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PubMed1987

Experimental studies on circulating antigen of Toxoplasma gondii in intermediate hosts: criteria for detection and structural properties.

The present study was performed to demonstrate circulating antigen (cag) of Toxoplasma gondii in the sera of orally and intraperitoneally infected rabbits and swine, to determine the time of their appearance after infection, and to characterize the antigenic components of the cag by means of affinity chromatography, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic blot onto nitrocellulose sheets. Cag, as detected by an indirect ELISA, was found in the sera of both rabbits and swine from week 5 to week 8 after infection. Electrophoretic separation of cag extracted from swine and human sera showed 5 and 8 distinct protein bands, respectively, the molecular weight of which ranged between 25 and 100 kD. After Western blot, 2 of the 5 protein bands of swine-cag (27 and 57 kD) and 3 of the 8 protein bands of human cag (27, 32, and 57 kD) reacted with the anti-Toxoplasma antibody used in the ELISA. On the basis of the data presented, the influence of the dose and mode of infection as well as that of the preparation method of antisera on cag detection is discussed.

Animals↗
PubMed1988

Evidence of structural proteins of Toxoplasma gondii in sera of experimentally infected mice.

The present study was performed to clarify whether structural proteins are constituants of the antigens of Toxoplasma gondii which circulate in the sera of experimentally infected mice. Rabbits were immunized with mice sera containing circulating antigen, the rabbit sera were then tested for antibodies against Toxoplasma gondii. Low titers of specific antibodies, directed against cell wall proteins, could be detected. Thus, the circulating antigen must at least partially consist of structural proteins.

Animals↗
PubMed1997

Prenatal diagnosis and treatment of congenital Toxoplasma gondii infections: an experimental study in rhesus monkeys.

The efficacy of treatment in fetuses in whom congenital Toxoplasma gondii infection has ben established has been investigated using rhesus monkeys as a model for humans. A polymerase chain reaction has been developed for the detection of Toxoplasma gondii. Using this polymerase chain reaction congenital infection can be established within 2 days of receiving an amniotic fluid sample. The polymerase chain reaction has subsequently been used to monitor the effect of treatment on fetal infection. The results show that early treatment with the combination of pyrimethamine and sulfadiazine was clearly effective in reducing the number of parasites in the infected fetus. The parasite was no longer detectable in the amniotic fluid 10 to 13 days after treatment was started. Spiramycin, on the other hand, has to be administered for at least 3 weeks to achieve the same effect. Moreover, pharmacokinetic studies revealed that spiramycin does not reach the brain. Pyrimethamine and sulfadiazine are able to pass the blood-brain barrier. Pyrimethamine appears to accumulate in the brain tissue and reaches concentrations which are also effective in vitro.

Amniotic Fluid↗
PubMed2001

Detection of IgG antibodies to Neospora caninum and Toxoplasma gondii in dogs examined in a veterinary hospital from Brazil.

A total of 163 dogs with neuromuscular, respiratory and/or gastrointestinal disorders, was admitted at the Veterinary Hospital, Federal University of Uberlândia, Brazil, and submitted to serology for Toxoplasma gondii and Neospora caninum. Assays for T. gondii included indirect haemagglutination (IHA), indirect fluorescent antibody (IFAT-Tg), immunoenzymatic (ELISA), and immunoblotting (IB-Tg). Assays for N. caninum included IFAT-Nc and immunoprecipitation (IP-Nc). Based on concordant results by three serological tests (IHA, IFAT-Tg and ELISA) for T. gondii, and divergent results further confirmed by IB-Tg for reactivity to TgSAG1, the 163 sera were divided into two groups: 59 (36%) Tg-seropositive samples and 104 (64%) Tg-seronegative samples. Antibodies to Neospora were detected in 11 (6.7%) out of 163 analyzed dog sera, with 5 (3.1%) samples reactive to both parasites (Tg+/Nc+), and 6 (3.7%) reactive only to Neospora (Tg-/Nc+). Antibodies only to T. gondii were found in 54 (33%) samples. Among the 11 Neospora-positive sera analyzed by IB-Tg, the five sera Tg+/Nc+ showed strong reactivity to Toxoplasma antigens, especially to TgSAG1 (p30). No reactivity was observed to TgSAG1 in the six samples Tg-/Nc+. By IP-Nc, two highly immunodominant antigens (29 and 35kDa proteins) were recognized by all 11 IFAT-Nc positive sera. Our results suggest that the infection by N. caninum can be concomitantly present in dogs from this area, although less common, and therefore should be considered in the differential clinical diagnosis with T. gondii in dogs presenting neuromuscular, respiratory and/or gastrointestinal disorders.

