 and 48 DAI each had 1 unique protein as well as numerous common proteins.

Biochemistry of the sarcocyst of Sarcocystis fusiformis of buffalo Bubalus bubalis.

Sarcocysts of Sarcocystis fusiformis from oesophageal muscles of naturally-infected Indian water buffalo (Bubalus bubalis) were analysed for total lipids, phospholipids, cholesterol, fatty acids and glycerides and total protein. Protein and phospholipids constituted the major portion of the sarcocyst. Acetylcholinesterase and glutamate-oxalo-acetate transaminase activities when assayed were higher than glutamate-pyruvate transaminase in sarcocysts.

Structure, isolation, and protein composition of the pellicle of Sarcocystis muris cystozoites (Protozoa, Coccidia).

The molecular organization of the Sarcocystis muris cystozoite pellicle has been investigated by freeze-fracture electron microscopy and by electrophoresis of the proteins of isolated pellicles. Freeze-fracture revealed a highly ordered organization of the inner membrane complex similar to the one described in other coccidian zoites. Purification of pellicles was achieved by French Press homogenization followed by sucrose gradient floatation. Electron microscopy of the pellicle fraction demonstrated the partial preservation of the triple-membrane structure whereas freeze-fracture showed the disorganization of the particle arrangements of the inner membrane complex. The SDS-PAGE of the fraction revealed a complex protein composition with one major protein of 31,000 daltons, not labeled by lactoperoxidase-catalyzed surface iodination of living cystozoites.

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.

Cytochemical studies on nuclear DNA of four eucoccidian parasites, Isospora (Toxoplasma) gondii, Eimeria tenella, Sarcocystis cruzi and Plasmodium berghei.

Feulgen-pararosaniline (SO2) staining was performed on stages in the life-cycle of Isospora (Toxoplasma) gondii, Eimeria tenella, Sarcocystis cruzi and Plasmodium berghei. The fluorescence emission of the stained DNA in nuclei of these stages was examined and compared with absorption microscopy measurements at 560 nm (green light) of the same specimens. Accurate identification of single cells, and especially discrimination between young schizonts and young gamonts was difficult after Feulgen staining. To overcome this problem preparations were first stained with Giemsa and the cells of interest precisely located with the aid of an England finder. The same preparations were then hydrolysed and stained with Feulgen-pararosaniline (SO2), after which the previously identified cells were investigated. The DNA distribution after Feulgen staining corresponded with the shape of nuclei after Giemsa staining. DNA was present in all investigated stages of the four parasites, including macrogamonts of I. (T.) gondii and E. tenella and peripheral blood gamonts of P. berghei. Macrogamonts could be recognized, even at a stage at which they can hardly be differentiated from young schizonts in Giemsa-stained preparations, by their typical distribution pattern of Feulgen fluorescence. Feulgen fluorescence was more granular and confined to the peripheral region of the nucleus, leaving a distinct nucleolus unstained. The horseshoe-shaped nuclei typical of macrogamonts could also be observed in some second generation merozoites of E. tenella, indicating that these merozoites are already sexually differentiated. The relationship between the present cytochemical observations and the ultrastructure of the four investigated parasites is discussed.

Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa, Coccidia).

A subcellular fractionation procedure has been developed, which allowed the recovery of purified fractions of micronemes, dense granules and pellicles from Sarcocystis tenella zoites. As expected, sodium dodecyl sulphate polyacrylamide gels electrophoresis of the pellicles showed a fairly heterogenous protein content. In contrast, only one major omponent (42 000) daltons) was found in the dense granules, whereas two major bands (20 000 and 22 000 daltons) were observed for the micromemes. These characteristic proteins were also major components of the whole zoite and might have important functions in the physiology of the organism.

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.