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PubMed2005

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures.

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.

Animals↗
PubMed2005

Performance of a Western immunoblot assay to detect specific anti-Toxoplasma gondii IgG antibodies in human saliva.

Toxoplasma gondii represents the most prominent infectious parasitic organism found in humans. While normally asymptomatic in healthy individuals, toxoplasmosis can cause abortion in patients during pregnancy, or can be fatal in immunosupressed individuals such as persons suffering from acquired immunodeficiency syndrom (AIDS). Toxoplasma gondii infection in humans is routinely assesssed by serological means. Here, we show that detection of anti-T. gondii IgG is also possible using a non-invasive methodology employing saliva. Sera and saliva of 201 healthy volunteers were investigated for the presence of anti-T. gondii-IgG antibodies by immunoblotting. The sera of 59 (29.4%) individuals showed IgG antibodies against T. gondii by ELISA, Vidas, and immunoblotting; 58 (98.3%) of these were also positive for anti-T. gondii IgG in the saliva immunoblot, with diagnostic relevant bands of Mr of 32-35 kDa and 40-45 kDa. The saliva immunoblot test exhibits a specificity of 100% and a sensitivity of 98.5%. Thus, saliva could be used as an alternative, non-invasive means for the detection of specific anti-T. gondii IgG in humans.

Adolescent↗
PubMed2005

Isolation and molecular characterization of Toxoplasma gondii from chickens from Sri Lanka.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 100 free-range chickens (Gallus domesticus) from Sri Lanka was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 39 chickens with titers of 1:5 in 8, 1:10 in 8, 1:20 in 4, 1:40 in 5, 1:80 in 5, 1:160 in 5, 1:320 in 2, 1:640 or more in 2. Hearts and brains of 36 chickens with MAT titers of 1:5 or more were bioassayed in mice. Tissues of 3 chickens with doubtful titers of 1:5 were pooled and fed to a cat; the cat shed T. gondii oocysts in its feces. Tissues from 61 chickens with titers of less than 1:5 were pooled and fed to 2 T. gondii-free cats; the cats did not shed oocysts. Toxoplasma gondii was isolated from 11 of 36 seropositive chickens by bioassay in mice. All 12 T. gondii isolates were avirulent for mice. Genotyping of 12 isolates using the SAG2 locus indicated that 6 were type III, and 6 were type II. This is the first report of genetic characterization of T. gondii from any host in Sri Lanka.

Agglutination Tests↗
PubMed2005

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from free-range chickens from Guatemala.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 50 free-range chickens (Gallus domesticus) from Guatemala was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 37 (74%) chickens with titers of 1:5 (11), 1:10 (7), 1:20 (11), 1:40 (1), 1:80 (1), 1:160 (3), 1:1,280 (2), and 1:2,560 (1). Hearts, pectoral muscles, and brains of 19 chickens with MAT titers of 1:20 or more were bioassayed individually in mice. Tissues from the remaining 31 chickens with titers of 1:10 or lower were pooled and fed to 4 T. gondii-free cats (13 chickens with titers of less than 1:5 to 1 cat, 11 chickens with titers of 1:5 to 2 cats, and 7 chickens with titers of 1:10 to 1 cat). Feces of cats were examined for oocysts; they did not shed oocysts. Toxoplasma gondii was isolated from 8 chickens with MAT titers of 1:20 or more (from 1 of 11 chickens with a titer of 1:20 and all 7 chickens with a titer of 1:80 or more) from the heart, brain, and pectoral muscle (3); heart and pectoral muscle (1); and heart alone (4). Genotyping of these 8 isolates with the SAG2 locus indicated that 5 were type III and 3 were type 1. This is the first report of isolation of T. gondii from chickens from Guatemala.

Agglutination Tests↗
PubMed2005

Prevalence of agglutinating antibodies to Toxoplasma gondii in adult and fetal mule deer (Odocoileus hemionus) from Nebraska.