Animals↗
PubMed2001

Toxoplasma gondii induction of interleukin-12 is associated with acute virulence in mice and depends on the host genotype.

The intracellular parasite Toxoplasma gondii can influence host resistance by modulating immune functions in various cell types. The stimulation of interleukin (IL)-12 production in macrophages, dendritic cells and neutrophils by T. gondii has been implicated to be important for skewing anti-parasite immunity early after infection as well as in mediating the pathologic effects induced by the parasite. The present study demonstrates secretion of IL-12 p40 and the bioactive p70 heterodimer by inflammatory macrophages following exposure to live Toxoplasma or tachyzoite lysate. Parasite induction of IL-12 occurred in a dose-dependent manner. Predigestion of T. gondii lysate with proteinase K abrogated its IL-12 inducing activity, thus indicating that a parasite protein(s) triggers this response. Macrophages from various mouse inbred strains showed a differential responsiveness: cells from T. gondii-susceptible mice released more IL-12 upon toxoplasmic challenge than those from resistant mice, although the infection rate and intracellular parasite growth were similar. In triggering macrophage production of IL-12, tachyzoites proved superior to bradyzoites prepared from the same T. gondii isolate. Furthermore, parasites of a mouse-virulent isolate became less efficient inducers of IL-12 following attenuation. The parallel loss in macrophage stimulation in vitro and acute virulence in vivo suggests a linkage of both parasite capacities. Together with the correlation on host side between the genotype-dependent mouse susceptibility to infection and cellular responsiveness to the parasite trigger, these findings indicate that an overproduction of parasite-induced IL-12 might represent a basic mechanism of T. gondii pathogenicity.

Animals↗
PubMed1997

Long-term humoral antibody responses by various serologic tests in pigs orally inoculated with oocysts of four strains of Toxoplasma gondii.

Antibody titers to Toxoplasma gondii were determined in 16 pigs orally inoculated with 1000 or 10,000 oocysts of one of the four strains (GT-1, ME-49, TS-2, TC-2) of T. gondii. Pigs were euthanized on postinoculation days 103-875 and their tissues were bioassayed for T. gondii. Antibody titers were measured in the modified agglutination test (MAT) using formalin-preserved (test A) or acetone-preserved (test B) tachyzoites, latex agglutination test (LAT), indirect hemagglutination test (IHA), enzyme-linked immunobsorbant assay (ELISA), and the Sabin-Feldman dye test (DT). Toxoplasma gondii was isolated from all but two (one with GT-1 strain and one with TC-2 strain) of the inoculated pigs. Results of the serologic tests varied by test used, by strain of T. gondii and from pig to pig within groups. One pig inoculated with the TC-2 strain was considered not to be infected because it remained seronegative in all tests and T. gondii was not isolated from its tissues by bioassay. The IHA and LAT did not produce consistently positive results with infected pigs and two pigs remained seronegative (< 1:64) in both tests. At the time of necropsy, IHA titers had declined to < 1:64 in five pigs and LAT titers had declined to < 1:64 in four pigs. The MAT (test A) and ELISA detected antibodies in all infected pigs but ELISA did not detect antibody at the time of necropsy in one pig. Antibody titers peaked earliest in the DT. Antibody titers in the MAT (test B) peaked at 2560 in all seropositive pigs by 6 weeks postinoculation (PI) but declined to < 1:160 by 15 weeks PI; this test may be useful in determining recency of T. gondii infection in pigs. Overall, the MAT (test A) gave most consistent results.

Agglutination Tests↗
PubMed1997

Clinical observations, pathology, bioassay in mice and serological response at slaughter in pigs experimentally infected with Toxoplasma gondii.

