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PubMed1983

Immune response to Toxoplasma gondii. I. Toxoplasma-specific proliferation response of peripheral blood lymphocytes from patients with toxoplasmosis.

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [3H]methylated thymidine. Analysis of Toxoplasma-specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma-specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma-specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.

Antibodies, Monoclonal↗

PubMed2001

Prevalence of congenital Toxoplasma gondii infection among newborns from the Poznań region of Poland: validation of a new combined enzyme immunoassay for Toxoplasma gondii-specific immunoglobulin A and immunoglobulin M antibodies.

We determined the value of a new serological assay detecting Toxoplasma-specific immunoglobulin M (IgM) and IgA antibodies at birth for use in mass neonatal screening. The incidence of congenital infection in newborns was compared with data from an epidemiological investigation on the seroprevalence of Toxoplasma in the studied population. Peripheral blood was collected on Guthrie cards during the first 3 days of life and tested for anti-Toxoplasma IgA and IgM using a noncommercial immunocapture enzyme-linked immunosorbent assay (ELISA). When the screening assay was positive, serum samples from the child and the mother were collected for use in Western blotting comparative immunological profile analysis and traditional serological tests for determination of specific IgG, IgM, and IgA antibodies. From December 1998 to April 2000, 17,653 filter paper samples from live-born neonates were successively screened. Congenital T. gondii infection was finally confirmed in 19 newborns. In traditional assays, 13 of 19 infants were IgM and IgA positive using filter paper eluates at birth, 1 child was positive only for IgM, 1 patient was positive for IgM and borderline for IgA, 1 had an equivocal level of IgA, and 3 cases were confirmed only by the Western blot assay. The prevalence of Toxoplasma-specific IgA and/or IgM in filter paper samples at birth was 1 per 929 live-born neonates (1.08/1,000) or about 1 per 523 children (1.9/1,000) born to nonimmune women with a potential risk of primary T. gondii infection during pregnancy, compared to the actual seropositivity rate of 43.7%. The diagnostic sensitivity of the combined IgA-IgM ELISA using neonatal filter paper specimens was not more than 95%, the positive predictive value of the test was 82.6%, and the diagnostic specificity was calculated to be 99.9%. The combined IgA-IgM ELISA is a valuable method for the diagnosis of congenital toxoplasmosis at birth and fulfills criteria for neonatal screening programs. The method showed a good diagnostic sensitivity in neonates untreated prenatally who were born in an area of high seroprevalence of T. gondii infection.

Animals↗

PubMedDate not supplied

[Evaluation of the Immulite 2000 Toxoplasma quantitative IgG et Toxoplasma IgM for the diagnosis of human toxoplasmosis].

Four hundred and ninety five human sera with clinical and biological data were tested for the evaluation of Immulite 2000 Toxoplasma Quantitative IgG and Immulite 2000 Toxoplasma IgM produced by Diagnostic Products Corporation (Los Angeles, USA) for the diagnosis of human toxoplasmosis. The results of these kits were compared to those of the University Hospital of Nancy where the reference assays were Enzygnost Toxoplasmosis IgG and Enzygnost Toxoplasmosis IgM (Berhing-Dade, Germany), Toxoscreen (bioMérieux, France), ISAgA Plus (IgM et IgA) (bioMérieux, France). The sensitivity and the specificity of IgG detection by Immulite 2000 Toxoplasma Quantitative IgG were 98% and 100%, respectively. The high sensitivity of IgM detection by Immulite 2000 Toxoplasma IgM was adapted to the early diagnosis of toxoplasmic primo-infection and to the pediatric diagnosis or follow-up of congenital toxoplasmosis but could reveal IgM a long time after primary infection.

Adult↗

PubMed1986

Antitumor effect of formalin-fixed Toxoplasma gondii organisms on EL4 lymphoma in Toxoplasma-infected mice.

