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PubMed1984

Immunoblot analysis of Toxoplasma gondii antigens by human immunoglobulins G, M, and A antibodies at different stages of infection.

The Toxoplasma gondii antigenic components eliciting the immunoglobulin G (IgG), IgM, and IgA antibody responses were studied by using follow-up sera from a laboratory worker who developed an acute glandular toxoplasmosis after an accidental infection with the protozoa. IgG toxoplasma antibodies reacted with multiple components over a wide molecular weight range from 6,000 to 150,000. In contrast, IgM toxoplasma antibodies reacted predominantly with polypeptides of 6, 25, and 35 kilodaltons, which might be useful in new diagnostic procedures. The general pattern of antigenic components in the IgA toxoplasma antibody response closely resembled that in the IgM response, even though some characteristic features were constantly observed. The possibility that the restricted IgM and IgA antibody responses relate to the pathogenetic events in human toxoplasmosis is considered.

Antibodies↗

PubMed1987

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.

Animals↗

PubMed1987

Comparative study of tissue culture and mouse inoculation methods for demonstration of Toxoplasma gondii.

Two methods for the isolation of Toxoplasma gondii were analyzed and compared. Bradyzoites or tachyzoites of three strains of T. gondii were injected into mice and introduced in parallel onto MRC5 fibroblasts cultured on cover slips. In the cultures, the parasites were more readily identified by an indirect immunofluorescence assay than by examination of unstained or Giemsa-stained cultures. With the RH strain, the tachyzoites replicated actively, and large foci of parasites were observed in 24 h. The bradyzoites or tachyzoites of the other strains could also be cultivated, but grew rather slowly; 2 days after inoculation, early stages of multiplication could be observed: from day +4, Toxoplasma clusters or foci were easily identified at a x100 magnification. The course of infection in mice was greatly dependent on the virulence of the strain and on the parasitic stage inoculated. In the chronically infected mice, evidence of Toxoplasma infection was only detected 45 days after inoculation through the demonstration of cysts in the brain or the presence of specific antibodies in the serum. The mean ratio of infected mice and positive cultures was compared in relation to the inoculum size. The tissue culture method was found to be at least as sensitive as mouse inoculation. Since Toxoplasma organisms may be isolated within a few days in tissue culture, it is proposed that this method should be used when early isolation of the parasite is crucial for the diagnosis of toxoplasmosis.

Animals↗

PubMed2000

Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes.

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify Toxoplasma gondii DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 x 10(6) to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to </=0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.

Adult↗

PubMed2004

Evaluation of a candidate international standard preparation for human anti-Toxoplasma immunoglobulin G.

A freeze-dried human serum preparation containing immunoglobulin G (IgG) to Toxoplasma gondii was assessed for its suitability as an international reference reagent in an international collaborative study by 24 laboratories from 17 countries. This candidate standard was compared with the third international standard (IS) for human anti-Toxoplasma serum, TOXM, with the previous second IS, TOXS, and with a range of other serum samples. Samples were tested with the Sabin-Feldman dye test and a range of agglutination assays and enzyme immunoassays. This study emphasizes the need for appropriate standards if intermethod agreement of estimates is to be achieved. On the basis of the results of this study, the preparation was established by the World Health Organization as the first IS for human anti-Toxoplasma IgG, with an assigned potency of 20 IU per ampoule of total anti-Toxoplasma antibodies.

Animals↗

PubMed2004

Detection of immunoglobulin M antibodies specific for Toxoplasma gondii with increased selectivity for recently acquired infections.

Toxoplasma gondii infections can cause serious complications in pregnant women, leading to miscarriage, stillbirth, and birth defects. Definitive diagnosis of T. gondii acute infection is therefore critical for the clinical management of a mother and her fetus. Positive immunoglobulin M (IgM) results are not sufficient as evidence of recent infection, as these antibodies are often present for many months. Further, IgG avidity and differential agglutination tests, two tests used by reference laboratories to distinguish between recent and past infections, are not always in agreement, and both methods yield a significant number of indeterminate results. We report the development of a new toxoplasma IgM immunoassay that is performed by using a bead-based immunoassay on an automated analyzer (BioPlex 2200). Initial validation included 204 samples from pregnant women and 198 samples from asymptomatic healthy adults. An overall specificity of 99% was observed. Further, 100% sensitivity for acute infections was observed for 10 well-characterized seroconversion panels. We then examined 50 samples from pregnant women, all of which were IgM positive by ELISA, which had been fully evaluated in a reference laboratory. Of the 50 samples, 34 (68%) tested positive in the BioPlex 2200 toxoplasma IgM assay, of which 32 of 34 (94%) exhibited an acute or equivocal pattern by differential agglutination. Of the 16 negative samples, 15 (94%) showed high-IgG-avidity antibodies. Collectively, these results suggest that this new toxoplasma assay shows a preferential response to IgM antibodies produced by recent infections, reducing the number of positive results for pregnant women that will require extensive additional clinical evaluation.

