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PubMed1992

Construction of a molecular karyotype for Toxoplasma gondii.

As an important step in developing genetic systems for the protozoan parasite Toxoplasma gondii, we have constructed a molecular karyotype based on separation of chromosomes by pulsed field gel electrophoresis and assignment of linkage groups by hybridization. Toxoplasma chromosomes were separated using transverse alternating field electrophoresis (TAFE) gels into 9 distinct bands that defined a minimum of 10 physical linkage groups with apparent sizes that range from approximately 2 Mb to more than 6 Mb. Individual chromosome sizes were stable with prolonged mitotic passage of a single strain but varied by approximately 15% for chromosomes III and V between three different strains of Toxoplasma. Preliminary physical linkage groups were defined by mapping 57 single or low copy number probes to specific chromosomes by hybridization. The majority of these probes consist of random DNA segments; however, a number of cDNAs encoding important structural and antigenic components were also mapped to specific linkage groups. Assuming random distribution, this set of probes should provide approximately 1 marker every 1.0-1.5 Mb over the 80 Mb haploid genome and should greatly aid in using genetics to study the biology, drug resistance, and virulence of this important opportunistic pathogen.

Animals↗

PubMed1995

Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection.

The ROP1 gene of Toxoplasma gondii encodes a rhoptry protein that has been implicated in host cell invasion by this obligate intracellular protozoan. To further explore the function of this protein, we created a ROP1 deletion mutant by transfection with a plasmid encoding the bacterial chloramphenicol acetyltransferase (cat) gene flanked by ROP1 genomic sequences. Selection for chloramphenicol resistance yielded the desired ROP1-deleted or 'knock-out' mutant. Analysis of this mutant both in vitro and in vivo shows no significant alterations in growth rate, host specificity, invasiveness or virulence and thus the ROP1 gene is not obligatory for the RH strain, at least under the conditions tested. However, electron microscopy reveals that the mutant strain's rhoptries are altered in ultrastructure; they are thinner and homogeneously electron-dense compared with the thicker and normally mottled or honeycombed appearance of wild-type rhoptries. The knock-out mutant was rescued using co-transfection of a cosmid carrying the ROP1 gene together with a plasmid encoding a new selectable marker for T. gondii, the bleomycin resistance gene (ble) from Streptoalloteichus. Southern blot analysis showed that both DNAs were stably integrated into the Toxoplasma genome, although not into the ROPI locus. The resulting strain showed wild-type levels of ROP1 expression and rescue of the ultrastructural phenotype (i.e., the rhoptries returned to their normal, mottled appearance), thus establishing a cause/effect relationship between the absence of ROP1 and the electron-opacity. These results demonstrate the utility of the reverse genetic approach in the study of Toxoplasma gene function and provide a further selectable marker for such manipulations.

Animals↗

PubMed1992

Prevalence of antibodies to Toxoplasma gondii and Trichinella spiralis in 509 pigs from 31 farms in Oahu, Hawaii.

Serum samples from 509 pigs from 31 farms in Oahu, Hawaii were examined for antibodies to Toxoplasma gondii using the agglutination test in dilutions of 1:25, 1:50, and 1:500 and for Trichinella spiralis using the enzyme-linked immunosorbent assay. Toxoplasma gondii antibodies were found in 48.5% of pigs. Antibody titers were: 5.1% positive at 1:25 dilution, 28.6% positive at 1:50 dilution and 14.7% positive at 1:500 dilution. The prevalence of Toxoplasma gondii antibodies in garbage-fed pigs (67.3% of 199 pigs) was higher than in grain-fed pigs (33.8% of 180 pigs). Antibodies to Trichinella spiralis were found on seven of 31 farms. On five of these farms only a single serum was positive for Trichinella spiralis antibodies. The two remaining farms each had three positive sera (three of ten and three of 25 pigs tested); both of these farms fed garbage to pigs.

Agglutination Tests↗

PubMed2005

Direct genotyping of animal and human isolates of Toxoplasma gondii from Colombia (South America).

Genetic analysis of the SAG2 locus was performed to determine the prevalence of the main genotypes of Toxoplasma gondii (SAG2 types I, II, and III) associated with humans, cats, birds and guinea pig toxoplasmosis in Colombia. This typing was directly performed on clinical samples and autopsy material from human or animals. A total of 50 from 146 samples were positive by specific B1 Toxoplasma PCR assay and then were analyzed by restriction fragment length polymorphism in PCR-amplified SAG2 products. Characterization of the SAG2 gene was successful in 33 (66%) of the samples. Genotyping indicated that 31 (93.9%) were SAG2 type I, 1 was SAG2 type III and 1 was atypical. In birds and cats all samples were SAG2 type I. Results support a predominance of the Toxoplasma SAG2 type I circulating in human and animals in South America.