Experimental infections of a total of 47 pigs with tachyzoites of the Toxoplasma gondii RH-strain, tissue cysts of the SSI-119 and R92 strains as well as oocysts of the SSI-119 strain were performed to determine the sensitivity of an indirect IgG-ELISA, using tachyzoite lysate of the RH-strain as antigen. The infections led to a dose dependent moderate clinical affection (inappetence, fever and poor general condition). Pigs infected with 10000 oocysts or with 1/2 mouse brain containing tissue cysts of the SSI-119 strain showed a significant decrease in weight gain compared to uninoculated pigs during the first 2 weeks p.i., followed, however, by compensatory growth during the next 6 weeks. At slaughter 3 to 4 months after inoculation 39/41 (95.1%) of pigs positive by bioassay in mice were seropositive in ELISA. Tissue cysts were not demonstrable by immunohistochemistry. ELISA OD-values obtained by analysis of meat juice from heart muscle and tongue (diluted 1:40) correlated strongly with OD-values by analysis of serum (diluted 1:400) (r heart juice = 0.942; r tongue juice = 0.915). Thus, meat juice samples were shown to provide a suitable alternative to serum for serological detection of Toxoplasma infection in pigs.

Abattoirs↗
PubMed1998

Shedding of Toxoplasma gondii oocysts by Felidae in zoos in the Czech Republic.

In 1995 and 1996, the shedding of Toxoplasma gondii oocysts was monitored in the faeces of Felidae in six zoos in the Czech Republic. In all, 2287 samples of faeces from 19 species of Felidae were examined. In Ostrava Zoo, four episodes of shedding of Toxoplasma-like oocysts were identified, using a flotation examination, in a pair of wild cats (Felis silvestris), six episodes in a wild cat held separately, and three episodes in a pair of Amur leopard cats (F. euptilurus). After the passage of sporulated oocysts through laboratory mice, T. gondii was confirmed in the pair of wild cats (three episodes), in the wild cat held separately (three episodes) and in the pair of Amur leopard cats (one episode). In Jihlava Zoo, one episode of shedding of T. gondii oocyst was identified in Geoffroy's cat (Oncifelis geoffroyi) using flotation and isolation examination. The possible sources of toxoplasmosis of the Felidae in zoos are discussed.

Animals↗
PubMed2000

Prospective study of IgM to Toxoplasma gondii on Beckman Coulter's Access(TM) immunoassay system and comparison with Zeus ELISA and gull IFA assays.

We compared a new assay for Toxoplasma IgM on the Access analyzer (Beckman Coulter, Inc., Chaska, MN, USA), a random access instrument based on the principle of paramagnetic particle enzyme immunoassay with an enzyme-linked immunosorbent assay (ELISA) (Zeus Scientific, Inc., Raritan, NJ, USA) and an immunofluorescent assay (IFA) (Gull Laboratories, Inc., Salt Lake City, UT, USA). Four hundred fresh, unfrozen clinical samples from pregnant women (n = 154), HIV positive patients (n = 41), and patients in whom infection with Toxoplasma gondii was suspected (n = 200) were collected and assayed over a three month period. The specificity of the Access assay was compared to the consensus results. Results that were discrepant between the ELISA and IFA were resolved using a third IFA (Zeus). Once resolved, the specificity for the Access assay, the Zeus ELISA and the Gull IFA were 99.22%, 97.91%, and 99.45%, respectively. We conclude that the Access assay specificity is comparable to consensus results, minimizing false positive results; and because it is a random access instrument, it may be preferable over batch methods.

Adolescent↗
PubMed1996

TNF-alpha enhances Toxoplasma gondii cyst formation in human fibroblasts through the sphingomyelinase pathway.

The cystogenesis event of Toxoplasma gondii is poorly understood. In order to throw light on it, the effect of tumor necrosis factor-alpha (TNF-alpha) was studied in the Prugniaud strain of the organism. This showed that TNF-alpha increased the number of cysts formed in vitro in human MRC5 fibroblasts. The sphingomyelinase pathway may be involved in mediating the TNF effect, since ceramide (natural form in permeabilized cells or cell-permeable analogue) could mimic the action of TNF. More precisely, our results strongly suggest the involvement of an acidic sphingomyelinase in mediating the effect of TNF; indeed, D609 inhibited both the TNF effect and cyst formation, while arachidonic acid had no effect. Moreover, protein kinase (PKC) seems also to play a role in the process, since phorbol-12-myristate-13-acetate (PMA) enhanced the cyst formation. However, chelerythrine chloride did not prevent the TNF effect, suggesting that several host-cell pathways can affect the cystogenesis event. Taken together, these results suggest the active participation of host-cell components in the cystogenesis of Toxoplasma gondii.