The antitumor effect of formalin-fixed Toxoplasma organisms (f-Tp) as an immunostimulant on EL4 lymphoma was examined in Toxoplasma-infected syngeneic female C57B1/6 mice. A potent antitumor effect, a marked suppression of tumor growth, as well as a prolongation of lifespan, was induced by an injection with 10(7) f-Tp in a mixture of 2.5 X 10(5) EL4 cells. The antitumor effect of f-Tp could be observed regardless of the time, route, or dose of infection. The peritoneal exudate cells induced by f-Tp in Toxoplasma-infected mice showed an antitumor activity when the cells were implanted with tumor cells in normal mice, indicating that the cells activated by f-Tp caused the antitumor effect of f-Tp. The effect of f-Tp on EL4 in Toxoplasma-infected mice was significantly stronger than that of 10(7) live bacillus Calmette-Guerin organisms (BCG) in BCG-sensitized mice.

Adjuvants, Immunologic↗

PubMed1979

The effect of Toxoplasma cell fractions and mycobacterial immunostimulants against virulent Toxoplasma gondii in mice.

Toxoplasma gondii tachyzoites were disrupted in a Ribi cell fractionator and separated into cell walls and protoplasm by differential centrifugation. These products were used alone or combined with a mycobacterial glycolipid (P3) and injected either as oil-in-water emulsions or incorporated in Freund's incomplete adjuvant. Mice were vaccinated by intravenous or intradermal routes and challenged intraperitoneally with a highly virulent strain of Toxoplasma gondii. A local granuloma formation was induced after i.d. inoculation of Toxoplasma vaccines containing P3 as this glycolipid enabled an adherence of the antigens on the mineral oil droplets. The adjuvant effect of P3 on antibody formation was also observed. Most of the fractions showed a low, but statistically significant prolongation of survival time. Vaccination by the i.v. route with homologous or heterologous antigens, including Trypanosoma cruzi, were not significantly effective, with the exception of a high dose of Toxoplasma protoplasm associated with P3.

Adjuvants, Immunologic↗

PubMed1995

[Direct detection of Toxoplasma gondii with polymerase chain reaction in diagnosis of fetal toxoplasma infection].

Primary infection with Toxoplasma gondii during pregnancy may affect the fetus and result in congenital toxoplasmosis. In Austria serological screening for detection of newly acquired infection during pregnancy was introduced in 1975. In this study we used polymerase chain reaction (PCR) for detection of fetal infection with Toxoplasma gondii. Amniotic fluid samples were analyzed from 11 women with serological indication of acute toxoplasmosis infection. Nine of these women had already received treatment prior to amnio-centesis and no evidence of Toxoplasma gondii DNA was detected with PCR in the respective amniotic fluid samples. Isolation of the organism by mouse inoculation was negative in these cases and follow-up serology as well as clinical examination of the infants confirmed these results. In 2 patients investigation of the amniotic fluid samples by means of PCR was positive; both women had not yet been treated at the time of amniocentesis. Our results indicate that identification of Toxoplasma gondii in amniotic fluid is a useful procedure for diagnosing or excluding fetal infection. Moreover, the current recommendations of the screening program appear to be successful in preventing congenital toxoplasmosis.

Amniotic Fluid↗

PubMed1977

The effect of chemo-immunotherapy with SDDS, pyrimethamine and anti-toxoplasma serum on toxoplasma gondii cysts in latent infected NMRI mice.

A substantial reduction of brain cyst numbers could be obtained through a six week therapy with SDDS-pyrimethamine combination or a high titer anti-Toxoplasma serum. NMRI mice experimentally infected three months earlier with cyst-forming Toxoplasma strains Witting, Alt or Gail were used. The effectiveness of the treatments could be increased considerably by simultaneous administration of anti-Toxoplasma serum and SDDS-pyrimethamine. A highly significant reduction of brain cyst numbers, upto 65%, could be obtained by using this mode of chemoimmunotherapy. The effectiveness of the treatments used varied amongst the three Toxoplasma strains.

Animals↗

PubMed1980

Caprine toxoplasmosis: abortion, clinical signs, and distribution of Toxoplasma in tissues of goats fed Toxoplasma gondii oocysts.