Animals↗

PubMed1992

Comparison of relative uses of commercial assays for Toxoplasma gondii IgM antibodies.

AIMS: To compare the sensitivity and user friendliness of seven commercially available enzyme linked immunoabsorbent assay (ELISA) kits for toxoplasma specific IgM. METHODS: Five antibody capture assays supplied by Abbott, Mercia, Northumbria, Organon and Sorin, and two indirect ELISA assays from Biostat and Mast, were assessed. Using defined dilutions of Toxoplasma gondii specific IgM, the performance and sensitivity of each assay was established. They were further assessed on a panel of 27 sera with a range of dye test and IgM results (as determined by the Scottish Toxoplasma Reference Laboratory). All of the assays were performed by three experienced operators and assessed for user satisfaction. RESULTS: The Mast, Organon, and Abbott assays were of low sensitivity; the Mercia and Northumbria were of high sensitivity; and the Biostat and Sorin assays produced too many false positive results. The Mercia kit provided most user satisfaction; the Mast and Abbott assays were most difficult to use. CONCLUSIONS: Local laboratories investigating toxoplasma infection should have three tests: one IgG and two IgM (high and low sensitivity) to help in the timing of infection. Alternatively, one sensitive IgM assay, such as that of Mercia, could be used by selecting appropriate high and low thresholds.

Animals↗

PubMed1998

Acute phase toxoplasma abortions in sheep.

Within 13 days of the experimental infection of 15 oestrus-synchronised ewes with 2000 sporulated oocysts of Toxoplasma gondii at 80 to 90 days of gestation 11 had aborted. The infection induced pyrexia and specific antibody in all the ewes. One ewe resorbed its fetus, 11 ewes aborted and three delivered, at full term, live congenitally infected lambs whose pre-colostral serum was antibody-positive. Tissues from the aborted fetuses and placentae from the live lambs were examined for toxoplasma infection by polymerase chain reaction (PCR) amplification of the B1 gene and by mouse inoculation. The live lambs were all shown to be infected by both methods, but there was no evidence of infection in any of the tissues from the acute phase abortions, suggesting that these fatalities occurred before the placenta or the fetus had been invaded by T gondii. Such toxoplasma-induced, acute-phase abortions are likely to be impossible to diagnose from fetal tissues. These results have implications not only for the diagnosis of naturally occurring ovine abortions but also for the understanding of the pathogenesis of toxoplasma-induced abortion.

Abortion, Veterinary↗

PubMed2005

The transcriptome of Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown. RESULTS: We have used serial analysis of gene expression (SAGE) to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (approximately 300,000 SAGE tags) representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity. CONCLUSION: This study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1-5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmission. Developmentally co-regulated genes were dispersed across all Toxoplasma chromosomes, as were tags representing each abundance class, and a variety of biochemical pathways indicating that trans-acting mechanisms likely control gene expression in this parasite. We observed distinct similarities in the specificity and expression levels of mRNAs in primary populations (Day-6 post-sporozoite infection) that occur prior to the onset of bradyzoite development that were uniquely shared with the virulent Type I-RH laboratory strain suggesting that development of RH may be arrested. By contrast, strains from Type II-Me49B7 and Type III-VEGmsj contain SAGE tags corresponding to bradyzoite genes, which suggests that priming of developmental expression likely plays a role in the greater capacity of these strains to complete bradyzoite development.

Animals↗

PubMed2002

Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii.

The tachyzoite stage of the protozoan parasite Toxoplasma gondii has two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work had established that the microtubule populations are individually nucleated by two distinct microtubule-organizing centers (MTOCs): the apical polar ring for the subpellicular microtubules and spindle pole plaques/centrioles for the spindle microtubules. When replicating tachyzoites were treated with 0.5 microM oryzalin or 1.0 mM colchicine they retained the capacity to form a spindle and undergo nuclear division. Although these parasites could complete budding, they lost the bulk of their subpellicular microtubules and the ability to reinvade host cells. Both nascent spindle and subpellicular microtubules were disrupted in 2.5 microM oryzalin or 5.0 mM colchicine. Under these conditions, parasites grew in size and replicated their genome but were incapable of nuclear division. After removal from 0.5 microM oryzalin, Toxoplasma tachyzoites were able to restore normal subpellicular microtubules and a fully invasive phenotype. When oryzalin was removed from Toxoplasma tachyzoites treated with 2.5 microM drug, the parasites attempted to bud as crescent-shaped tachyzoites. Because the polyploid nuclear mass could not be correctly segregated, many daughter parasites lacked nuclei altogether although budding and scission from the maternal mass was able to be completed. Multiple MTOCs permit Toxoplasma tachyzoites to control nuclear division independently from cell polarity and cytokinesis. This unusual situation grants greater cell cycle flexibility to these parasites but abolishes the checks for coregulation of nuclear division and cytokinesis found in other eukaryotes.

