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The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans.

The attachment of Toxoplasma gondii to target cells is mediated by recognition of cellular heparan sulfate proteoglycans (HSPGs). The present study was performed to determine whether SAG1 and SAG3, two of the parasite surface antigens anchored to the membrane via glycosylphosphatidylinositol groups (GPIs), are involved in the tachyzoite binding to proteoglycans. The use of recombinant soluble forms of these proteins allowed us to demonstrate that SAG3, but not SAG1, interacts specifically with cellular HSPGs. Indeed, soluble recombinant SAG3 protein (recSAG3) was found to bind to immobilized heparin, whereas recSAG1 did not interact with this glycoaminoglycan. The specific adherence of recSAG3 to CHO cells was inhibited by soluble glycoconjugates, of which heparin, fucoidan and dextran sulfate were the most effective. Moreover, binding of recSAG3 to two HSPGs-deficient cell mutants was reduced by up to 80%. Proteoglycan sulfation was critical for SAG3 adherence to HSPGs as incubation of cells in the presence of sodium chlorate drastically reduced the recSAG3 binding. Finally, preincubation of CHO cells with recSAG3 blocked the adsorption of radiolabelled Toxoplasma tachyzoites. Taken together, these results indicate that SAG3 is a first glycoaminoglycan-binding protein associated with Toxoplasma, and SAG3-HSPGs interactions are involved in the parasite attachment to target cells.

Animals↗

The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein.

Genes for the Toxoplasma gondii dense granule and rhoptry proteins nucleoside triphosphate hydrolase 3 and ROP2 were expressed at high levels in the closely related parasite N. caninum. The protein products were processed appropriately and were targeted to their correct secretory organelles. NTPase 3 was secreted into the parasitophorous vacuole. The utility of this system was demonstrated in the analysis of a new open reading frame identified upstream of two identical copies of the ROP2 gene. The unknown open reading frame was introduced into Neospora, and transfected parasites were analyzed by immunoblot with antibodies to known T. gondii rhoptry protein families. The transfected Neospora expressed a novel 52 kDa protein, designated ROP8, which localized in the rhoptries. These results illustrate that transfection of known Toxoplasma genes into N. caninum can be used to study their expression, processing and targeting in an immunologically distinct background. They also illustrate the usefulness of N. caninum transfection in the identification and subcellular distribution of proteins encoded by previously uncharacterized Toxoplasma genes.

Acid Anhydride Hydrolases↗

Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases.

As an initial effort to dissect the signaling pathways responsible for pathogenesis of Toxoplasma gondii infection, we report the cloning and in vitro functional studies of TPK3 (Toxoplasma protein kinase-3), a homologue of shaggy/glycogen synthase kinase-3 (GSK-3) kinases. The shaggy/GSK-3 family of kinases are highly conserved protein kinases that play important roles in cell fate determination, nuclear signaling and hormonal regulation. The TPK3 gene was isolated by RT-PCR with degenerate primers corresponding to highly conserved regions of serine/threonine protein kinases. The complete sequences of genomic and cDNA clones indicated the open reading frame, 1185 bp in size, is interrupted by five introns. The predicted protein sequence of TPK3 shows 54% identity to shaggy/GSK-3 over the catalytic domains. Southern analysis revealed TPK3 is a single copy locus in the Toxoplasma genome. Antisera to other GSK-3 proteins from other species recognized GST-TPK3 and a protein of the predicted size in parasites lysates. In vitro kinase assays with GST-TPK3 indicated that TPK3 autophosphorylates and phosphorylates protein phosphatase inhibitor-2 (I-2), a specific substrate of GSK-3 kinase.

Amino Acid Sequence↗

Risk factors for the presence of antibodies to Toxoplasma gondii in Norwegian slaughter lambs.

In a study of lambs from 194 sheep flocks from different areas of Norway, 44.3% of the flocks were found seropositive against Toxoplasma gondii using an ELISA test, with antibodies found in 16.2% of the 1940 individual animals. The risk factors for the occurrence of antibodies to Toxoplasma gondii found in the multivariate logistic regression were: daily presence of a young cat in the sheep house (Odds ratio, OR = 4.11, 95% CI = 1.01-19.7); 'atypical' grazing of lambs (OR = 6.35, CI = 2.36-17.11); use of mouse poison in the sheep house (OR = 2.26, CI = 1.02-5.00); farm situated at an altitude > 100 meters above seal level (101-250 m: OR = 1.20, CI = 0.49-2.92; 251-500 m: OR = 4.97; CI = 2.04-12.0; > 500 m: OR = 3.66, CI = 1.33-10.1). A lower risk was found for flocks with perforated metal floors in the sheep house (OR = 0.47, CI = 0.23-0.96) and timber construction of the sheep house (OR = 0.34, CI = 0.15-0.80). Based on these findings it was recommended that farmers avoid keeping young cats in the sheep houses, that close-to-farm grazing be kept to a minimum and that perforated metal floors be used in the sheep houses. However, with such a high seroprevalence, the proposed measures alone would not reduce the occurrence of Toxoplasma in lambs to a level where undercooked lamb can be consumed without posing an unacceptable risk for some consumer groups.