Alkaloids↗
PubMed2003

From cells to signaling cascades: manipulation of innate immunity by Toxoplasma gondii.

The intracellular opportunistic protozoan Toxoplasma gondii is a potent stimulus for cell-mediated immunity, and IL-12-dependent IFN-gamma induction is vital in resistance to the parasite. Dendritic cells, neutrophils and macrophages are important sources of IL-12 during infection. T. gondii possesses two mechanisms for triggering IL-12. One is dependent upon the common adaptor protein MyD88, and is likely to involve Toll-like receptors. The other is a more unusual pathway that involves triggering through CCR5 by a parasite cyclophilin molecule. Countering these potent pro-inflammatory activities, T. gondii has several mechanisms to down-regulate immunity. Intracellular infection causes a blockade in the NFkappaB macrophage signaling pathway, correlating with reduced capacity for IL-12 and TNF-alpha production. The parasite also prevents STAT1 activity, resulting in decreased levels of IFN-gamma-stimulated MHC surface antigen expression. Furthermore, infection also induces resistance to apoptosis through inhibition of caspase activity. Extracellular pathways of suppression involve soluble mediators such as IL-10 and lipoxins that have potent IL-12 down-regulatory effects. The balance of pro-inflammatory and anti-inflammatory signaling which T. gondii engages is likely dictated by requirements for a stable host-parasite interaction. First, there is a need for Toxoplasma to induce an immune response robust enough to allow host survival and establish long-term chronic infection. Second, the parasite must avoid immune-elimination and induction of pro-inflammatory pathology that can cause lethality if unchecked. The widespread distribution of T. gondii and the normally innocuous nature of infection indicate the skill with which the parasite achieves the two seemingly contrary goals.

Adaptor Proteins, Signal Transducing↗
PubMed1990

A rapid and simple method of purification of Toxoplasma gondii trophozoites originating from tissue culture for use in the indirect immunofluorescent antibody test.

A simple and convenient purification method for Toxoplasma trophozoites from tissue culture cells by density centrifugation is described. Separation of cells treated with formaldehyde is achieved by means of a Percoll solution with a density of 1.056 g/ml permitting removal of more than 99% of the cells and cell debris from the tissue culture. Thus highly purified trophozoites of Toxoplasma gondii raised in tissue cultures become available for serological tests, particularly for the indirect immunofluorescent antibody test.

Animals↗
PubMed2003

Migration of Toxoplasma gondii across biological barriers.

The molecular mechanisms underlying migration of pathogens across biological barriers remain poorly characterized. Following oral infection, the apicomplexan parasite Toxoplasma gondii actively crosses non-permissive biological barriers such as the intestine, the blood-brain barrier and the placenta, thereby gaining access to tissues where it causes severe pathology. Recently, enhanced migration was found to be associated with virulent strains of Toxoplasma, suggesting that this phenotype contributes to pathogenesis. The migratory machinery appears to be morphologically and functionally well conserved within the phylum of apicomplexan parasites, however, the mechanisms for cellular traffic to breach biological barriers remain to be elucidated. As penetration of host tissue is a prerequisite for the establishment of infections by most apicomplexan parasites, understanding parasite migration is crucial for the development of new approaches to combat disease.

Actin Cytoskeleton↗
PubMed2001

Tachyzoite-specific isoform of Toxoplasma gondii lactate dehydrogenase is the target antigen of a murine CD4(+) T-cell clone.