Animals↗

PubMed2001

Modified protection against Toxoplasma gondii lethal infection and brain cyst formation by vaccination with SAG2 and SRS1.

Numerous studies have supported the importance of immunity to SAG1, the most predominant antigen of Toxoplasma tachyzoite, in protection against Toxoplasma gondii infection. Nevertheless, vaccination with SAGI provides insufficient protection when compared with that of Toxoplasma lysate (TL). In order to screen the Toxoplasma antigens for immunogenic potential shown by modified protection or induction of specific immune response after infection, recombinant antigens were prepared in Eschericha coli using DNA fragments corresponding to SAG1, SAG2, SAG3, SRS1 and P54 of T. gondii RH strain maintained in our laboratory. Each of the recombinant antigen products or a mixture of the five antigens (Mix) was used to vaccinate mice. Mice then received a lethal dose of T. gondii. Up to 25% of the mice vaccinated with SAG2, SRS1, P54 and Mix survived, whereas there were no survivors in gene 10- (negative control), SAG1- and SAG3- vaccinated groups. In all the survivors, brain cysts were not observed. Conversely, vaccination with TL almost completely protected mice in the acute phase but permitted brain cyst formation and resulted in gradual decrease of survivors to 33% during 4 months of experiments. Western blot analysis on convalescent sera showed an extensive IgG induction to a 30 kDa antigen in TL-vaccinated mice, a 22 kDa in SAG2-vaccinated mice and a 55 kDa in P54-vaccinated mice. The protection modified by boost in specific antibody is suggestive of the immunogenic potential of SAG2, SRS1 and possibly P54 against T. gondii infection.

Animals↗

PubMed1990

Effects of cyclosporin A on susceptibility and interferon-generating capacity in mice infected with Toxoplasma gondii.

Cyclosporin A (Cs-A), a potent immunosuppressant, was administered to mice to evaluate the role of T lymphocytes for the development of a protective immunity to an obligate intracellular protozoan parasite, Toxoplasma gondii. Although daily administration of various amounts of Cs-A for 7 days enhanced the host susceptibility at all doses employed, a dose-dependent manner of Cs-A treatment was not observed as far as the dosage regimens applied here are concerned; mice died at the same rate (40%) among the groups receiving various amounts of Cs-A. Cs-A treatment had a differential effect on the course of disease depending on how it was given in relation to infection. All mice receiving 50 mg of Cs-A per kg per day for 10 days from the beginning of infection eventually died of toxoplasmosis. Cs-A did not suppress the production of intereferon(IFN)-alpha/beta that was induced shortly after the infection, whereas it reduced greatly the ability of Toxoplasma-infected mice to produce IFN-gamma induced by stimulation with Toxoplasma lysate antigen (TLA). Moreover, the decrease in IFN-gamma production correlated with an increase in the parasitic growth in the peritoneal cavities of Cs-A-treated mice. These results suggest that the immunosuppressive effect of Cs-A on the primary Toxoplasma infection in mice is expressed by inhibiting the development of effector T cells responsible for the production of IFN-gamma.

Animals↗

PubMed1990

Effects of pyrimidine salvage inhibitors on uracil incorporation of Toxoplasma gondii.

Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro-dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses de novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reflect the specific proliferation of Toxoplasma. It can be exploited to characterize the effects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis.

Animals↗

PubMed1993

Inhibition of entry of Toxoplasma gondii into MDCK cells by fetal bovine serum.

We experienced the partial inhibition of entry of Toxoplasma gondii into MDCK cells when the FBS was depleted from media. MDCK cells and Toxoplasma (RH strain) were co-cultured, the penetration was inhibited up to 60-80% with concentration-dependence of FBS. Inhibitory effect was clear when the conc. of FBS was over 1% (v/v) with 50% inhibition conc. of 5%. When Toxoplasma was pre-incubated with FBS and then applied to MDCK cells, there were no inhibitory effect, but when FBS was added to Toxoplasma-MDCK co-culture, the time of adding was critical with rapid inhibition. And when FBS was further treated with heat (95 degrees C, 10 min), the inhibitory effect was decreased slightly in both raw and inactivated FBS. The FBS factor(s) might participate to neutralize secreted materials which enhancing penetration or intervene between receptor-ligand binding at the moment of entry through sterically rather than functionally.