Animals↗

PubMed2006

Toxoplasma gondii as a risk factor for early-onset schizophrenia: analysis of filter paper blood samples obtained at birth.

BACKGROUND: Infections during fetal life or neonatal period, including infections with Toxoplasma gondii, may be associated with a risk for schizophrenia and other mental disorders. The objectives of this study were to study the association between serological markers for maternal and neonatal infection and the risk for schizophrenia, related psychoses, and affective disorders in a national cohort of newborns. METHODS: This study was a cohort-based, case-control study combining data from national population registers and patient registers and a national neonatal screening biobank in Denmark. Patients included persons born in Denmark in 1981 or later followed up through 1999 with respect to inpatient or outpatient treatment for schizophrenia or related disorders (ICD-10 F2) or affective disorders (ICD-10 F3). RESULTS: Toxoplasma gondii immunoglobulin G (IgG) levels corresponding to the upper quartile among control subjects were significantly associated with schizophrenia risk (odds ratio [OR] = 1.79, p = .045) after adjustment for urbanicity of place of birth, year of birth, gender, and psychiatric diagnoses among first-degree relatives. There was no significant association between any marker of infection and other schizophrenia-like disorders or affective disorders. CONCLUSIONS: Our study supports an association between Toxoplasma gondii and early-onset schizophrenia. Further studies are needed to establish if the association is causal and if it generalizes to cases with onset after age 18.

Animals↗

PubMed2006

Toxoplasma gondii: cross-immunity against the enteric cycle.

Eight of nine cats inoculated with strain ME-49 and challenged with three different strains of Toxoplasma were immune to oocyst shedding, as ascertained with bioassays of their feces. In a second experiment, only toxoplasma asexual stages were seen in H&E stained gut sections of cats treated with suppressive doses of sulfamerazine and pyrimethamine starting 2 days after oral inoculation with cysts of the strain ME-49 and killed 6 days later. In a third experiment, four cats were similarly inoculated and treated for 20 days. Six weeks later, the cats received an oral homologous challenge with cysts, and none shed toxoplasma oocysts. An acceptable level of cross-protection was achieved with strain ME-49, and therefore, it can be used as a candidate strain from which antigens could be tested for enteric protection.

Animals↗

PubMed2004

Neutrophils, dendritic cells and Toxoplasma.

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.

Animals↗

PubMed2006

Proliferation of Toxoplasma gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production in the host cell.

While reactive oxygen species (ROS) can kill Toxoplasma gondii in vitro the role these molecules play in vivo is not known. We used a flow cytometry-based assay to investigate the relationship between intracellular infection and ROS production during acute peritoneal toxoplasmosis in mice. A distinct population of ROS(+) inflammatory macrophages, detected by the oxidation of hydroethidine, was observed to increase progressively in frequency during the course of infection, and to be inversely correlated with the degree of cell parasitization. These data imply that either intracellular parasites inhibit ROS synthesis or, alternatively, ROS-producing cells contain anti-Toxoplasma activity. The latter interpretation was supported by the finding that uninfected ROS-producing inflammatory macrophages were resistant to infection in vivo. However, in the same animals, ROS-producing macrophages that had previously been parasitized could readily be infected with additional parasites, suggesting that the difference in ROS production between highly infected and less infected cells was not due to ROS-associated killing of parasites within these cells. In addition, macrophages infected with T. gondii in vitro and then briefly transferred to acutely infected mice upregulated ROS production in a manner that was again inversely correlated with the degree of intracellular parasitization. Taken together, these findings suggest that both ROS-associated anti-Toxoplasma activity and parasite-driven inhibition of ROS production underlie the observed pattern of ROS production. ROS function and parasite evasion of this function may contribute significantly to the balance between host defense and disease progression during acute infection.

Acute Disease↗

PubMed2005

Intracranial Toxoplasma gondii granuloma in a cat.

An 8-year-old cat with recent-onset generalized seizures was diagnosed with a right forebrain mass using magnetic resonance imaging. The mass was excised and upon histologic and immunohistochemical examination shown to be a Toxoplasma gondii granuloma. Serology supported active T gondii infection. The cat was treated with phenobarbital to control seizures. After definitive diagnosis of toxoplasma granuloma, clindamycin was administered for approximately 1 month. Seizures recurred 8 months after initial presentation, and the cat was euthanased at the owner's request. This is a previously unreported manifestation of feline central nervous system toxoplasmosis. When a mass lesion is present in the brain of a cat and serologic test results support active infection with T gondii, toxoplasma granuloma must be a differential diagnosis. If the patient is suffering from clinical disease, surgical resection of the mass (if possible) can be complimented with medical treatment until definitive diagnosis is obtained. Immunocompromising factors should be identified and addressed if possible.