Altitude↗

Detection of Toxoplasma gondii in cured meats.

Congenital toxoplasmosis is associated with acute maternal infection acquired during or shortly before the pregnancy. The mother's infection is initiated by the ingestion of one of the life forms of the parasite but the relative importance of the different sources of toxoplasmosis are not established. Recent epidemiological studies have confirmed ingestion of raw meats as a risk factor but also identified consumption of cured meats as being associated with acute toxoplasmosis in pregnancy. There is little existing information concerning the efficiency of commercial curing processes for inactivating Toxoplasma gondii. We sought to detect the presence of T. gondii in ready-to-eat cured meat samples by amplification of the parasite's P30 gene using the polymerase chain reaction (PCR). In addition, tissue culture was used in order to isolate viable parasites. Laboratory inoculated specimens were used to assess the sensitivity of each method. PCR was able to detect parasite contamination down to a level of 5 x 10(3) trophozoites/g while viable toxoplasma could be detected in tissue culture at a level of 10(3) trophozoites/g cured meat. The high salt content of some cured meats limited sensitivity of the PCR assay by inhibition of the polymerase enzyme and reduced the sensitivity of tissue culture due to osmotic pressure causing cytopathic effect. However viable T. gondii was detected in one out of 67 ready-to-eat cured meat samples. Our results highlight the need for improved methods for detecting toxoplasma contamination of food. Health implications of consuming cured meats in pregnancy require careful consideration.

Animals↗

Modulator effect of Toxoplasma lysate antigen in mice experimentally infected with Plasmodium berghei.

Normal mice were pretreated twice at an interval of 2 weeks with an emulsion of TLA (Toxoplasma lysate antigen), PLA (Plasmodium lysate antigen) or both in LMO (light mineral oil) or with a combination of the emulsion and Obioactin or Tp-LKs (Toxoplasma lymphokines) as an immunopotentiator. They were then given Obioactin or Tp-LKs 3 and 25 days after the first treatment and were further given parasitized erythrocytes with 1 X 10(2)-10(4) P. berghei 2 weeks after the second treatment. Thirty (3/10, number of survival/number of examined) per cent of mice treated with TLA, 50 (5/10)% of those treated with a combination of TLA and Tp-LKs and 60 (6/10)% of those treated with a combination of TLA and Obioactin survived as long as 20 days postinfection while none of untreated controls survived more than 15 days postinfection. Only 18.2 (2/11)% of mice treated with PLA or TLA + PLA survived and 20 (2/10), 18.2 (2/11) and 60 (6/10)% of those treated with TLA + Obioactin, PLA + Obioactin or TLA + PLA + Obioactin survived throughout the experiment, respectively while none of controls survived more than 13 days postinfection. Five mice of each group were killed right before infection, and 5, 10 and 15 days postinfection. In mice treated with TLA + Obioactin, more macrophage phagocytosis and macrophage migration inhibition induced by sensitized T-cells were observed than in those treated otherwise. No appreciable differences were noted according to the method of treatment in blood examination values. Cross immunities between Toxoplasma and Plasmodium antigens were tested by counter-immunoelectrophoresis and indirect fluorescent antibody technique. By using counter-immunoelectrophoresis, a specific precipitin line was observed between TLA and anti-PLA which was absorbed by mouse erythrocytes, leucocytes and liver powder. By the indirect fluorescent antibody technique, anti-Plasmodium IgM and IgG titers were detected in sera from mice treated with TLA or TLA-Obioactin before infection.

Adjuvants, Immunologic↗

Attenuated Toxoplasma gondii ts-4 mutants engineered to express the Leishmania antigen KMP-11 elicit a specific immune response in BALB/c mice.