Toxoplasma gondii is an apicomplexan parasite of mammals and birds. Herbivores acquire postnatal infection by ingesting oocysts from contaminated food or water. Toxoplasma gondii infection is common in white-tailed deer, Odocoileus virginianus, but little is known about the prevalence of infection in mule deer, O. hemionus. We examined sera from 89 mule deer from Nebraska for agglutinating antibodies to T. gondii using the modified direct agglutination test (MAT) with formalin-fixed tachyzoites as antigen. Thirty-one (35%) of the samples were positive at dilutions of > or = 1:25. Samples were examined from 29 fetuses from these mule deer and none were positive in the MAT. Sera from 14 white-tailed deer from Nebraska were also examined and 6 (43%) were positive for T. gondii. Samples were examined from 5 fetuses from these white-tailed deer and none was positive in the MAT. Our results in both deer species from Nebraska are similar to studies conducted in white-tailed deer from other regions of the United States. Our findings indicate that mule deer are frequently infected with T. gondii and that mule-deer meat may be a source of human infection.

Agglutination Tests↗
PubMed2006

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.

Agglutination Tests↗
PubMed2006

Characterization of Toxoplasma gondii isolates in free-range chickens from Amazon, Brazil.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 50 free-range chickens (Gallus domesticus) from Amazon, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 33 (66%) chickens with titers of 1:5 in 3, 1:10 in 2, 1:20 in 1, 1:40 in 1, 1:80 in 2, 1:160 in 5, 1:200 in 9, 1:400 in 5, 1:800 in 2, 1:1,600 in 2, and 1:3,200 or higher in 1. Hearts and brains of 33 seropositive chickens were bioassayed individually in mice. Tissues from 17 seronegative chickens were pooled and fed to 2 T. gondii-free cats. Feces of cats were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 24 chickens with MAT titers of 1:5 or higher. Genotyping of these 24 T. gondii isolates by polymorphisms at the SAG2 locus indicated that 14 were type I, and 10 were type III; the absence of type II strains from Brazil was confirmed. Fifty percent of the infected mice died of toxoplasmosis, irrespective of the genotype.

Agglutination Tests↗
PubMed2006

Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies.

Cats are important in the natural epidemiology of Toxoplasma gondii, because they are the only hosts that can excrete environmentally resistant oocysts. Cats are infected with T. gondii via predation on infected birds and rodents. During 2005, 238 rats (Rattus norvegicus) were trapped in Grenada, West Indies, and their sera along with tissue samples from their hearts and brains were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test (MAT, titer 1:40 or higher); only 2 (0.8%) of 238 rats were found to be infected. Brains and hearts of all rats were bioassayed in mice. Toxoplasma gondii was isolated from the brain and the heart of only 1 rat, which had a MAT titer of 1:320. All of 5 mice inoculated with the heart tissue, and the 5 mice inoculated with the brain tissue of the infected rat remained asymptomatic, even though tissue cysts were found in their brains. Genetically, the isolates of T. gondii from the heart and the brain were identical and had genotype III by using the SAG1, SAG2, SAG3, BTUB, and GRA6 gene markers. These data indicate that rats are not important in the natural history of T. gondii in Grenada.

Agglutination Tests↗
PubMed1990

Immune responses against Toxoplasma and Sarcocystis infections in ruminants: diagnosis and prospects for vaccination.

Toxoplasma gondii and a variety of Sarcocystis species are common cyst-forming coccidian parasites of domestic ruminants worldwide. T. gondii infection is a major cause of ovine and caprine abortion and perinatal mortality, but does not appear commonly to cause significant disease in other ruminants. Sarcocystis infections are probably most important in growing ruminants in which they can cause subclinical anaemia and reduced weight gain. The life cycles, pathogenesis and current knowledge of immunity to these protozoa, in ruminants, are reviewed. Although clinical signs may suggest a diagnosis, confirmation requires the use of specific immunological methods such as serology and immunohistochemistry. These are discussed and the necessity for using characterised antigens and antisera is emphasised. Whereas live Toxoplasma "vaccines" may be effective in sheep and goats, the eventual preparation of a killed vaccine capable of inducing long-term protection is of the utmost importance. To achieve this goal it will be necessary to identify the specific antigens capable of stimulating protective immunity and to conduct further research on both the pathogenesis of toxoplasmosis and fundamental aspects of the immune response of pregnant ruminants to T. gondii.

