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Cell cycle-dependent entry of Toxoplasma gondii into synchronized HL-60 cells.

The degree of attraction of Toxoplasma gondii to vertebrate cells varies with cell type and cell phase. Human promyelocytic leukemia cells, HL-60, were synchronized by double thymidine block method and co-cultured with Toxoplasma for 1 hr at each cell stage to investigate the cell cycle specific susceptibility of parasites to host cells. For 30 hr the average number of Toxoplasma that invaded was a little changed except at 3 hr from G1/S phase boundary which concurred with the peak point of DNA synthesis. At 3 hr which is a relatively short interval compared to whole S phase, modification of cells by parasitic invasion was most remarkable. The number of Toxoplasma that penetrated was increased to more than six times. The shape of the cells became sludgy and almost indiscernible by strong accessibility of parasites only for an hour of mid-S phase. The same fluctuation was also observed at the second peak of S phase but weakly. This suggests that there be surface molecules concerning with the attachment of Toxoplasma to the host cells, which is expressed at special point of S phase. Further studies on the specific protein or similar molecules related could be carried out using synchronized HL-60 cells.

Animals↗

[Immunological properties of the 30 kDa antigen of toxoplasma gondii].

The molecular weight 30 kDa membrane protein of Toxoplasma gondii (Toxoplasma 30 kDa) apparently conserved in most strains of T. gondii and sera of infected hosts. The present study aimed to elucidate Toxoplasma 30 kDa as a useful diagnostic antigen for serodiagnosis of toxoplasmosis by ELISA and for induction of protective immunity. Murine spleen cells immunized with the membrane antigen of T. gondii were fused with mouse Sp2/O-Ag14 myeloma cells. Out of 8 clones selected, five were IgG2b, the others belonged to IgG1 and IgG2a. The 30 kDa antigen was distributed mainly on the surface membrane of tachyzoites by indirect fluorescence method. Murine peritoneal macrophages which were activated by 30 kDa antigen produced more amounts of NO2 compared with crude antigen-treated group, however there were no significant differences in toxoplamacidal activity between the two groups. Higher specificity of Toxoplasma 30 kDa antigen was recognized for serodiagnosis of toxoplasmosis than the crude antigen. From these results, Toxoplasma 30 kDa antigen enhances the cytotoxic effect of macrophages as well as a more reliable means for the serodiagnosis of toxoplasmosis by ELISA.

Animals↗

Intrathecal production of antibodies against Toxoplasma gondii in patients with toxoplasmic encephalitis and the acquired immunodeficiency syndrome (AIDS).

We attempted to determine whether patients with the acquired immunodeficiency syndrome (AIDS) and toxoplasmic encephalitis produce Toxoplasma antibodies in the central nervous system, and whether these antibodies would be useful for diagnosing toxoplasmic encephalitis. Thirty-seven patients with AIDS and toxoplasmic encephalitis and 11 patients with AIDS alone were studied. All patients had serum IgG but not IgM Toxoplasma antibodies. Twenty-three of the patients with AIDS and toxoplasmic encephalitis had Toxoplasma antibody in cerebrospinal fluid compared to none of the patients with AIDS alone. Of the patients with AIDS alone who had serum antibodies to T. gondii, none had Toxoplasma antibodies in cerebrospinal fluid. Production of antibodies in the central nervous system was seen in 11 of 16 patients with AIDS and toxoplasmic encephalitis but not in 4 patients in the control group who only had Toxoplasma antibody in their cerebrospinal fluid. These results suggest that production of T. gondii antibodies in the central nervous system may be diagnostic of toxoplasmic encephalitis.

Acquired Immunodeficiency Syndrome↗

Is there more than one species in the genus Toxoplasma??