In order to test recombinant Toxoplasma as adjuvant and live vaccine carrier in the infectious disease model of murine experimental leishmaniasis, we engineered the attenuated, temperature-sensitive Toxoplasma gondii strain ts-4 to express the heterologous Leishmania antigen kinetoplastid membrane protein-11 (KMP-11). Transgenic ts-4 clones were obtained which express KMP-11 as cytoplasmatic protein or target it to the secretory pathway of the tachyzoites. Immunization of BALB/c mice with these stably transformed parasites elicited proliferative responses to both T. gondii antigen and recombinant KMP-11. When challenged with Leishmania major, we observed significant protection in animals that had been vaccinated with the KMP-11-expressing ts-4 mutants. The adjuvant attenuated only the onset of the Leishmania infection, but animals were ultimately not able to control the disease. Thus, our findings demonstrate that recombinant Toxoplasma has the potential to serve as an efficient vaccine carrier for cutaneous leishmaniasis. Furthermore, they establish a protective role for the antigen KMP-11 when given in such a vaccine formulation.

Animals↗

Correlation of duration of latent Toxoplasma gondii infection with personality changes in women.

Many parasites induce characteristic changes in behavior of their hosts. In humans latent toxoplasmosis is associated with changes in personality profiles. It has been already shown that a decrease in superego strength is correlated with duration of toxoplasmosis in men. Here we studied changes in personality profiles with Cattell's 16 PF questionnaire in Toxoplasma-infected women. The changes were measured as differences in personality factors between Toxoplasma-infected subjects and uninfected controls of the same age. The low-rate changes were studied in 230 women diagnosed with acute toxoplasmosis during past 14 years. The results showed the correlation between duration of toxoplasmosis and level of factors G (high superego strength) and Q3 (high strength of self sentiment). The high-rate changes were estimated by measuring the correlation between level of Toxoplasma-antibody titers (which rapidly decline after the end of acute phase of toxoplasmosis) and personality factors in an experimental set of 55 young mothers with latent toxoplasmosis. Again, certain factors, namely A (affectothymia), F (surgence), G (high superego strength), H (parmia), and L (protension), correlated with the length of the infection. We suggest that the parasite induced the changes in the personality profiles of the women because of our observation of an increasingly different personality profile over time between women with latent infection and controls. The same evidence questions the view that women with a particular personality profile are more prone to acquisition of T. gondii infection.

Acute Disease↗

Immune responses against excreted/secreted antigens of Toxoplasma gondii tachyzoites in the murine model.

In the present study, excretory secretory antigens (ESA) of Toxoplasma gondii were evaluated in immunization of 8-10 week inbred female Balb/c mice. Tachyzoites of the parasite were cultured in cell-free incubation medium (RPMI-1640), and then supernatant of the medium was loaded on an ion-exchange chromatography column. Two fractions (ESA-F(1) and ESA-F(2)) were collected from the column. For immunization of the mice, 50 were allocated into 5 groups of 10. The first, second, third, and fourth groups were immunized, twice with total-ESA, ESA-F(1), ESA-F(2) or toxoplasma lysate antigen (TLA), respectively. The fifth group was selected as a negative control group (non-immunized). The virulent RH strain of Toxoplasma gondii was used to challenge. Delayed-type hypersensitivity responses (DTHs) were measured by intra-footpad injection measuring induration at timed intervals. Lymphocyte transformation tests (LTTs) were done on lymph node cells using [3H] thymidine incorporation as an indication of reactivity. Peritoneal macrophages from sensitized mice were stimulated and nitric oxide was measured by Griess method. The ESA-F(1) and ESA-F(2) fractions were separated on poly acrylamide gel electrophoresis (PAGE) and SDS-PAGE. ESA-F(1) had 4 bands on PAGE and 14 bands on SDS-PAGE. ESA-F(2) had one band on PAGE and two bands on SDS-PGE. Sensitized mice showed DTH and lymphocyte transformation responses to total-ESA, ESA-F(1), and ESA-F(2) and peritoneal macrophages produce nitric oxide following stimulation. In challenge experiments, all non-immunized mice died within 10 days, whereas immunized mice survived for longer time periods (P<0.05). The highest survival rate was observed in mice that immunized with ESA-F(2). We suggest that these antigens especially ESA-F(2) should be of value for the development of new strategies for immunization against toxoplasmosis.

Animals↗

Specific anti-toxoplasmic IgG antibody immunoblot profiles in patients with AIDS-associated Toxoplasma encephalitis.

Among 186 suspected cases of Toxoplasma encephalitis (TE) in HIV-infected patients, 113 were classified as TE and 73 as non-TE. Serum Toxoplasma gondii (T.g.) antibodies were detected by ELISA in 97% of TE vs 71% of non-TE cases (p < 0.001). In the 164 patients positive for T. g. antibodies, the IgG 27 and 32 bands were more frequently present in TE than in non-TE (p = 0.003, p = 0.002, respectively). Among patients with positive T.g. serology, multivariate analysis showed that the presence of an IgG 32 (OR 3.1) or IgG 27 band (OR 2.7) on Western blot was highly indicative of TE independently of each other. Positive T.g. serology, but not anti-T.g. IgG antibody titres, was predictive. Thus, the positivity of IgG 27 and 32 bands on Western blot analysis provides useful data for improving the diagnosis of presumptive TE in HIV-infected patients with suspected TE and positive Toxoplasma serology.

