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PubMed1997

Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study.

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo.

Amylopectin↗
PubMed1998

Neurons in primary culture are less efficiently infected by Toxoplasma gondii than glial cells.

Infection by Toxoplasma gondii is asymptomatic, leading to an immune response that controls the disease. In immune-compromised patients, however, quiescent cysts can reactivate, leading to toxoplasmic encephalitis. We studied the infection of cells of the central nervous system in an attempt to understand the process leading to this complication. Primary cultures of rat hippocampal glial cells and neurons were infected with the virulent strain RH and examined by immunofluorescent microscopy after fixation of cells and staining with antibodies specific to the different cell types. After 24 h of infection, glial cells were highly infected and showed active division of the parasite. Neurons, on the other hand, were much less efficiently infected than glial cells, but actual penetration of the parasites was demonstrated by confocal microscopy. Whereas glial cells contained vacuoles with several parasites, the vacuoles observed in neurons usually contained one parasite or, rarely, two, indicating that the parasites inside neurons did not undergo active division. This was corroborated by determination of the incorporation of [3H]-uracil. Little is known about the mechanism of neuronal infection by Toxoplasma. The experimental setup used in this study should help to improve our understanding of neuronal infection and bring insight into the physiopathology of toxoplasmic encephalitis.

Animals↗
PubMed1999

Performance of a Western blot assay to compare mother and newborn anti-Toxoplasma antibodies for the early neonatal diagnosis of congenital toxoplasmosis.

The aim of this study was to retrospectively evaluate the performance of a Western blot assay to compare mother and newborn anti-Toxoplasma gondii antibodies for the early neonatal diagnosis of congenital toxoplasmosis. Since specific anti-Toxoplasma IgM or IgA is detected inconstantly at birth in the neonate, the diagnosis of congenital toxoplasmosis is often delayed until 6-9 months, after IgG titers have been observed persistently. In this study, 81 paired samples from 60 mother/child pairs were tested for IgG and IgM patterns. All mothers had (or were strongly suspected to have) acquired toxoplasmosis during pregnancy. Specific IgM and IgA were simultaneously detected by immunocapture tests, and IgG was titrated. A serological and clinical follow-up of infants was conducted during the first year of life until the diagnosis of congenital toxoplasmosis could be either confirmed or ruled out. Seventeen of the 60 newborns were congenitally infected. Specific IgM or IgA was detected by immunocapture at birth in 76.5% and 70.6% of cord sera from infected neonates, respectively, with an equal specificity of 77.5%. Comparative Western blot allowed the detection of neosynthesized IgG and IgM in the cord blood of 50% and 78.6% of infected infants, respectively, with a specificity of 100%. The combination of IgA and IgM immunocapture tests, the analysis of IgG and IgM Western blot patterns, and the combination of both techniques allowed the detection of 94%, 94%, and 100% of cases within the first 3 months of life, respectively. In conclusion, Western blotting seems to be a useful complementary tool for the early postnatal diagnosis of congenital toxoplasmosis.

Animals↗
PubMed1999

Cell-culture system for continuous production of Toxoplasma gondii tachyzoites.

The aim of this study was to identify a sustainable cell line and culture method that could continuously provide a sufficient quantity of Toxoplasma gondii tachyzoites to serve the needs of a general hospital laboratory. Three continuous cell lines (HeLa, LLC and Vero) and three cell-culture methods (culture in conventional flasks, culture in membrane-based flasks and an automated culture system) were investigated. In multiplicity-of-infection and time-course experiments, HeLa was the cell line of choice. Harvests from HeLa cells had significantly higher tachyzoite yields than those from LLC cells (P<0.00005) or Vero cells (P<0.05). Membrane-based flasks gave higher yields (6.15x10(6) tachyzoites/ml) than conventional flasks (1-2x10(6) tachyzoites/ml) initially, but these were not sustained. The automated cell-culture system was unsuitable for parasite culture. Continuous passage in 25 cm2 flasks was successful, yielding 1x10(6) tachyzoites/ml; viability exceeded 90% after 96-120 h of infection throughout 38 passes, during which time the viability improved and the time to harvest became more consistent. Toxoplasma gondii grown in continuous culture in HeLa cells can provide a regular supply of viable tachyzoites. Demonstration that HeLa-derived tachyzoites could be used for the dye test confirms the potential of this in vitro system for use in general hospital laboratories.