A complete life cycle for the ubiquitous protozoan parasite Toxoplasma was proposed over 25 years ago. Since that time, despite attempts to make the genus polyspecific, there has been only one species, Toxoplasma gondii, consistently recognised in the genus. Recent studies on taxa in genera closely related to Toxoplasma such as Neospora, Hammondia, Frenkelia, Isospora and Sarcocystis, have convincingly showed the need for a reclassification of many of the species in these genera. However, in addition to these genus level studies, over the last 10 years several laboratories have used molecular techniques including isoenzyme electrophoresis, restriction fragment length polymorphism analyses, random amplified polymorphic DNA - polymerase chain reaction, and comparisons of the small subunit ribosomal RNA gene, DNA polymerase alpha intron, and 70 kDa heat shock protein gene nucleotide sequences to investigate the genetic diversity among strains in the species T. gondii. Overall, the results of these analyses confirm that the strains in the genus Toxoplasma comprise a limited number of clonal lineages, directly correlated with their virulence in mice. The aim of this presentation is to review the molecular research in this area in order to raise the hypothesis that there may be more than one species in the genus Toxoplasma, which may contain taxa with distinct and different life cycles.

Animals↗

[Usefulness of quantitative assessment of Toxoplasma gondii genome using PCR-TaqMan in amniotic fluid, maternal and neonatal blood in selected complications in pregnancy].

UNLABELLED: Routine serological diagnosis of Toxoplasmosis provides high sensitivity, but not high specificity. The high sensitivity combined with high specificity offered by PCR-TaqMan as well as the degree of infection led us to investigate the presence and levels of Toxoplasma gondii genome in amniotic fluid, maternal and neonatal blood in cases of pregnancy where infection with this agent was suspected. MATERIALS AND METHODS: Samples of amniotic fluid and blood were taken from 28 women between the 16th and 40th week of gestational age. Postnatal blood samples were also taken from their infants. Included in the study group were women with IUGR, PROM, preterm delivery imminent, Toxoplasmosis in previous pregnancies, raised IgG or IgM anti-Toxoplasma antibody titers and with amniotic fluid disturbances (oligohydramnios and polyhydramnios). Presence and levels of Toxoplasma genome was investigated using PCR-TaqMan. PCR products were detected by electrophoresis on polyacrylamide gel. RESULTS: Toxoplasma gondii genome was detected in blood from 13 women, 3 newborns and in amniotic fluid from one other women. Toxoplasma genome was detected in blood from one newborn, but was not detected in sample from its mother. CONCLUSIONS: The PCR-TaqMan test is highly sensitive and specific method allowing detection of the parasite genome and assessment of its level. Limitations of this method are its relatively high cost and poor access to ABI PRISM 7700 (TaqMan) sequence detector. The PCR TaqMan is useful in cases, where serological tests for the presence of infections are ambiguous.

Amniotic Fluid↗

Serological study of Toxoplasma gondii in kidney recipients.

Toxoplasma gondii IgG antibodies were determined in 200 kidney recipients by the Sabin-Felmand dye test. Twenty-two (11%) cases were positive for antibody detection. There was a statistically significant difference in the history of taking under-cooked meat, between the number of sero-positive cases and that of sero-negative subjects (63.6% vs 28.8%, p = 0.02). No such significant difference was evident regarding cat ownership (13.6% vs 22.0%, p = 0.3). Sixteen (72.6%) of the 22 subjects with positive Tgondii antibody had undergone kidney transplantation less than one year ago during which a high dose of immunosuppressive drugs were prescribed. The remaining six (27.3%) cases had had transplantation more than one year ago and were on a lower dosage of immunosuprresants. Toxoplasma reactivation seemed to be higher in the former group, which should thus be closely followed-up. Preventive chemoprophylaxis should be considered if there is any indication of toxoplasma reactivation. Since there have been occasional reports of donor-to-host transmission of toxoplasmosis in kidney transplant recipients, serological screening of toxoplasma antibody in kidney donors is advisable. Potential donors with positive toxoplasma antibody should be rejected; but if that is unavoidable, 6-week prophylactic treatment of primary infection in kidney recipients should be administered.