AIDS-Related Opportunistic Infections↗

Detection and characterization of circulating antigens in acute experimental infections of mice with four different strains of Toxoplasma gondii.

Acute infections with four different strains of Toxoplasma gondii, all of them being highly pathogenic for mice, were provoked by intraperitoneal injection of 2.2 x 10(7) trophozoites. The times of the appearance of circulating antigens in the sera of the infected mice were determined and the amounts of these antigens were measured by an enzyme-linked immunosorbent assay. The molecular weight of the circulating antigens was determined by gel filtration and by Western blot following PAGE. The isoelectric points of these antigens were determined by immunoblotting after isoelectric focusing. Circulating antigens were detectable up to ng amounts/ml serum from the 1st day p.i. onwards. The circulating antigens consisted of a number of proteins with molecular weights of greater than 10(4), 300, 65, 25, and less than 5 kd. The isoelectric points of these proteins were situated between pH 3.5 and pH 7. Time of appearance and structure of the circulating antigens were very similar in all four Toxoplasma strains. Circulating antigens are apparently generated regularly in experimental acute Toxoplasma infections, and lysis of parasite cells appears to be the major mechanism of their formation.

Animals↗

Age-sex distribution of Toxoplasma antibody in the South Australian population.

Sera from 1071 patients in nine age categories were screened for Toxoplasma antibody by indirect immunofluorescence. Thirty per cent of the sera contained antibody at a titre greater than or equal to 1/16. The percentage of sera containing antibody rose from 3% in the 6 months--5 years age group to a maximum of about 40% in the 31--40 years age group. It remained constant thereafter. Eleven per cent of the 84 sera with Toxoplasma antibody titres greater than or equal to 1/128 had Toxoplasma IgM titres greater than or equal to 1/32. No significant difference was found in the possession of antibody between the sexes.

Adolescent↗

Prevalence of toxoplasma antibodies according to age with comments on the risk of prenatal infection.

Sera from 1661 persons in 12 age groups from 0 to 79 years were titrated for toxoplasma antibodies in the indirect immunofluorescence test. The sera were collected from patients with symptoms suggestive of acute, mainly respiratory, viral infections. After the first year of life, the prevalence of antibodies started to rise, reaching 59% between 40 and 79 years of age. From the prevalence of antibodies in different age groups the annual infection risk, i.e, the risk of a non-immune person acquiring toxoplasma infection, was estimated for successive age periods. The estimated annual infection risk increased from 0 . 5% in early childhood to 3% during adolescence and early adult life. Approximately 70--80% of females entered the age of reproduction without evidence of seroimmunity to toxoplasma. The risk of primary infection during pregnancy was estimated from the age distribution of parturient women in The Netherlands in 1975 and the age-specific incidence of primary infections, i.e. the incidence in the total population of susceptible and immune persons. This incidence of primary infection decreased from 1 . 62% per 9 months at the age of 17 1/2--20 years to 0 . 37% at the age of 37--45 years. The incidence of primary infections in pregnant women was estimated to be 1 . 25%.

Adolescent↗

Neutrophil chemotactic factors secreted from Toxoplasma gondii.

Neutrophil chemotactic activity was detected in the fluid of Toxoplasma gondii cultures by the agarose plate and the Boyden chamber methods. Toxoplasma culture fluid was obtained by incubating the tachyzoites at 37 degrees C in a 5% CO2 atmosphere for 6 h in Dulbecco's modified Eagle's minimum essential medium containing 10% heat-inactivated foetal calf serum. Soluble extracts from tachyzoites had negligible activity, indicating that the chemotactic factors were metabolites secreted from tachyzoites. The chemotactic activity in Toxoplasma culture fluid depended on the number of tachyzoites and the period of incubation. The substances responsible for neutrophil chemotaxis were two heat-labile peptides with estimated Mr 4.5 and 14 kDa with N-terminal groups free.

Agglutination Tests↗

Naturally occurring immunoglobulin M antibodies: enhancement of phagocytic and microbicidal activities of human neutrophils against Toxoplasma gondii.

