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PubMed1997

Genetic analysis in Toxoplasma: gene discovery with expressed sequence tags and rapid mapping of natural polymorphisms.

Genetic analysis of the protozoan parasite Toxoplasma gondii has undergone a rapid expansion in recent years. This is due to effort in a number of laboratories that have worked on the development of molecular genetic techniques. It is also due, however, to the natural biology of this system (including a well-described sexual cycle) that makes possible genetic mapping of the F1 progeny from a cross. In this article, we present a detailed methodology for rapidly mapping natural polymorphisms between the ME49 and CEP strains for which extensive restriction fragment length polymorphism analysis has already been performed. The example we present shows that the failure to detect expression of bradyzoite-specific surface antigens in the CEP strain under conditions that promote differentiation in vitro is not a result of a general failure to express such genes; instead, it is apparently due to antigenic polymorphism in the gene products concerned. This conclusion was reached rapidly and definitively by genetic mapping, whereas molecular approaches would have taken considerably longer. We also show how the recent effort to create an extensive database of expressed sequence tags for this parasite can promote the very rapid discovery of genes that reveal much about the biology of Toxoplasma. The example presented deals with the expression of a family of closely related surface antigens in the tachyzoite stage.

Alleles↗
PubMed1990

Prevalence of Toxoplasma gondii antibodies in gravidas and recently aborted women and study of risk factors.

Two hundred seventeen parturients and eighty six recent aborters were screened for IgM and IgG toxoplasma antibodies. Age, profession, educational level, residence (urban/rural), presence of cat and other domestic animals were recorded for each subject. None of the subjects was IgM-positive. Prevalence of IgG positivity was 52.3% in the parturients and 50.2% in the recently aborted women. None of the personal or social characteristics investigated could be related to IgG positivity. However, the frequency of toxoplasma antibodies was found to be higher in recent aborters from rural areas where contact with soil is common regardless of whether cats are kept as pets or not. This study confirms other investigators' conclusion on the importance of soil contact as a risk factor for infection.

Abortion, Spontaneous↗
PubMed1993

Risk factors for severe disease due to Toxoplasma gondii in HIV-positive patients.

The objective of this study was to assess the risk of toxoplasmosis in HIV-positive subjects as a basis for primary prophylaxis. A retrospective chart review of 400 consecutive patients was carried out and clinical and laboratory markers at first presentation and follow-up data on the occurrence of toxoplasmosis were recorded. Independent variables were identified, laboratory parameters were stratified, and estimates for the risk of toxoplasmosis and the impact of different variables on its occurrence were made using conventional statistical methods. An increased risk of toxoplasmosis was strongly associated with a positive Toxoplasma gondii IgG EIA in conjunction with a CD4+ cell cont below 0.15/nl (the estimated risk of toxoplasmosis was 20% and 35% after 12 and 24 months, respectively) or a history of one or more opportunistic infections (the estimated risk was 12% and 30% after 12 and 24 months, respectively). Toxoplasma gondii-seropositive patients with CD4+ cell counts below 0.15/nl and those with antecedent opportunistic infections are most likely to develop toxoplasmosis and thus might benefit from primary prophylaxis. The risk of disease probably outweighs the risk of medication in these subjects. Prospective clinical trials are needed to define the optimal choice of drugs.

AIDS-Related Opportunistic Infections↗
PubMed1977

[Freeze fracture study of Toxoplasma and Sarcocystis infective stages (author's transl)].

Infective stages of Toxoplasma and Sarcocystis have been studied by the freeze fracture technique. The outer membrane of the pellicle is continuous and shows an apical 8 + 1 particles rosette in the P fracture face. The inner membrane complex is made of rectangular flattened vesicles aligned in longitudinal rows and joined in a puzzle like fashion. Sarcocystis has 11 of these rows whereas their number varies in Toxoplasma. A posterior interruption is present. Anteriorly is one truncated conical plate apically opened by a vertical ridge. The membranes of the inner complex are characterized by parallel alignment of particles (in P faces) some of which are joined and continuous with double rows radiating in the apical cap. Those rows correspond in number and arrangement with the underlying microtubules. The rhoptries membranes show periodic circular arrays of particles.

