Use of mouse macrophage cell lines for in vitro propagation of Toxoplasma gondii RH tachyzoites.

In most laboratories, Toxoplasma gondii is maintained in mice and is studied in vitro using nonlymphoid cell lines or primary mouse macrophages. In this study, three rapidly dividing mouse macrophage cell lines (J774 A.1, P388D1, RAW264.7) were evaluated for their suitability for studying the RH strain of T. gondii. For comparison, tachyzoites were also grown in two slowly dividing epithelial cell types: a rat lung cell line (L2) and a bovine turbinate cell line (BT). Various inocula of T. gondii were added to the above cells and tachyzoites were harvested from the culture supernatants after 2-8 days of infection. The mouse macrophage cell lines supported rapid growth of T. gondii RH allowing up to a 300-fold increase of the inoculum in 2-4 days. L2 and BT supported slower growth of T. gondii (10- to 90-fold increase of inoculum in 5 to 8 days) and, thus, may be more suitable for assessment of host cell-parasite interactions and drug activity. Toxoplasma gondii RH isolated from each of the cell cultures described were able to multiply in all cell types used. Protein profiles of whole tachyzoite isolated from mice or cell cultures and protein profiles of the corresponding soluble and membrane fractions of the intraphagosomal membrane network were similar as seen after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In mice, intraperitoneal injection of 10(6), 10(5), and 10(3) tachyzoites isolated from the cell cultures or from infected mice caused death after 4, 5, and 8 days, respectively, indicating that parasites grown in vitro retained virulence.

Characterization of the lipid content of Toxoplasma gondii rhoptries.

Quantitative and qualitative analysis of lipids has been performed on a rhoptry fraction purified from Toxoplasma gondii tachyzoites. The lipid to protein ratio was estimated to be 0.26. The cholesterol to phospholipid ratio was unusually high at 1.48. Phosphatidylcholine was the major phospholipid; phosphatidylserine, phosphatidylinositol and sphingomyelin were absent whereas significant amounts of phosphatidic acid and lysophospholipids were found. This pelicular composition could be related to functional involvement of the organelle in host-cell invasion.

Characterization of the protein contents of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies.

A subcellular fractionation procedure has been established to isolate the rhoptries of Toxoplasma gondii tachyzoites on a self-generating Percoll gradient. The rhoptry fraction also contains dense granules. Monoclonal antibodies have been raised against the fraction and used to identify major proteins in the organelles by immunoelectron microscopy and Western blotting. Six major rhoptry proteins (60.5 kDa, Pi 5.8; 55, 57, 59, 60 kDa, all of Pi over 8; 42 kDa, Pi 4.8) and 3 dense granule proteins (30 kDa; 28kDa, Pi 7.5; 27 kDa, Pi 4.5) together with 5 other proteins of 57, 90, 120, 168, 220 kDa that have been located in the rhoptry area by indirect immunofluorescence have been identified.

Localization of lectin-binding sites and sugar-binding proteins in tachyzoites of Toxoplasma gondii.

Lectins and neoglycoproteins labeled with colloidal gold particles were used for the ultrastructural localization of carbohydrate residues and sugar-binding sites, respectively, in thin sections of tachyzoites of Toxoplasma gondii embedded in the Lowicryl K4M resin. Incubation of the sections in the presence of gold-labeled Canavalia ensiformis (Con A), Arachis hypogaea (PNA), Ricinus communis I (RCA I), Triticum vulgaris (WGA), and Limax flavus (LFA) agglutinins showed significant labeling of the rhoptries. However, no labeling of the parasite's surface was observed. Incubation of tachyzoites in the presence of gold-labeled albumin-N-acetyl-D-glucosamine or albumin-galactose, but not in the presence of albumin-mannose, led to labeling of the rhoptries in a pattern similar to that observed with the lectins. The results obtained are discussed in relation to the possible role played by secretion of rhoptry macromolecules during the process of T. gondii-host cell interaction.