Thirteen goats (9 does and 4 bucks) were each inoculated orally with 10,000 infective Toxoplasma gondii oocysts. Three does and one buck were used as noninoculated controls. In 2 to 4 days after inoculation (DAI), inoculated goats became dull, pyrectic (40 to 41 C), and anorectic. Three goats died (10, 10, and 14 DAI) and two goats were killed (7 and 32 DAI) because they were moribund; also, 3 does aborted, 2 had weak kids, and 2 had dead fetuses. Toxoplasma was isolated from the placenta of three goats, and the fetal tissues of four goats. The control goats remained asymptomatic. The distribution of T gondii in blood and other tissues was studied by inoculation of mice with caprine tissues. Parasitemia was detected in 7 of 7 goats--beginning 4 DAI in 1 goat, 5 DAI in 5 goats, and 8 DAI in 1 goat. The parasitemia lasted 3 to 10 days. Toxoplasma was isolated from the milk of 2 goats at 12 and 14 DAI. Toxoplasma was isolated from 15 or more tissues of 5 goats killed 7 to 35 DAI and from 10 tissues of 2 goats killed 69 and 95 DAI.

Abortion, Veterinary↗

PubMed2006

Identification and expression analysis of ABC protein-encoding genes in Toxoplasma gondii. Toxoplasma gondii ATP-binding cassette superfamily.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

ATP-Binding Cassette Transporters↗

PubMed1997

Growth inhibitory effect of bovine lactoferrin on Toxoplasma gondii tachyzoites in murine macrophages: role of radical oxygen and inorganic nitrogen oxide in Toxoplasma growth-inhibitory activity.

To study the effector pathway of Toxoplasma growth-inhibitory activity induced by lactoferrin in murine macrophage, the role of reactive oxygen intermediates (O2-) and inorganic nitric oxide (NO) was examined. Production of O2- was diminished in cultures of macrophages supplemented with lactoferrin and the effect of lactoferrin was dose and time dependent. Production of NO was enhanced in cultures of macrophages supplemented with interferon-gamma, but not with lactoferrin. These findings suggest that this Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages is not mediated by O2- or NO molecules. A competitive inhibitor of the L-arginine dependent effector pathway, NG-monomethyl-L-arginine (NG MMA), virtually abolished the inhibitory effects induced by interferon-gamma. Similarly, the inhibitory activity induced by lactoferrin was also diminished in cultures supplemented with NG MMA. From these findings, it appears that the Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages may be mediated by an L-arginine-dependent effector pathway that does not involve NO production.

Animals↗

PubMed2005

Protective effect of vaccination with Toxoplasma lysate antigen and CpG as an adjuvant against Toxoplasma gondii in susceptible C57BL/6 mice.

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems to both humans and livestock and of great economic impact worldwide. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote Th1 responses, an adjuvant activity that is desirable for vaccination against intracellular pathogens. We investigated the feasibility of using CpG as an adjuvant combined with Toxoplasma lysate antigen (TLA) as a vaccine against toxoplasmosis. Genetically susceptible C57BL/6 mice were vaccinated with TLA with or without CpG ODN as an adjuvant and then challenged with 85 cysts of the moderately virulent RRA (Beverley) strain of T. gondii. Prior to challenge infection, immunization with TLA plus CpG ODN directed cellular and humoral immunity toward a Th1 pattern, characterized by enhanced INF gamma production by splenic cells in response to TLA, and enhanced production of toxoplasma-specific IgG and IgG (2a) antibodies. Consequently, CpG/TLA-treated mice showed prolonged survival and 64% reduction in brain parasite burden compared to non-CpG/TLA treated group. Our results suggest that CpG ODN would provide a stable and effective adjuvant for use in vaccination against toxoplasmosis.

Adjuvants, Immunologic↗

PubMed1995

The use of PCR in detecting toxoplasma parasites in the blood and brains of mice experimentally infected with Toxoplasma gondii.

Polymerase chain reaction (PCR) has been extensively used for diagnosis recently because of its very high sensitivity and specificity. We studied the applicability of PCR to the early diagnosis of toxoplasmosis in a murine model orally infected with Toxoplasma gondii (S-273). PCR was performed using EH24 and HE27 primers synthesized by the phosphoramidite method. Mice blood and brains collected on various post infection days (PID) were analysed by PCR (35 cycles). A portion of the brain tissue from each mouse was examined microscopically for the presence of parasite cysts. Blood and brain PCR were positive on the 9th and 12th day post-infection (DPI). Toxoplasma cysts in brain tissue appeared only on the 18th PID. The results showed that Toxoplasma parasites can be detected earlier in the blood than in the brain during primary infection, indicating that blood PCR is the more useful procedure.