Animals↗

PubMed2005

The Toxoplasma surface protein SAG1 triggers efficient in vitro secretion of chemokine ligand 2 (CCL2) from human fibroblasts.

Chemokines play an important role in the physiopathology of toxoplasmosis in murine models. Infection of different human cell types by Toxoplasma gondii induces the secretion of these immune mediators. The aim of our study was to identify parasite molecules that could be involved in the triggering of chemokine ligand 2 (CCL2) secretion during T. gondii host cell invasion: surface, micronemal, rhoptry and dense granule proteins. The secretion of CCL2 was studied 1) after infection of human fibroblasts with mutants of Toxoplasma RH strain deficient either for GRA5, GRA2-GRA6, ROP1 or SAG1; 2) after stimulation by micronemal proteins or by the immunodominant surface antigen 1 of T. gondii. CCL2 secretion was quantified by ELISA at 3 h and/or 24 h after infection or stimulation. Infection by Deltagra2-Deltagra6, Deltagra5 or Deltarop1 mutants did not modify the level of CCL2, as compared with the level measured after infection with the wild-type strain. Moreover, stimulation with micronemal proteins did not increase the secretion of this chemokine. By contrast, the level of CCL2 was increased 3 h post-stimulation by purified or recombinant SAG1. Specificity of this effect was confirmed by the decrease in CCL2 secretion when human fibroblasts were infected with the Deltasag1 mutant (48%) as compared with the wild-type strain (100%). In conclusion, this major Toxoplasma surface protein SAG1, specific to the tachyzoite stage, is directly or indirectly involved in the cellular mechanisms triggering CCL2 secretion after T. gondii infection. These results could explain the parasitic mechanisms leading to cell infiltrates detected only in the presence of tachyzoites, a phenomenon observed in toxoplasmic reactivation.

Animals↗

PubMed2006

The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1.

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28alpha/beta complex.

Animals↗

PubMed2005

Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall.

Recent outbreaks of waterborne toxoplasmosis have stimulated the development of sensitive methods to detect Toxoplasma gondii oocysts in samples suspected to be contaminated. The immunomagnetic separation (IMS) have been standardised to detect waterborne protozoa, but it did not exist for Toxoplasma oocysts. In this study, we describe two monoclonal antibodies (mAbs 3G4 and 4B6) produced against the oocyst wall, and the incorporation of mAb 3G4 in an IMS procedure. We found that an indirect IMS method gave better mean recoveries than a direct one (69.4% and 25.2%, respectively). Dissociation of oocyst_magnetic bead complexes was greatly improved by using a 2% aqueous H2SO4 solution instead of a 0.1 N HCl solution (82.8% and 17.4%, respectively). With these parameters, mean recoveries of less than 1000 oocysts ranged from 44.6% to 82.9%, depending on incubating temperature and buffer. Age of oocysts (1 or 12 months old) does not influence IMS performances. Results of this study indicate that the described IMS is an efficient technique to recover Toxoplasma oocysts.

Animals↗

PubMed2004

A cross-sectional survey of anti-Toxoplasma gondii antibodies in Jerusalem cats.

A cross-sectional seroprevalence study of anti-Toxoplasma gondii IgG antibodies was performed in Jerusalem domestic and stray cats. An enzyme linked immunosorbent assay (ELISA) immunodiagnostic technique was used and results analyzed according to specific variables. The overall seroprevalence was 16.8%. After an initial high seroprevalence of 29.7% in kittens after 1-10 days of age, average positive titers dropped to a low of 7.0% at ages 11-60 days. Thereafter, average positivity increased continuously and was observed in 50% of cats more than 5 years of age (P = 0.0081). Toxoplasma seroprevalence was highest in the month of summer (25.1%, P < 0.0001); domestic indoor cats had a higher seropositivity (39.0%) than stray cats (14.2%, P = 0.0004). On average, cats from the mainly Arab inhabited areas of Jerusalem showed a higher positive seroprevalence to Toxoplasma than Jewish inhabited areas (34.1% and 16.0%, respectively, P = 0.0032). There were no significant differences in positivity rates between sexes, and between rates of those with the presence/absence of clinical symptoms similar to those of the disease.

Age Factors↗

PubMed2006

Seroprevalence of Toxoplasma gondii infection in stray and household cats in Tehran.