Isolation of the pellicle of Toxoplasma gondii (Protozoa, Coccidia): characterization by electron microscopy and protein composition.

Two methods were compared for the isolation of the Toxoplasma gondii pellicle: centrifugation using a discontinuous sucrose gradient and affinity purification by microspheres activated with protein A. Electron microscopy showed that these two means of purification gave similar results, although better yields were obtained with the former than with the latter. Proteins of membrane fractions isolated using sucrose gradient were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands in the molecular-weight regions of 19, 22, 35, and 79 kDa appeared to be associated with the T. gondii pellicle.

Analysis of Toxoplasma gondii proteins after Triton X-114 solubilization and hydrophobic chromatography.

The distribution of the surface proteins of Toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Two polypeptides with 15,000 and 70,000 distributed in both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities. Two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of T. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.

Characterization of a family of rhoptry proteins of Toxoplasma gondii.

A monoclonal antibody (McAb 4A7) raised against a rhoptry enriched subcellular fraction of Toxoplasma gondii tachyzoites reacted in immunofluorescence studies with rod-like organelles located in the anterior part of the organisms and gave specific labeling of rhoptries in immunoelectron microscopy. On immunoblots, two major proteins of 55 and 60 kDa were identified by McAb 4A7. Similar results were obtained both by immunodetection and immunoblotting with tachyzoites, bradyzoites and sporozoites. Pulse chase analysis of [35S]methionine labeled tachyzoites demonstrated that the 55 and 60 kDa rhoptry proteins derived from a 66-68 kDa doublet which was processed approximately 30 min after biosynthesis. Two other monoclonal antibodies (McAb 2F8, McAb 2H3) respectively specific for rhoptry proteins of 55 kDa having a 66 kDa precursor and 60 kDa having a 68 kDa precursor were also obtained; we suggest that they recognize separately the two components of the 55-60 kDa rhoptry protein family of Toxoplasma.

Immunocytochemical localization of actin in Toxoplasma gondii.

The localization of actin in the trophozoites of Toxoplasma gondii was examined by means of immunogold staining for electron microscopy. The thin-sectioned specimens were incubated with the IgG fraction of polyclonal antibodies raised against Ascaris body-wall smooth-muscle actin following colloidal gold-conjugated protein A. Electron-dense gold particles were confined to the anterior polar region in the trophozoites: they were found in the conoid, preconoidal rings, possibly in the polar ring, and in the space between the anterior terminal of the inner membrane complex and conoid. The present experiments also suggest interactions of actin with subpellicular microtubules, leading to speculation that the association of actin with microtubules provides a link to myosin, a potential source of power for microtubule-dependent movements.

Detection and localization of actin in Toxoplasma gondii.

The immunological cross-reactivity of the protein from whole-cell extracts of Toxoplasma gondii with antisera against actins from chicken-gizzard smooth muscle and from Ascaris body-wall muscle was demonstrated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE) and immunofluorescence studies. A protein of about 42 kDa, with the same mobility as that of rabbit smooth-muscle actin in SDS-PAGE showed cross-reactivity with anti-actin antisera on the electroblotted nitrocellulose sheet. Indirect fluorescent antibody staining of the parasites clearly showed that actin localized strongly in the anterior third of the organism in both extra- and intrahost cellular stages. From both a biochemical as well as structural point of view, the results obtained in the present experiments might explain the locomotive features of the parasite such as twirling and sliding, with most of the activity at the anterior end.

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study.

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.

Isolation and characterization of a monoclonal anti-P30 antibody resistant mutant of Toxoplasma gondii.