Animals↗
PubMed1995

Seroprevalences of Toxoplasma, malaria, rubella, cytomegalovirus, HIV and treponemal infections among pregnant women in Cotonou, Republic of Benin.

Seroprevalences for toxoplasmosis, malaria, rubella, cytomegalovirus, HIV and treponemal infections were evaluated among 211 pregnant women residing in the Cotonou area, Republic of Benin. One hundred and thirteen women (53.6%) had toxoplasma antibodies, 185 (87.7%) malaria antibodies and 181 (85.8%) rubella antibodies. Among the 205 (97.2%) women with cytomegalovirus antibodies, 6 presented recent or current infection. No HIV seropositivity was detected. Five (2.4%) of these women had a positive treponematosis serology corresponding to previous infection or reinfection. These results were compared with previous studies conducted in Africa. Routine serological screening should be recommended in young age and in pregnancy for rubella, only in pregnant women for HIV and toxoplasma infections, in order to control their possible consequences on women and newborns.

Adolescent↗
PubMed1983

Serologic evidence for acute toxoplasmosis in polymyositis-dermatomyositis. Increased frequency of specific anti-toxoplasma IgM antibodies.

Several case reports have suggested an association between acquired toxoplasmosis and polymyositis-dermatomyositis. Because the presence of anti-Toxoplasma IgM antibodies suggests recent infection, 58 patients with polymyositis-dermatomyositis (from two medical centers) were examined for the presence of IgM antibodies using a specific indirect immunofluorescent technique. Serum samples were also examined for antibodies using the Sabin-Feldman dye test and complement fixation methods. Of 58 patients with polymyositis-dermatomyositis, 29 (50 percent) had positive Sabin-Feldman dye test results and 14 (24 percent) had positive IgM immunofluorescent findings. This is higher than the expected frequency. None of the patients with negative Sabin-Feldman dye test results had IgM immunofluorescent antibodies. Furthermore, IgM immunofluorescent antibodies were associated with the presence and titer of both Sabin-Feldman dye test and complement fixation antibodies. Evidence that the presence of antinuclear antibody and rheumatoid factor did not influence these results is presented. Patients with muscular dystrophy and systemic lupus erythematosus (with or without myositis) did not have an increased frequency of anti-Toxoplasma IgM immunofluorescent antibodies.

Acute Disease↗
PubMed1984

Toxoplasma gondii: growth in ovine fetal kidney cell cultures.

Serial, in vitro passage of Toxoplasma gondii (Rh strain) was successfully performed in a cell line derived from ovine fetal kidney cells. Invasion of this parasite into the kidney cells was easily discernible 1 hr after inoculation. The subsequent proliferation of the parasite was followed in the cytoplasm of the kidney cells. Very active endodyogeny and rosette formations, as many as 13 in a cell, were observed in the cytoplasm of the kidney cells 48 hr postinoculation. After 96 hr of incubation, the parasite population had increased about 132-fold. The virulence of T. gondii against mice was not attenuated after 2 years of in vitro growth which represented 100 serial passages through the kidney cell cultures. Although no "exotoxin" was produced by T. gondii grown in vitro, a Toxoplasma sp. agar gel immunodiffusion test antigen was isolated from the cell-free supernatant fluid of the kidney cell cultures which was identical to an antigen isolated from "toxogenic" organisms harvested from infected mice.

Animals↗
PubMed1990

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites.

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.

Animals↗
PubMed1995

A ubiquitous intracellular parasite: the cellular biology of Toxoplasma gondii.

Toxoplasma gondii shares many features with other apicomplexan parasites but is unusual in its extremely broad host and tissue specificity. The parasite exhibits typical 'zoite' morphology, its highly polar structure being dictated by the complex cytoskeleton. Molecules on the surface of the zoite are prime candidates for interaction with the host cell and in vitro assays have implicated 2 of the 5 tachyzoite surface molecules in invasion: SAG1 as a ligand mediating host cell invasion, and SAG2 in enabling reorientation prior to invasion. The functional roles of other molecules, secreted from internal organelles during invasion and intracellular development, are also becoming clear through immuno-EM and biochemical studies, and from sequence data. Molecules from the rhoptries including the penetration enhancing factor ROP1 are secreted at the point of invasion and are integral to the newly formed parasitophorous vacuole membrane. Release of the dense granule molecules GRA 1-6, appears to be calcium regulated and occurs within 10 min of invasion leading to formation of the tubular membranous network and stabilization of the vacuole. The interaction between Toxoplasma and the host cell is stage specific. The tachyzoite divides rapidly and synchronously forming rosettes and causing host cell lysis, while the bradyzoite exhibits slow asynchronous division secreting a granular matrix and becoming enclosed within a cyst wall. This altered phenotype is a reflection of changes in gene expression. Bradyzoite specific molecules are found internally, on the parasite surface, and in the cyst matrix while important tachyzoite proteins such as SAG1 and SAG2 are downregulated. Differentiation between the 2 stages is reversible and is influenced by immunomodulatory agents. However a strong genetic element is involved and it is notable that virulent strains show a very low frequency of cyst production.