Naturally occurring immunoglobulin M antibodies to Toxoplasma gondii in human sera were examined for their ability to enhance toxoplasmacidal activities of human neutrophils to show a role of natural antibodies in immunity to the early stage of Toxoplasma infection in humans. Neutrophils were mixed with tachyzoites at a ratio of 1:5 in the presence or absence of antibodies and were cultured for 1-18 h for microscopical examination. The count of tachyzoites phagocytosed in 200 neutrophils within 1 h was significantly higher in the presence of sera with natural IgM antibody levels of greater than 0.8 than those of less than 0.1, with 2.4- to 2.9-fold differences (P less than 0.02). The total tachyzoite counts at 18 h decreased to 26-39% of those at 1 h in the presence of natural IgM antibody levels of greater than 0.8 (P less than 0.01), while the counts at 1 h consistently increased by 18 h in the absence of natural IgM antibodies. These results indicate that relatively high levels of natural IgM antibodies enhanced phagocytic and microbicidal activities of neutrophils against Toxoplasma. The enhancement was dose dependent, and was also weaker than that obtained with the same dilution of sera from individuals with chronic or acute infections. Live and dead tachyzoite counts in infected neutrophils suggested a quicker effect of natural IgM antibodies than IgG antibodies elicited by infection.

Animals↗

Induction of changes in human behaviour by the parasitic protozoan Toxoplasma gondii.

Toxoplasma gondii, the coccidian parasite, is known to induce changes in the behaviour of its intermediate hosts. The high prevalence of this parasite in the human population (20-80%) offers the opportunity of studying the influence of the parasite on human behaviour by screening of a normal population. A total of 224 men and 170 women were tested for toxoplasmosis and their personality profiles were measured by Cattell's questionnaire. Highly significant differences between Toxoplasma-infected and uninfected subjects were observed (P < 0.01). For men the factors G (low superego strength), L (protension), O (guilt proneness), and Q2 (group dependency) were positively influenced in infected subjects. For women the prevailing factors were A (affectothymia, P < 0.01), L (alaxia), O (untroubled adequacy) and Q2 (self-sufficiency). To reveal whether toxoplasmosis induces personality factor-shifts or whether certain combinations of personality factors influence the probability of acquiring Toxoplasma infection, we examined the personality profiles of 164 male patients diagnosed with acute toxoplasmosis during the past 13 years. The existence of a positive correlation between the duration of latent toxoplasmosis and the intensity of superego strength decrease (P < 0.02) suggested that the decrease of superego strength (the willingness to accept group moral standards) was induced by T. gondii infection.

Animals↗

Prevalence and transmission of Toxoplasma gondii in wild brown rats, Rattus norvegicus.

Prevalence of Toxoplasma gondii within 6 UK farmstead wild rat populations, and 1 population maintained within a captive cat-free environment for 2.5 years, was examined. The mean prevalence found was 35% (n = 235), which is more than 3 times as high as all other wild rat prevalence levels previously reported in the UK. There were no significant age, sex or site differences in prevalence between or within populations irrespective of habitat type or presence of cats. Toxoplasma was also maintained within the captive rat population in the absence of cats at a prevalence, intensity, age and sex distribution similar to that of the farmstead rat populations. These results suggest, firstly, that Toxoplasma can be perpetuated within wild rat populations without the sympatric presence of cats and secondly, that the congenital route is the predominant route of transmission in wild rats. This study concludes that wild rats represent a significant and persistent wildlife intermediate host reservoir for toxoplasmosis.

Age Factors↗

Toxoplasmosis in rats (Rattus norvegicus): congenital transmission to first and second generation offspring and isolation of Toxoplasma gondii from seronegative rats.

To study congenital transmission of Toxoplasma gondii during acute and chronic infections, 4 pregnant Sprague-Dawley rats were each fed 10,000 oocysts of the VEG strain. Toxoplasma gondii was recovered from 33, 55, 83 and 57% of rats (F1) when dams were inoculated at 6, 9, 12 or 15 days of gestation, respectively. Progeny of 15 congenitally infected female rats were examined for T. gondii. Toxoplasma gondii was recovered from tissues of 1 of 155 rats (F2) born to congenitally infected dams. A total of 4 (F2) females were mated; 0 of 40 (F3) rats born to them were infected. None of the acutely infected 4 dams that had given birth to congenitally infected litters produced congenitally infected offspring during the second pregnancy. Thus, unlike mice, evidence for repeated congenital transmission of T. gondii in the rat was found in < 1% of cases. Of the 16 congenitally T. gondii infected pups with demonstrable tissue cysts, 5 were seronegative (< 1:4) in the Sabin-Feldman dye test and 5 were seronegative (< 1:20) in the modified agglutination test by the use of whole formalinized tachyzoites and mercaptoethanol.

Animals↗