Of the possible iodine-labelled Toxoplasma gondii surface proteins, P30 (apparent Mr 30,000) is the principal one recognized by acute and convalescent anti-toxoplasma sera. This protein which comprises from 3 to 5% of the total parasite protein was used to raise a panel of parasiticidal monoclonal anti-P30 antibodies. One of these monoclonal antibodies was able to select a resistant mutant from a large population of chemically mutagenized wild-type P strain parasites. This mutant retained the wild type sensitivity to other non-P30 parasiticidal monoclonal antibodies as well as polyclonal anti-P30 rabbit sera. Analysis of surface radioiodinated wild type and mutant parasites showed that the mutant had a quantitative reduction in the amount of P30. A comparison of surface biotin labelled wild type and resistant parasites by two dimensional electrophoresis showed that the mutant lacked one and possibly two of several proteins that make up wild type P30. Western blot analysis indicated that the mutant was devoid of antigenically reactive P30. These findings further support the hypothesis that antigenic variants of T. gondii can be induced and may involve the major surface membrane antigens of the parasite.

Histoenzymological study of selected dehydrogenase enzymes in Pneumocystis carinii.

The metabolic activity of Pneumocystis carinii cysts was studied histochemically by a tetrazolium dye technique to assess substrate-specific dehydrogenase activity. Lactate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase produced moderate-to-strong reactions in the cysts, whereas glucose-6-phosphate dehydrogenase had little if any reactivity. These results suggest that pneumocystis cysts have some of the enzymes necessary for glycolysis, Krebs cycle activity, and intermediary protein metabolism. These studies provide a method of directly assessing metabolic pathways in P. carinii which circumvents the uncertainties of specificity inherent in previous investigations with partially purified suspensions.

Immune response to Toxoplasma gondii--alteration of antigen specificity of peripheral blood leukocyte proliferative responses in acute toxoplasmosis.

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoite extract was chromatographed on Sephadex G-200, and one main peak (93,000 M.W.) and three small peaks (greater than 160,000 M.W., 110,000 M.W., and 20,000 M.W.) were eluted. Toxoplasma-specific proliferative T cell responses of peripheral blood leukocytes (PBL) from a patient with acute toxoplasmosis caused by accidental injection of tachyzoites of the protozoa were sequentially examined by using these fractioned antigens. As early as one week after the accidental injection of the protozoa, significant proliferative responses of PBL could be detected. The reaction of proliferative T cells was observed occurring mainly with Fr. II antigen. Then T cells began to respond to Fr. I and III in addition to Fr. II 3 weeks after the injection. Thus, expansion of antigen specificity in Toxoplasma-specific T cell responses was observed at the initial stage of acquired acute toxoplasmosis.

[Use of immunoenzyme analysis in the diagnosis of toxoplasmosis]. / Ispol'zovanie immunofermentnogo analiza v diagnostike toksoplazmoza.

The method for the preparation of antigen for ELISA and the technique of this assay are described. The comparative data obtained in the examination of 50 donors and 95 children suspected to have toxoplasmosis, carried out with the use of immunofluorescence and ELISA, are presented. A good correlation between the results of the both tests has been shown.

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.

[Rheumatoid factor activity as a disturbing factor in the serological diagnosis of specific IgM antibodies]. / Rheumafaktoraktivität als Störfaktor bei der serologischen Diagnostik spezifischer IgM-Antikörper.

Rheumatoid factors (RF) are autoantibodies mainly directed against autologous IgG. They belong at most to the IgM class antibodies. It is demonstrated at groups with unsolved hepatitis B, rubella, syphilis and toxoplasmose infection that RF do occur not rarely at these patients even without rheumatoid arthritis. This is probably due to stimulation by antigen-IgG-complexes. During serologic detection of specific IgM antibodies they present an antigen independent mu-specificity. So the test for specific IgM might even loose its diagnostic and possibly therapy indicating value. It is shown how the disturbance by RF can be calculated after adsorption with aggregated IgG. Also RF can be titrated by an enzyme immunoassay (ELISA). With IgG coated latex particles RF can be eliminated prior to the IgM-test. Solid phase techniques which are applied with enzyme-coupled antigen instead of marked anti-IgM cannot be disturbed by RF significantly.

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.