Animals↗
PubMed1995

Calibration of an in vitro assay system using a non-adherent cell line to evaluate the effect of a drug on Toxoplasma gondii.

A TG 180 non-adherent cell culture with Toxoplasma gondii and spiramycin was performed in order to calibrate TG 180 cells used in a therapeutic assay system. TG 180 cells were infected with the RH strain of T. gondii, and spiramycin was added at final concentrations of 1, 2, 4, 8, 12, 16, 20 and 25 micrograms/ml. A least-squares linear regression with confidence interval was calculated from the experimental curve and allowed the determination of the 50% inhibitory concentration of spiramycin. The IC50 of 13.7 micrograms/ml obtained is comparable with the results of the literature. The system including TG 180 cells (recently cultured in vitro) provides a rapid, accurate and economical determination of the effects of a drug on Toxoplasma gondii.

Animals↗
PubMed1986

Experimental infection of pregnant sheep with Toxoplasma gondii: pathological and immunological observations on the placenta and foetus.

The pathogenesis of Toxoplasma gondii infection in pregnant sheep was investigated. In one experiment, sheep were infected at 40, 60 and 90 days gestation and examined 30 days later, while in a second experiment, pregnant sheep were infected at either 60 or 90 days gestation and examined 10, 15 and 20 days later. By 10 days post infection (dpi), the parasite had invaded the gravid uterus and caused focal damage in the caruncular septa and, by 15 days, the degenerative changes had spread to the crypt wall and trophoblast. At this time, toxoplasma were isolated from all challenged foetuses. Foetuses did not produce any significant immune response until 20 dpi when increased numbers of IgM positive cells were found in lymphoid tissues. However, at 30 dpi, the numbers were normal while numbers of IgG positive cells and circulating IgG were increased. It was concluded that, while the foetal immune system need not be fully mature to respond to T. gondii infection, its ability to mount a response is limited at 70 days gestation but becomes increasingly competent after this. Furthermore, while it is able to respond more comprehensively with increasing maturity, maturation itself appears to be accelerated by the stimulus of infection.

Animals↗
PubMed1985

Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30).

A double-sandwich ELISA, developed for detection of IgM antibodies to the major surface protein of Toxoplasma gondii (P30), is proposed for the diagnosis of acute acquired toxoplasmosis. The method is based on the capture of serum IgM antibodies, which are revealed indirectly by the sequential addition of a Toxoplasma extract and a beta-galactosidase-conjugated anti-P30 monoclonal antibody. All 57 patients tested with serological characteristics of recently acquired toxoplasmosis showed high levels of IgM anti-P30 antibodies. In addition, 5 out of the 24 patients with chronic toxoplasmosis and all 7 patients with a clinical acute infection in which the classical IgM serology was negative, also presented significant anti-P30 IgM antibodies. Patients with either rheumatoid factor or antinuclear antibodies were all negative. In view of its simplicity, specificity and sensitivity, this method is recommended for the current diagnosis of T. gondii infection.

Animals↗
PubMed1991

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.

Amino Acid Sequence↗
PubMed1988

Human toxoplasmosis in Somalia. Prevalence of Toxoplasma antibodies in a village in the lower Scebelli region and in Mogadishu.