Animals↗
PubMed1977

Studies on production of biologically active substance which inhibits the intracellular multiplication of Toxoplasma within mouse macrophages.

In vitro assessments were carried out to study some biological aspects of immune lymphocytes producing a new lymphokine, called by the authors Toxoplasma growth inhibitory factor (Toxo-GIF), which inhibits the intracellular multiplication of Toxoplasma gondii within nonimmune mouse macrophages. Concanavalin A and phytohemagglutinin-P were found to induce vigorous production of Toxo-GIF, whereas bacterial lipopolysaccharide did not. In vitro treatment of splenic lymphocytes with rabbit anti-mouse thymocyte serum plus complement abolished almost completely their ability to produce Toxo-GIF. Treatment of splenic lymphocytes with inhibitors of protein synthesis, cycloheximide or puromycin resulted in a remarkable reduction of the ability of sensitized lymphocytes to produce this lymphokine. Thus the production of Toxo-GIF seems to be dependent on the cellular metabolic events of sensitized T-lymphocytes. The significant activity of Toxo-GIF was demonstrable even in the supernate of lymphocyte cultures incubated in serum-free medium and was also evident after immune lymphocytes and homologous antigen were incubated for the relatively short period of 10 h.

Animals↗
PubMed1983

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies.

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with [125I]-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.

Animals↗
PubMed1987

An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice.

The ultrastructural features of the early development and tissue cyst formation of Toxoplasma gondii were examined in the brains of mice at various intervals from 7 days to 22 months post inoculation (PI). At 11 days PI toxoplasmas, with the ultrastructural features of the proliferative (endozoite) form, were identified undergoing multiplication within both inflammatory and neural cells. Early tissue cyst formation was also observed, predominantly within neurons. By 21 days PI the proliferative forms had disappeared and only developing tissue cysts containing densely packed cystozoites were present. The proportion of dividing cystozoites decreased with increasing size and age of the cysts. The wall of the tissue cyst developed as an adaptation of the lining of the parasitophorous vacuole. In the majority of older cysts, numerous tubular structures were present beneath the cyst wall. All the cysts observed were retained within intact host cells. The only morphological change with increasing age was that a proportion of the older cysts contained loosely packed cystozoites in an electron lucent ground substance. There was no evidence of any degenerative changes within the cystozoites.

Animals↗
PubMed1979

Response of white mice to inoculation of irradiated organisms of the Toxoplasma strain RH.

Chemotherapeutic agents available for use against toxoplasmosis are usually not suitable for prophylactic purposes because of their toxicity. The observed increasing number of activated latent infections with Toxoplasma, especially in immune suppressed patients, requires that safe techniques are available for use during the patient's regression period. Pretreatment of mice with Toxoplasma killed by irradiation appeared to induce resistance to challenge with virulent organisms. Survival times of six months have been observed to date. Increasing effectiveness was seen after more than one administration. Further investigation into the duration of effective resistance is needed; the question of at which intervals subsequent inoculations should be performed in order to acquire a booster effect, if any, has still to be solved before application to man can be recommended.

Animals↗
PubMed1980

[Dynamics of splenic lymphocytes and immunosuppression in Toxoplasma infected mice (author's transl)].

Mice infected with high-virulent strains of T. gondii sustained a marked decrease of splenic lymphocytes within 7-10 days after infection, just prior to death. When infected with a low-virulent strain, the numbers of splenic lymphocytes and of T-lymphocytes fluctuated, but gradually increased over a period of 30 days. To evaluate their immunologic potential, the mice infected with a low-virulent strain of Toxoplasma were immunized with sheep erythrocytes on different days after infection, and antibody levels were measured by direct hemagglutination. Antibody response was minimal when sheep cells had been injected 10 days after infection with Toxoplasma; subsequently immunologic reactivity improved, but even after 30 days anti-sheep agglutinin titers were still below pre-infection levels.