In two-dimensionally separated Toxoplasma gondii lysate, mouse Th1 clone 3Tx15 detects two proteins of apparent molecular weight 40000 and pI of 5.8 and 5.9. Microsequencing of peptide fragments from tryptic digestion of one of these proteins yielded partial sequences of T. gondii lactate dehydrogenase (LDH)1. As shown by Western blot, toxoplasmic LDH co-migrates in two-dimensional gel electrophoresis with both T-cell antigenic proteins. With synthetic peptides spanning the complete primary structure of T. gondii LDH1, the T-cell epitope was mapped to a nine amino acid partial sequence which exhibits a motif for binding to I-E(k), the class II restriction element of antigen recognition by clone 3Tx15. From the two known isoforms of T. gondii LDH, clone 3Tx15 specifically recognises tachyzoite LDH1, but not bradyzoite LDH2, as shown with the corresponding epitope peptides and recombinant proteins. Antigen-presenting cells infected with live bradyzoites stimulate 3Tx15 T cells, while killed bradyzoites provide no antigenic stimulus. This finding implies that a transformation into the tachyzoite stage occurs in cells challenged with bradyzoites. Although LDH1 represents one major constituent of the tachyzoite proteome, the protein does not seem to be immunogenic in T. gondii infection of mice. This is evident from the lack of serum anti-LDH immunoreactivity and the failure of adoptively transferred 3Tx15 T cells to protect against lethal challenge. In conclusion, a T-cell-stimulatory Toxoplasma antigen is identified by means of a novel, high-resolution T-cell blot technique, the clones antigenic fine specificity allowing detection of parasite-stage conversion.

Amino Acid Sequence↗
PubMed2002

Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii.

A number of cysteine and serine protease inhibitors blocked the intracellular growth and replication of Toxoplasma gondii tachyzoites. Most of these inhibitors caused only minor alterations to parasite morphology irrespective of the effects on the host cells. However, three, cathepsin inhibitor III, TPCK and subtilisin inhibitor III, caused extensive swelling of the secretory pathway of the parasite (i.e. the ER, nuclear envelope, and Golgi complex), caused the breakdown of the parasite surface membrane, and disrupted rhoptry formation. The disruption of the secretory pathway is consistent with the post-translational processing of secretory proteins in Toxoplasma, and with the role of proteases in the maturation/activation of secreted proteins in general. Interestingly, while all parasites in an individual vacuole (the clonal progeny of a single invading parasite) were similarly affected, parasites in different vacuoles in the same host cell showed different responses to these inhibitors. Such observations imply that there are major differences in the biochemistry/physiology between tachyzoites within different vacuoles and argue that adverse effects on the host cell are not always responsible for changes in the parasite. Treatment of established parasites also leads to an accumulation of abnormal materials in the parasitophorous vacuole implying that materials deposited into the vacuole normally undergo proteolytic modification or degradation. Despite the often extensive morphological changes, nothing resembling lysosomal bodies was seen in any treated parasites, consistent with previous observations showing that mother cell organelles are not recycled by any form of autophagic-lysosomal degradation, although the question of how the parasite recycles these organelles remains unanswered.

Animals↗
PubMed2002

Toxoplasma gondii tachyzoite-bradyzoite interconversion.

During infection in the intermediate host, Toxoplasma gondii undergoes stage conversion between the rapidly dividing tachyzoite that is responsible for acute toxoplasmosis and the slowly replicating, encysted bradyzoite stage. This process of tachyzoite-bradyzoite interconversion is central to the pathogenesis and longevity of infection. Recent research has identified several stage-specific genes and proteins. However, despite recent advances in the understanding of Toxoplasma cell biology, more research is necessary to elucidate the complex events occurring during tachyzoite-bradyzoite interconversion. Here, a brief summary of this process is provided and a new method to characterize gene expression during interconversion is introduced.

Animals↗
PubMed2003

Toxoplasma gondii, sex and premature rejection.

Adaptive sex ratio theory explains why gametocyte sex ratios are female-biased in many populations of apicomplexan parasites such as Plasmodium and Toxoplasma. Recently, Ferguson has criticized this framework and proposed two alternative explanations--one for vector-borne parasites (e.g. Plasmodium) and one for Toxoplasma. Ferguson raises some interesting issues that certainly deserve more empirical attention. However, it should be pointed out that: (1) there are theoretical and empirical problems for his alternative hypotheses; and (2) existing empirical data support the application of sex ratio theory to these parasites, not its rejection.

Animals↗