Animals↗
PubMed2005

Quinolone-based drugs against Toxoplasma gondii and Plasmodium spp.

Owing to the rapid emergence of multi-resistant strains of Plasmodium spp. (the causative agents of malaria) and the limitations of drugs used against Toxoplasma gondii (an important opportunistic pathogen associated with AIDS and congenital birth defects), the discovery of new therapeutical targets and the development of new drugs are needed. The presence of the prokaryotic-like organelle in apicomplexan parasites (i.e. plastids), which comprise these major human pathogens, may represent a unique target for antibiotics against these protozoa. Quinolones which are known to be highly potent against bacteria were also found to specifically disrupt these parasites. They inhibit DNA replication by interacting with two essential bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. There are some clues that quinolones act on plastids with a similar mechanism of action. After a brief presentation of plasmodium and toxoplasma dedicated to their life cycle, the chemotherapies presently used in clinics to fight against these protozoa and the potential new targets and drugs, we will focus our attention on their plastid which is one of these promising new targets. Then, we will present the various drugs and generations of quinolones, the leading molecules, and their inhibitory effects against these parasites together with their pharmacological properties that have been established from in vitro and in vivo studies. We will also discuss their possible mode of action.

Animals↗
PubMed2002

Prospects for a human Toxoplasma vaccine.

Human toxoplasmosis is usually benign, but may occasionally lead to severe or lethal damages when combined with immunosuppressive states or when transmitted to the fetus during pregnancy. Only a vaccine could prevent these harmful effects. The oral route is the natural portal of entry of T. gondii. A protective immune response at the mucosal level is required to kill the parasite as soon as it penetrates the intestinal barrier thus preventing toxoplasma from invading the host and settling into tissues. The probable major roles played by both CD8 T cells and antibodies, specially IgA, suggest that the best strategy would be to stimulate both the cellular and humoral arms of the mucosal immune system. Mucosal dendritic cells have been shown to induce good protection against oral toxoplasma challenge. Our hypothesis is that an acceptable and effective human vaccine would have to carry the optimized synthetic vaccine (subunit, DNA or replicon) plus an appropriate adjuvant and to target the mucosal dendritic cells by means of an inert delivery system such as polymer microparticles, which can be endocytosed by M cells of the gut or nasal-associated lymphoid tissues.

Animals↗
PubMed1985

Detection of Toxoplasma-specific IgM in cord blood sera by antibody-class capture enzyme-linked immunosorbent assay.

An antibody class capture (ACC) enzyme-linked immunosorbent assay (ELISA) utilizing an F(ab)2 conjugate was established and its specificity and reproducibility were determined. The test was further validated by comparing its results with those obtained by 2 overseas reference laboratories which used the indirect immunofluorescence antibody test (IIFT) for Toxoplasma-specific IgM. It was found that the F(ab)2ACC-ELISA was not affected by the presence of rheumatoid and antinuclear factor in serum, that it was reproducible (coefficients of variation less than 10.0%), and that it compared well with tests currently used overseas. ELISA was then used to test for the presence of Toxoplasma-specific IgM in cord blood sera. The results obtained to date are consistent with the hypothesis that the prevalence of congenital toxoplasmosis in the population of Adelaide is less than 1/2,000.

Antigens, Protozoan↗
PubMed1997

Nucleotide sequence of ToxPK1 gene from Toxoplasma gondii.

We report here for the first time a complete nucleotide sequence (6.8 kb) of a protein kinase gene (ToxPK1) from the obligate intracellular protozoan parasite of man, Toxoplasma gondii. This gene comprising putatively of 9 exons and 8 introns forms the Toxoplasma gene with the largest number and size of introns reported so far. The predicted protein with 508 amino acids contains the 15 invariant residues as well as the characteristic motifs specific to protein serine/threonine kinases. Homology-based computational comparisons suggested that TOXPK1 belongs to or closely resembles the SNF1 subfamily of protein-serine/threonine kinases. Based on the functions of SNF1 homologs in other organisms and our RT-PCR results, it is likely that TOXPK1 may be transiently expressed to up-regulate glycogen biosynthesis during the development of tachyzoites into bradyzoites.