Animals↗

PubMed1997

Characterization of anti-Toxoplasma activity of SDZ 215-918, a cyclosporin derivative lacking immunosuppressive and peptidyl-prolyl-isomerase-inhibiting activity: possible role of a P glycoprotein in Toxoplasma physiology.

The immunosuppressive agent cyclosporin A (CsA) also possesses broad-spectrum antimicrobial activity. Previous investigators have reported that the obligate intracellular protozoan Toxoplasma gondii is sensitive to CsA. We have measured the sensitivity of Toxoplasma to 26 CsA derivatives that maintain only a subset of the parent compound's activity. We identified one compound, SDZ 215-918, that is a particularly potent inhibitor of parasite invasion and replication, with a 50% inhibitory concentration of 0.45 microg/ml, which is 10-fold lower than that of CsA. Kinetic studies demonstrate that activity has a rapid onset (half-life, < or = 20 min) and is initially reversible, although long-term exposure (> 24 h) to 5 microg/ml is lethal; in contrast, this concentration had no effect on host cell protein synthesis or cell division. SDZ 215-918 acts directly on the parasite, as demonstrated by inhibition of macromolecular synthesis in host-free extracellular parasites. Inhibition of invasion is due to a reduction in parasite motility. SDZ 215-918 does not bind to cyclophilins, the ubiquitous cyclosporin-binding proteins, but is a potent inhibitor of the mammalian P glycoprotein, a member of the ATP binding cassette transporter superfamily and the pump responsible for multidrug resistance in cancer and parasite cell lines. SDZ 215-918 blocks the efflux of rhodamine 123 from extracellular parasites, consistent with inhibition of a P glycoprotein-like pump. We suggest that a P glycoprotein or a related transporter plays a crucial role in the biology of Toxoplasma and may be a novel target for antiparasitic compounds. Preliminary studies with animals indicate that SDZ 215-918 inhibits parasite growth in vivo; its relationship to CsA may make it suitable for clinical development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1975

Interactions between Toxoplasma gondii and its host cells: function of the penetration-enhancing factor of toxoplasma.

A protein with a molecular weight of 70,000 to 150,000 which was extracted from merozoites of Toxoplasma gondii enhanced the host cell penetration of the merozoites. The optimal pH and temperature for penetration of merozoites coincided with those favoring the action of the penetration-enhancing protein. In addition, a dependence on Ca and Mg existed for penetration of merozoites, either in the presence or absence of this protein. No evidence was found that indicated that the enhancing effect on penetration elicited by the protein was due to increased phagocytic capacity of host cells (HeLa) or improved motility of the merozoites. Electron microscopy demonstrated that the protein, in high concentration, caused disruption of cytoplasmic membranes. In a 100-fold-lower concentration, which still caused a marked enhancement of penetration, no such effect was observed. However, the vacuoles surrounding the penetrated parasites seemed smaller than for merozoites penetrating in cultures to which no penetration-enhancing factor was given, and the membranes limiting the vacuoles demonstrated discontinuities more often. The penetration-enchancing effect of some known enzymes was studies. However, none of these enzymes seemed to correspond to the penetration-enhancing protein of toxoplasma. The mode of entry of toxoplasma merozoites into host cells is discussed. It is concluded that phagocytosis must play a less important role and that merozoites actively penetrate the cytoplasmic membranes of the host cells. The penetration is proposed to be a result of combined mechanical and chemical actions. It is suggested that an enzymatic function of the penetration-enhancing factor released by the merozoites is of importance. The membrane limiting the vacuole of a penetrated merozoite seems to be newly formed in the cell after penetration is completed.

Acid Phosphatase↗

PubMed1999

[The protective effect against Toxoplasma infection in mice immunized with laser-irradiated Toxoplasma tachyzoites].