Adolescent↗

[Detecton of circulating antigen in sera from mice infected with Toxoplasma tachyzoites].

AIM: To explore an assay method for the early diagnosis of Toxoplasma infection. METHODS: Serum samples collected from three groups of mice infected experimentally with different doses of Toxoplasma trophozoites were detected for the presence of Toxoplasma circulating antigen by using fast ELISA. RESULTS: Toxoplasma circulating antigen was detected on days 4, 4 and 3 after infection in light, moderate and heavy infection groups, respectively. CONCLUSION: The level of circulating antigen was in parallel with the duration of infection. The determination of circulating antigen is useful in the early diagnosis of Toxoplasma infection.

Animals↗

Toxoplasma infection of stray cats in Egypt.

A seroepidemiological survey of cats for Toxoplasma antibody revealed a high infection rate. No significant difference was detected between males and females or between rural and urban areas, although acute infection seems to be more prevalent in males and in rural areas as revealed by the high titres of the dye test. Toxoplasmosis in cats, as revealed by serologic surveys, seems to prevail more during the warm rather than the hot season of the year being favoured by milder temperature and higher relative humidity and rainfall in the former season. Futhermore, it was concluded that infection rate with Toxoplasma increases with the age of the host. Toxoplasma antibody in the young age groups is to a great extent of maternal origin. Study of the relationship of Toxoplasma to Isospora showed no cross immunity. They follow a reversed pattern with increasing age of the host. Data also shows the high specificity of dye testing for detection of Toxoplasma antibody. Feeding experiments show that a relatively high percentage of cats shed oocysts in their faeces. Not only seropositive but also seronegative cats excrete oocysts, though at a lower rate in the latter case. The majority of cats shedding oocysts are those with either low positive titres or seronegative cats.

Age Factors↗

Application of ECCLS guidelines to the analysis of Toxoplasma IgG and Rubella IgG using laboratory kits based on the Meia method.

The Meia method is an enzymo-immunoassay involving fluorometric detection, which is used in the Abbott IMx automatic analyzer. The purpose of this report was to analyse the Meia Toxoplasma gondii IgG antibody and Rubella IgG antibody assays, following ECCLS guidelines for the analysis of laboratory kits. The results showed that between-run imprecision for Rubella IgG was close to 15%; for Toxoplasma IgG the percentage was 13%. The mean recovery for Rubella IgG was 104% and 94% for Toxoplasma IgG. The carry-over for Rubella IgG was 0.64% and 0.26% for Toxoplasma IgG which, in both cases, was less than the analytical variability. Both Meia and Elisa showed a linear relationship in the analytical range of the method. Comparing Meia with the Elisa method, constant and proportional differences were found for IgG Rubella and proportional differences for IgG Toxoplasma. The Meia method has many positive analytical features to recommend and it can easily be used in a multidisciplinary laboratory, needing only a small number of serum samples.

Animals↗

[Isolation of Toxoplasma gondii by cellular culture in HIV infected patients].

Nine hundred and eight biological samples obtained from HIV-infected patients were examined for Toxoplasma gondii by a tissue culture method. Sixty-two isolations of Toxoplasma gondii were made in 43 patients. Fourteen isolations were made from tissue biopsies (brain 4, striated muscle 2, liver 1), bone marrow aspirates (4 cases), pericardial or pleural effusions (3 cases) and vitreous body (1). In 6 cases, a parasitic dissemination was evidenced by the demonstration of Toxoplasma in blood or other tissues or body fluids. Toxoplasma organisms were demonstrated in the bronchoalveolar lavage fluid of 19 patients; this was associated with a parasitaemia in 6 of the 10 patients whose blood sample was cultured at the same time. Among 273 blood samples cultured, 29 were positive for Toxoplasma (24 patients); all patients with parasitaemia had clinical symptoms, but only 8 of them had clinical or radiological symptoms suggestive of a cerebral localization; this suggests that the presence of a parasitaemia is fairly indicative of an extra cerebral localization of toxoplasmosis. In 11 patients, blood was examined after treatment was initiated; in 8 patients parasitaemia was undetectable at the first examination after treatment (1 to 15 days after initiation of therapy), whereas in 3 patients sampled 1, 4 or 6 days respectively after treatment a persistent parasitaemia could be demonstrated. These results clearly show that the tissue culture method is efficient and sensitive to provide an early evidence of infection (4 days), and may prove useful to evaluate therapeutic effectiveness.