Animals↗
PubMed1978

[Effects of a latent Toxoplasma infection on the learning ability in white laboratory rats and mice (author's transl)].

1. Both white laboratory rats and white laboratory mice had a diminished learning capacity in connection with a latent Toxoplasma infection. 2. Five infected couples of rats out of 12 pairs were not able to learn their task by the double-training program in a given period of time. A second group of five rats was able to learn this program but later than the noninfected control-group of brothers and sisters did. 3. Mice seem to be more damaged by Toxoplasma infections than rats in their learning capacity. All infected mice did not reach the same ability than their control brothers and sisters. The reason for this differences could reflect the higher infection rate of the brain of mice than rats during the latent phase of infection.

Animals↗
PubMed1989

Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study.

The incidence and effect of tissue cyst rupture in the brains of mice chronically infected with Toxoplasma gondii was studied by immunocytochemistry and electron microscopy. Cyst rupture was extremely rare (2 of 750 tissue cysts) irrespective of the interval post-infection. The event was associated with a rapid cell-mediated immune response, giving rise to microglial or inflammatory nodules. Macrophages were observed to engulf and degrade the cystozoites and cyst debris. Initially, the nodules contained large amounts of immunologically reactive material, but this was degraded with the majority (94%) of lesions containing no recognizable parasites or Toxoplasma antigens. There was little evidence of parasite multiplication or new cyst formation associated with cyst rupture. This study shows that although intermittent cyst rupture occurs, in immunocompetent individuals the immune response limits the potential damage from the release of large numbers of infective organisms to small microglial/inflammatory nodules.

Animals↗
PubMed1995

Genetic comparison of Neospora caninum with Toxoplasma and Sarcocystis by random amplified polymorphic DNA-polymerase chain reaction.

To determine the relationship of Neospora caninum to protozoa classified in the family Sarcocystidae of the phylum Apicomplexa, the genomes of N. caninum, three Toxoplasma gondii strains (RHa, CEP, TPR) and three Sarcocystis species (S. tenella, S. muris, S. gigantea) that were thought to be closely related coccidia were compared by the random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) technique. The genomic DNAs were amplified by the use of seven 10-mer arbitrary sequence primers to generate polymorphic DNA. Significant DNA polymorphisms were observed among Neospora, Toxoplasma and Sarcocystis. It appears that one primer tested may have value in a diagnostic RAPD-PCR to differentiate T. gondii from other closely related protozoa. The high level of genetic divergence of N. caninum from T. gondii strains and several Sarcocystis species observed in this study is consistent with the hypothesis that N. caninum is indeed an independent species of protozoan parasite. As compared with the Sarcocystis species tested, a closer genetic relationship of N. caninum to T. gondii was not observed. By contrast, a closer genetic relationship of S. muris to T. gondii was revealed in this study.

Animals↗
PubMed1993

Coexistence of heterogeneous populations of Toxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody.

The expression of bradyzoite-specific antigens (Bsa) of Toxoplasma gondii was studied in murine bone-marrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication of Toxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchronous event for a vacuole community.

Animals↗
PubMed1991

Human T-cell clones against Toxoplasma gondii: production of interferon-gamma, interleukin-2, and strain cross-reactivity.

The soluble fraction from a sonicate of Toxoplasma gondii tachyzoites (termed F3) was shown to induce dose-dependent blastic transformation of peripheral-blood mononuclear cells (PBMC) from seropositive individuals only and was used to isolate a panel of T-cell clones from the PBMC of an immune donor. Proliferation assays using F3 showed that 15 (14 CD4+ and 1 CD8+) of the 18 isolated clones were specific for T. gondii. In response to antigen stimulation, 5 of the 15 clones produced detectable levels of interleukin-2 (IL-2, 0.2-15 u/ml) and 9 clones produced significant levels of interferon-gamma (IFN-gamma, 17.5-1400 IU/ml). Seven of the 7 T-cell clones tested reacted with two different Toxoplasma strains (RH and Wiktor). When used as antigen-presenting cells, an autologous B-lymphoblastoid cell line could efficiently present the antigen to only three of the six T-cell clones tested. This study identifies and characterizes cellular probes that could be useful for future vaccine design.