The prevalence of anti-Toxoplasma gondii specific IgG in stray and household cats in Tehran was determined by Indirect Fluorescent Antibody Test (IFAT) on serum samples from 100 cats (50 stray and 50 households). Overall infection rate was 63%. The infection rate in stray cats (90%) was significantly higher (p<0.001) than that of household cats (36%). Last serum positive dilutions varied from 1: 32 to 1: 512 titres in which the highest percentage (27%) was for 1:256 and the least (4.8%) was at 1:32. The rate of infection between male and female cats of both groups was not significantly different; 90.3% versus 89.5% for male and female in stray cats, respectively. Different sexes of household cats were seropositive at the same rate (36%). A high positive correlation (r(2)=0.97) between age and the rate of infection was observed. The prevalence of Toxoplasma gondii infection in cats in Tehran was high, especially in stray cats which are probably the main source of Toxoplasma infection in this area.

Age Factors↗

PubMed1986

The ultrastructural pathology of congenital murine toxoplasmic retinochoroiditis. Part I: The localization and morphology of Toxoplasma cysts in the retina.

This study describes the ultrastructural characteristics of retinal parasitization by Toxoplasma gondii in a congenital mouse model. Forty-two eyes from infected mice, 18-22-weeks-old, and 24 control eyes were initially studied by light microscopy of semithin sections. Twenty-six eyes from infected animals and six from the controls were further investigated by transmission electron microscopy. A total of 13 Toxoplasma cysts was found in samples of the retinas of six eyes from five infected animals. These were located in the inner retina, particularly the ganglion-cell layer, but in no other ocular tissue. The cyst wall interdigitated with the host cell which in most cases was probably glial in origin (Müller cell). Two cysts showed evidence of parasitization of neural cells. The individual Toxoplasma cystozoites demonstrated characteristic ultrastructural features. There was no evidence of morphological changes indicative of toxicity to surrounding retinal tissues, and the associated inflammatory cell reaction (described in Dutton, McMenamin, Hay and Cameron, 1986b) was remote from the parasite. There was no morphological evidence of rupture or degeneration of cysts. No free parasites (endozoites) or pseudocysts were observed.

Animals↗

PubMed1998

Association of host cell intermediate filaments with Toxoplasma gondii cysts in murine astrocytes in vitro.

Toxoplasma gondii is an intracellular parasite that is a common opportunistic infection of AIDS patients where it causes a severe and often fatal encephalitis. Toxoplasmic encephalitis in AIDS patients results from a reactivation of the cyst stage of Toxoplasma gondii in the brain. A previous study found an association of host cell intermediate filaments with parasitophorous vacuoles and some studies have suggested the host cell cytoskeletal elements are incorporated into the cyst wall. In this study, the interaction of glial filaments with Toxoplasma gondii cysts was studied in cysts derived in vitro in mouse astrocytes and in cysts isolated from mouse brains. Glial filaments, detected by immunostaining of the glial fibrillary acidic protein, were found to accumulate around the perimeter of the cysts as they developed in mouse astrocytes. Transmission electron microscopy revealed a layer of glial filaments was wrapped around the cytoplasmic side of the cyst. The glial filaments were present in close apposition to the cyst wall and arranged around the cysts in a concentric layer, measuring 5-10 microns in thickness. The layer of glial filaments excluded host cell mitochondria and endoplasmic reticulum from the cytoplasmic surface of the cyst. Colocalisation of glial fibrillary acidic protein and the cyst wall via confocal and immunoelectron microscopy, confirmed that there was no glial fibrillary acidic protein present within the cyst wall. The cyst wall of cysts isolated from mouse brains were also found to be negative for glial fibrillary acidic protein. In conclusion, we found no evidence of structural integration of the host cell intermediate filaments in the cyst wall, but glial filaments were found to encase the cysts in the host cell during cyst development in host cells in vitro. The glial filaments wrapping of cysts may play a role in bradyzoite differentiation and/or cyst stabilisation in the host cell cytoplasm.

Animals↗

PubMed2000

Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR.

We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.

Animals↗

PubMed1997

Assessment of enzyme linked immunosorbent assay based diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii in human serum.

An enzyme linked immunosorbent assay (ELISA) using penicillinase was developed in the form of diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii. The performance of both the kits was compared with commercially available diagnostic kits, i.e. Enzygnost-Toxoplasmosis/IgG (Behring Co., Germany), TOXOTEK-G (Flow Lab., U.K.) and Toxoplasma IgM Microassay (Diamedix Corp., U.S.A.) by testing toxoplasma-suspected human serum samples. The results indicate a good reliability between these diagnostic kits. Toxokit-G has 86.66 and 96.05% sensitivity and specificity respectively. The main advantage of Toxokit-G is that the end result can be assessed visually without using sophisticated instruments. Toxokit-M has 100% sensitivity and specificity and test results were not affected by the presence of antitoxoplasma IgG antibodies, rheumatoid factor or antinuclear antibodies.

Animals↗