Amino Acid Sequence↗
PubMed1979

The prevalence of toxoplasma antibodies among 11,736 pregnant women in Norway.

A serologic screening for toxoplasma antibodies by dye test (DT) and IgG indirect fluorescent antibody test (IgG-IFAT) has been carried out among 10729 pregnant women in the Oslo area and 1 007 pregnant women from the county of Møre and Romsdal situated on the west coast of Norway. The frequency of toxoplasma antibodies was found to be higher in women living in rural areas and a higher prevalence was observed among women living in the western districts of Oslo than among women from the eastern districts of the city. The mean frequency of DT antibodies was found to be 12.5% in the Oslo area and 13.3% in Møre and Romsdal, whereas the yearly incidence of acquired infections in the two areas was found to be 5 and 2.3%, respectively. The predicted incidence of congenital toxoplasmosis in the Oslo area, and presumably also in the whole country, would represent 2% of all pregnancies. No serologic differences were found when women with prior histories of either sporadic or habitual abortions were compared to a total group of multigravidae among the Oslo women.

Antibodies↗
PubMed1991

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

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

Animals↗
PubMed1989

Characterization of proteases of Toxoplasma gondii.

The proteases of Toxoplasma gondii were purified partially and characterized for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteases working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion exchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotors. The protease working at pH 6.0 was inactivated by iodoacetamide with LD50 of 10(-3) M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10(-5) M and was promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.

Adenosine Triphosphate↗
PubMed1992

[Changes in blastogenic responses and antibody titers of mice infected with Toxoplasma gondii].

This study was performed to observe the cell-mediated and humoral immune responses in mice which were infected with Beverley, Fukaya and ME49 strain of Toxoplasma gondii, respectively. The blastogenic responses of splenocytes using [3H]-thymidine and serum antibody titers were measured weekly up to 10 weeks after infection. The blastogenic responses of splenocytes treated with concanavalin A and Toxoplasma lysate were significantly declined in the 3 strain groups as compared with the non-infected group (p less than 0.05), however lipopolysaccharide-treated blastogenic responses were not significantly different between infected and non-infected groups. The serum IgG antibody titers in the three infected groups increased from 2 weeks after infection, and the serum IgM antibody titers increased until 4 weeks after infection. No significant differences were revealed in blastogenic responses and serum antibody titers among the 3 groups. The present study suggested that cell-mediated immune responses were involved in T. gondii infected mice and blastogenic responses of T lymphocytes were inhibited in acute T. gondii infection.

Animals↗
PubMed1992

Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme-linked immunosorbent assay.

Sensitivity of anti-Toxoplasma antibody (IgG) test by enzyme-linked immunosorbent assay (ELISA) was evaluated in comparison with indirect latex agglutination (ILA) using 2,016 paired human samples of serum and cerebrospinal fluid (CSF). The samples were collected from neurologic patients in Korea with mass lesions in central nervous system (CNS) as revealed by imaging diagnosis (CT/MRI). When the sera were screened for anti-Toxoplasma antibody by ILA, 76 cases(3.8%) were positive (1:32 or higher titers). In the paired samples of CSF, no positive reactions were observed. When ELISA was performed using PBS extract of Percoll purified tachyzoites as antigen, cut-off absorbance was determined as 0.40 for serum and 0.27 for CSF tests. The antibody positive rates by ELISA were 7.0% in serum and 5.6% in CSF. Of them, 40 cases (2.0%) showed positive reactions in both serum and CSF. The antibody positive rates were higher in groups older than 40 years. The rates were higher in male (4.7% by ILA, 8.3% by ELISA) than in female (2.2% by ILA, 5.0% by ELISA). The rates in CSF showed no such sex difference. ELISA showed twice higher positive rates when serum was tested, and was sensitive enough to detect specific antibodies in CSF. Etiologic relations between positive antibody tests and CNS lesions remained unknown.

Age Factors↗