AIDS-Related Opportunistic Infections↗

[The role of Toxoplasma gondii in the etiology of chronic tonsillitis in children].

Toxoplasma gondii is known to have an affinity to lymphatic tissue. A series of 506 children with chronic tonsillitis were examined serologically for toxoplasmosis by means of CBR, IIR and ELISA. In 7.7% of the children examined low toxoplasma antibody titers were recorded. Isolation experiments performed on tonsillar tissue of the children with the highest toxoplasma antibody titers were negative. Direct microscopic evidence of tissue toxoplasma cysts failed to be established and the cytopathologic picture characteristic of nodal toxoplasmosis was not present in smears of cells aspirated from lymphatic nodes regional to palatine tonsils. The authors assumed that chronic toxoplasma tonsillitis did not occur in the series studied. In our ecological conditions, T. gondii may not be involved in the etiology of chronic tonsillitis, or it may participate to an extent which is epidemiologically negligible. (Tab. 4, Ref. 14).

Adolescent↗

Detection of Toxoplasma gondii DNA and specific antibodies in high-risk pregnant women.

Primary maternal infection with toxoplasmosis during pregnancy is frequently associated with transplacental transmission to the fetus. This study was conducted to test the utility of a polymerase chain reaction (PCR) assay to detect recent infections with Toxoplasma in pregnant women. One hundred forty-eight women with high-risk pregnancies who had abnormal pregnancy outcomes (cases) and 100 with normal pregnancies (controls) were tested for the presence of Toxoplasma DNA in their blood by a nested PCR and specific antibodies to Toxoplasma by an enzyme-linked immunosorbent assay. The IgG results of the cases differed significantly from those of the controls (54% and 12%, respectively; P < 0.02). Four (2.7%) of the cases were IgM positive, but none of the controls were positive. Detection of Toxoplasma DNA in 20 (8.1%) of the IgG-positive cases suggests a recent infection. The risk factors associated with the infection were eating raw meat and contact with soil. The diagnostic serology of recent infection in early pregnancy could be confirmed by a positive Toxoplasma-specific PCR result in blood samples collected in the first half of pregnancy, even in the presence of serologic results difficult to interpret due to the lack of sequential follow-up during pregnancy.

Adult↗

[Development of immunoblot kit for the detection of anti-Toxoplasma antibodies].

OBJECTIVE: To develop a diagnostic kit for the detection of anti-Toxoplasma antibodies in human sera by immunoblot technique. METHODS: The cytoplasm proteins of Toxoplasma gondii were extracted from collected ascites in Kunming strain mice infected by tachyzoites of Toxoplasma gondii (RH strain). Toxoplasma gondii soluble antigens were separated by SDS-PAGE electrophoresis and transferred to pyroxylin membrane. Using nonpoisonous tetramethylbenzidine (TMB) as horseradish peroxidase substrate for immunoblot, the optimal experimental conditions were selected through comparison of antigen preparation, reagents for blocking or washing, dilution concentration, reaction time, and frequencies of reacting band. Sensitivity, specificity, Youden index and stability were evaluated as the standard for the kit. RESULTS: In 30 sera with anti-Toxoplasma [gM antibodies and 28 with IgG antibodies, the sensitivity for IgM and IgG antibody detection was 90.0% (27/30) and 85.7% (24/28) respectively, the specificity was all 100% in examining 40 healthy control sera, and the Youden index was 0.9 and 0.86 respectively. The kit was stable at 49 degrees C for 6 months. CONCLUSION: The immunoblot kit shows an easy operation, fast reaction and reliable result, and may be practical.