AIM: To observe the level of immune responses and protective immunity in mice induced by laser-irradiated Toxoplasma tachyzoites (LIT). METHODS: ICR mice were immunized with LIT. After one month, the mice were challenged with live Toxoplasma tachyzoites to observe the effect of LIT. Indirect immunofluorescence assay and ELISA were used to detect CD4+/CD8+ lymphocyte subpopulation and IgG antibody. RESULTS: Immunization with LIT could partially protect the mice from Toxoplasma infection, prolong the survival time, enhance the CD4+/CD8+ ratio and produce specific IgG antibody. CONCLUSION: Intraperitoneal injection with LIT can induce partial protective immunity and specifically elicit ICR mice to generate humoral and cell-mediated immune responses.

Animals↗

PubMed1977

[The effect of toxoplasma antibodies after reinfection with T. gondii. III. Communication: investigations on the question of placental transmission of toxoplasma in immunised pregnant animals (author's transl)].

Investigations on whether theprotective effects of maternal defence mechanisms against T. gondii could prevent a placental transmission of the parasite to the foetus were carried out on pregnant rabbits and mice. With rabbits which were infected 6 to 1 month before the conception and showed a high infection immunity, there was never a transmission to the foetus and likewise at no time after reinfection of such maternal animals during the pregnancy. In mice which were latent infected with 11 different Toxoplasma strains, placental transmission succeeded with only 6 strains intrauterine infection of the foetuses were confirmed in 1 to 3%. After active immunisation of rabbits with non-multiplying antigen before the conception and later primary infection during the pregnancy, intrauterine transmission to the foetus could be detected in 55%. After passive immunisation with hyperimmune serum, infected foetuses could be ascertained in 79% of rabbits and 24% of mice. Possible intrauterine transmission to foetus protected by maternal defence mechanisms is likely to be dependent on various factors. 1. On the host species, 2. the state of immunity (infection immunity), 3. on Toxoplasma strain. An infection immunity with the presence of living parasites in maternal organism is probably dependent on Toxoplasma strain in mice and offers in rabbits an apparently certain protection against congenital infection. On the contrary, serologically detectable humoral antibodies after active or passive immunisation do not prevent a transplacental transmission. On the basis of experimental findings it has been concluded that at least the immunoglobulins of type IgG, IgA and IgM in lower titer ranges offer no protection against congenital transmission after reinfection during pregnancy. Also, the protective effect can not be based on the structure of respective placenta. The roles of cellular immune defence and other possible factors in this connection are discussed.

Animals↗

PubMed1977

Immunization of NMRI mice against virulent Toxoplasma gondii. Differing efficacy of eleven cyst-forming Toxoplasma strains.

Mice were infected with eleven cyst-forming Toxoplasma strains of varying virulence and challenged 1 month later with highly virulent BK strain parasites. The early cellular reaction in vivo was estimated by collecting the peritoneal exudates 24 h after challenge. This consisted of 54.34--77.61% lymphocytes and 0-8.88% infected macrophages in the eleven immunized groups in contrast to 27.17% lymphocytes and 18.64% infected macrophages in the control group. The peritoneal exudate 72 h after challenge comprised of 55.88-73.86% lymphocytes and 0-6.97% infected macrophages compared to 25.88% lymphocytes and 92% infected macrophages in the control group. Following the virulent challenge, deaths occurred in seven of the eleven groups immunized with live strains and ranged 5-42% at the end of a 6-week observation period. The most virulent of the Toxoplasma strains used - Alt and Gail - gave higher mortality while the least virulent ones - 558, 1070, K8 and KSU - were solidly resistant. The significantly different mortality obtained with strains Alt, Gail and Witting in a similar experiment performed at an interval of 7 months is discussed.

Animals↗

PubMed2000

Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences.

The Toxoplasma gondii (TGR) genes constitute a family of non-coding sequences, three of which have been previously described as possible tools for typing of Toxoplasma gondii isolates. We obtained new isolates of T. gondii from domestic and wild animals, and used these to evaluate the possibility of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T. gondii isolates could be separated into seven groups. Sequencing the amplified products showed that at least 20 TGR sequences not hitherto described had been found, demonstrating that the TGR gene family comprises a large number of different yet highly homologous sequences. Each isolate had its own unique TGR sequence. The TGR gene family therefore seems a promising target for typing individual T. gondii isolates and for studying the genetic distance between two isolates, which can be used for tracing routes of infection.

Animals↗