The acquisition of Toxoplasma antibodies in various age groups was studied in 2 populations in Somalia, inhabitants of a village in the southern part of the country and residents in Mogadishu. The overall prevalence of antibodies was 56% in the village and 40% in Mogadishu. In both populations, antibodies were acquired early in life. At the age of 10 years, 44% of the villagers and 31% of the Mogadishu children were seropositive. In Europe and the USA the highest rate of antibody acquisition has been recorded in young adults. This difference may indicate different routes of transmission. In Europe and the USA Toxoplasma is transmitted mainly by the ingestion of undercooked pork or lamb. Undercooked meat is seldom consumed in Somalia, and pork not at all. However, the soil in Somalia is heavily contaminated with cat faeces and the humid climate in the southern part of the country may contribute to long survival of oocysts. In the villages all household activities are performed on the ground and in Mogadishu children play mainly outdoors on the ground. It therefore seems that conditions in Somalia favour transmission by oocysts rather than by infected meat. The early acquisition of antibodies in Somalia suggests that infection during pregnancy and, therefore, congenital toxoplasmosis are rare.

Adolescent↗
PubMed1983

Application of an indirect carbon immunoassay (CIA) for the rapid diagnosis of antibody to Toxoplasma gondii in sheep.

Carbon immunoassay (CIA), a novel indirect rapid test for Toxoplasma antibody in sheep, was compared with indirect fluorescent antibody assay (IFA). CIA relies on the adherence of carbon particles of India-ink to rabbit immunoglobulin G.l Carbon labelled anti-sheep rabbit IgG was used for the detection of sheep antibody when attached to tachyzoites of Toxoplasma gondii. The result was read in an ordinary light microscope and there was a clearcut difference between negative and positive reactions. Out of a total of 97 sheep sera tested, 15 sera were negative in both tests and 12 were negative in CIA but showed low positive titres in IFA. The remaining 70 sera were positive in both tests but the titres were usually about 3 dilution steps lower when investigated with CIA as compared to IFA.

Animals↗
PubMed1988

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.

Animals↗
PubMed1992

Purification and properties of a pyrophosphate-dependent phosphofructokinase from Toxoplasma gondii.

Inorganic pyrophosphate-dependent phosphofructokinase was identified in toxoplasma gondii and purified to near homogeneity from the crude extracts. The purified enzyme displayed one major protein band which coincided with enzyme activity on nondenaturing polyacrylamide gel electrophoresis. This phosphofructokinase had a molecular weight of 100,000 determined by gel filtration and was composed of one type of subunit with the molecular weight of 45,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a homodimer. Some kinetic parameters of the purified enzyme were investigated in the forward and the reverse directions. The substrate saturation curves for fructose 6-phosphate and pyrophosphate were all hyperbolic. The apparent Km values for fructose 6-phosphate and for pyrophosphate were 2.7 x 10(-4) M and 3.3 x 10(-5) M respectively. Kinetics for Fru-1,2-P2 and for Pi in the reverse reaction were also hyperbolic. The activity of this enzyme was magnesium-dependent. Nucleoside triphosphates and polyphosphates did not serve as phosphate donor and the enzyme activity was not altered in the presence of any of these nucleotides. As in the case of pyrophosphate-dependent phosphofructokinases from other anaerobic eukaryotes the Toxoplasma enzyme was not activated by fructose 2,6-biphosphate.

Animals↗
PubMed1992

A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry.

The monoclonal antibody Tg49 both recognizes a Toxoplasma gondii rhoptry protein (ROP1) and inhibits penetration enhancing factor. The latter is a proteinaceous factor found in Toxoplasma lysates or conditioned media that increases the efficiency with which parasites invade host cells. Tg49 was used to screen a lambda gt11 cDNA library and the clone obtained was identified as the cognate gene for ROP1 by several criteria: (1) recombinant protein reacted with the monoclonal; (2) antiserum against the recombinant reacted with the same bands on Western blots as did Tg49; and (3) antiserum against the recombinant recognized a protein in the rhoptries. The ROP1 gene is a single copy gene with a message of approximately 2.1 kb. The predicted polypeptide sequence of ROP1 shows an unusual charge and amino acid asymmetry. There is a highly acidic, proline-rich domain in the amino-terminal portion of the predicted protein, followed by a strongly basic carboxy-terminal domain. An octapeptide repeat is found almost midway through the peptide sequence toward the end of the acidic domain. The ROP1 gene was expressed in a bacterial system, and the resulting polypeptide exhibited anomalous migration on polyacrylamide gel electrophoresis. Given that Tg49 inhibits penetration enhancing factor, it seems likely that the ROP1 protein is a component of that factor, and that the unusual sequence of this protein plays some role in host cell penetration by T. gondii.

Amino Acid Sequence↗