Animals↗
PubMed1992

Analysis by immunoblotting of Toxoplasma gondii exo-antigens and comparison with somatic antigens.

Two soluble Toxoplasma gondii antigen preparations were compared using the immunoblotting technique. The first preparation, which is commonly employed in ELISA tests, corresponds to a lytic extract of the parasite. The second known as exo-antigen, has yielded good results in cell immunity research and is obtained from Toxoplasma gondii culture supernatants. In both preparations, components exhibiting same molecular weight of 57-52 (doublet), 43, 38, 35, 30 and 20 kDa were revealed. In addition, two major 27- and 78-kDa components were detected in exo-antigens. The 30 kDa protein was intensely recognised by all sera, confirming the advantage of its use as an antigen in serological reactions. Exo-antigens could be employed as a soluble antigenic solution because they are easy to obtain, they display good antigenicity and their antigenic composition has been defined. Moreover their preparation does not require mouse inoculation. An evaluation of the enzyme-linked immunosorbent assays (ELISAs) involving these antigens has yet to be performed.

Animals↗
PubMed1991

Detection of specific antibodies to Toxoplasma gondii by a competitive enzyme immunoassay using a monoclonal antibody against the P30 antigen.

An inhibition EIA using a monoclonal antibody against the major P30 Toxoplasma gondii surface protein was designed for detection of specific antibodies in human sera. The assay was based on the inhibition of binding of peroxidase labelled monoclonal antibody to Toxoplasma gondii crude antigen coated plates by the corresponding antibodies present in human sera. This rapid and simple assay was compared to indirect immunofluorescence, direct agglutination and an immunosorbent agglutination assay using 435 human sera. The specificity and sensitivity were 100% and 97% respectively. This test was found to be as sensitive as the dye test.

Animals↗
PubMed1992

Improved sensitivity of the polymerase chain reaction for detection of Toxoplasma gondii in biological and human clinical specimens.

The aim of the present study was to improve the sensitivity of the polymerase chain reaction for detection of Toxoplasma gondii in biological and clinical specimens. Using a pair of primers amplifying a 634 bp fragment of the B1 gene of this parasite, it was possible to detect ten parasites in 100 microliters of sample suspensions containing a high concentration of concomitant host cells. A comparison of different DNA purification methods indicated that cell-rich clinical specimens intended for use as samples for the polymerase chain reaction should be digested with proteinase K prior to DNA amplification. By using the described sample preparation methods and the polymerase chain reaction, Toxoplasma gondii DNA was demonstrated in ten of 52 clinical specimens of patients with clinical or serological indications of toxoplasmosis.

Animals↗
PubMed1993

Comparison of a commercial enzyme immunoassay and an immunoblot technique for detection of immunoglobulin A antibodies to Toxoplasma gondii.

A commercial enzyme immunoassay (Platelia-Toxo IgA) and an immunoblot technique were compared with regard to their ability to detect IgA antibodies to the major surface protein P30(SAG1) of Toxoplasma gondii in 105 serum samples from patients with suspected or proven acquired toxoplasmosis. Comparison of the IgA-EIA with the immunoblot technique showed a concordance of 81.0%, with a sensitivity of 92.6% and a specificity of 78.4%. Due to its high sensitivity the IgA-EIA might detect IgA antibodies against Toxoplasma gondii at an early stage of infection, although excessive sensitivity could lead to detection of IgA antibodies for an extended period of time following the onset of infection.

Acquired Immunodeficiency Syndrome↗