Animals↗

PubMed2000

Recombinant attenuated Toxoplasma gondii expressing the Plasmodium yoelii circumsporozoite protein provides highly effective priming for CD8+ T cell-dependent protective immunity against malaria.

The protozoan parasite Toxoplasma gondii elicits strong cell-mediated immunity against itself as well as nonspecific resistance against other pathogens and tumors. For this reason, we asked whether recombinant Toxoplasma could be utilized as an effective vaccine vehicle for inducing immunity against heterologous microbial infections. The circumsporozoite protein (PyCSP) of Plasmodium yoelii was engineered into a T. gondii temperature-sensitive strain (ts-4), a mutant that induces complete protection against virulent Toxoplasma challenge. When administered to mice in a single dose, a recombinant ts-4 (CSC3) that both secretes and expresses surface PyCSP induced strong anti-CSP Ab responses, with an isotype distribution pattern similar to that stimulated by the T. gondii carrier. When challenged with P. yoelii sporozoites during the first month after CSC3 vaccination, these animals displayed substantial levels of nonspecific resistance attributable entirely to the T. gondii carrier. Nevertheless, after the nonspecific protection had waned, high levels (up to 79%) of specific immunity against sporozoite challenge were achieved by boosting the animals with recombinant vaccinia virus expressing PyCSP. These CSC3-primed PyCSP-vaccinia-boosted mice displayed high frequencies of splenic PyCSP-specific IFN-gamma-producing cells, as well as CD8+ T cell-dependent cytolytic activity. In vivo depletion of CD8+ lymphocytes at the time of challenge completely ablated protective immunity in the T. gondii-primed/vaccinia-boosted animals, while neutralization of IFN-gamma or IL-12 caused a partial but significant reduction in resistance. Together these findings establish the efficacy of recombinant attenuated Toxoplasma as a vaccine vehicle for priming CD8+-dependent cell-mediated immunity.

Animals↗

PubMed2004

Toxoplasma gondii interferes with lipopolysaccharide-induced mitogen-activated protein kinase activation by mechanisms distinct from endotoxin tolerance.

We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-alpha and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IkappaBalpha degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.

Animals↗

PubMed2004

Inhibition of the growth of Toxoplasma gondii in immature human dendritic cells is dependent on the expression of TNF-alpha receptor 2.

An effective immunity to Toxoplasma gondii in humans is dependent on the cellular immune response. Toxoplasma can infect and replicate in almost all nucleated cells, and the most important cytokine regulating the growth in humans is IFN-gamma; however, the role of TNF-alpha has to date been largely described to be synergistic. We show that, compared with mature human dendritic cells (mDC), immature human DC (iDC) demonstrate a reduced parasite proliferation when infected with Toxoplasma. This toxoplasmostasis was only present in iDC after 11 days of culture and was not present in DC that had been matured ex vivo using a cytokine mixture (mDC). Spontaneous toxoplasmostatic activity has previously only been described in fresh human monocytes, and the mechanism involved is as yet unclear. We show that, in comparison with an absence of expression in mDC, TNF-R2 is expressed in both iDC and monocytes infected with Toxoplasma, and furthermore, that blocking the TNF-R2 with Abs abrogates the toxoplasmostasis in the iDC. These findings demonstrate a functional role for TNF-R2 in the newly described spontaneous toxoplasmostasis of iDC.

Animals↗

PubMed1988

Isoenzymic characterization of seven strains of Toxoplasma gondii by isoelectrofocusing in polyacrylamide gels.

Toxoplasma gondii strains are usually defined by biological parameters such as pathogenicity in mice. A characterization of toxoplasma strains by biochemical techniques has not been reported. In this study, extracts of tachyzoites of 7 toxoplasma strains were compared on the basis of their isoenzyme patterns for 39 enzymes by means of isoelectrofocusing in polyacrylamide gels. Eighteen enzymes gave clear and reproducible bands. Of these, 14 had identical electrophoretic patterns for all strains. Two different isoenzyme types were found for the enzymes aspartate aminotransferase, glutathione reductase, glucose phosphate isomerase, and amylase. This allowed the description of 3 isoenzyme pattern groups among the 7 toxoplasma strains. The possible relationship between biological behavior and isoenzyme pattern groups is discussed.

Amylases↗