Animals↗

[Application of staphylococcal protein A for immunodiagnosis of early toxoplasma infection].

RH strain Toxoplasma gondii was used to inoculate mice and rats. Sera collected at intervals were assayed by the indirect fluorescent antibody test (IFAT) and the latex agglutination test (LAT) to monitor the time-course change in titers of anti-toxoplasma IgM and IgG antibodies. In addition, the above sera and some anti-toxoplasma seropositive human sera (with IFAT antibody titers greater than or equal to 1:16) were adsorbed with staphylococcal protein A (SpA) and assayed for changes in IgM and IgG antibody titers so as to evaluate the usefulness of SpA adsorption in detecting anti-toxoplasma IgM which shows up early in toxoplasma infections. Samples assayed included 262 sera from mice, 65 sera from 5 rats and 85 human sera. The results revealed that parasite specific IgM-IFAT antibodies were detectable in 40% of the sera from mice 3 days after infection. After SpA adsorption, however, the IgM-IFAT antibody could be detected in a few specimens as early as 2 days post infection. IgG-IFAT and LAT antibodies first appeared in the sera on the 5th day of infection. In mice inoculated with freeze-killed tachyzoites (immunized mice), IgG-IFAT and LAT antibodies remained high from 16-35 days after inoculation, whereas IgM-IFAT antibodies were undetectable. Even after SpA treatment, only about half of the 16-day samples showed IgM antibody titers and the other specimens still remained negative. In general, IgM antibody titers increased 2-6 fold after SpA adsorption while IgG antibodies were almost completely removed after the treatment with a residual IgG-IFAT titer of less than or equal to 1:4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Expression and bioactivity of S1 scFv antibody against SAG1 of Toxoplasma gondii fused with green fluorescent protein].

OBJECTIVE: To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma gondii and fused to green fluorescent protein (GFP), and observe its binding capacity to tachyzoite of Toxoplasma gondii. METHODS: The GFP gene amplified from vector pEGFP-N1 was subcloned into procaryotic expression vector pET-26b (+), then the S1 scFv antibody gene amplified from phagmid pIT-2-S1 was cloned into downstream of GFP gene. The recombinant plasmid pET-26b-GFPS1 proved by DNA sequencing was transformed into E. coli BL21, and induced for fusion expression of GFPS1 with IPTG, the green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was observed under the fluorescence microscope. The expressed GFPS1 was purified with Ni2+ chelating HiTrap HP column, and detected with SDS-PAGE. Toxoplasma tachyzoites were incubated with the recombinant GFPS1, and the binding bioactivity was observed under the fluorescence microscope. RESULTS: The fused gene of S1 and GFP was successfully cloned into procaryotic expression vector pET-26b proved by DNA sequencing. The green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was catched under the fluorescence microscope. The recombinant GFPS1 protein about Mr 53 000 was expressed in E. coli as inclusion body. The immunofluorescence detection verified that anti-rSAG1 scFv antibody S1 could specifically bind outer membrane of Toxoplasma tachyzoite. CONCLUSIONS: The purified rGFPS1 shows a strong binding capacity to outer membrane of Toxoplasma tachyzoite using GFP as a labeling protein, and the constructed pET-26b-GFP can also be used for research on other targeting molecules.

Animals↗

[Experimental animal studies for the detection of circulating Toxoplasma antigens].

Rabbits and mice were infected by Toxoplasma gondii (RH and HanR) and Hammondia hammondi. An enzyme immunoassay with monoclonal antibodies was used to reveal circulating Toxoplasma antigens. They were found only in mice and rabbits that had been infected by the cyst-forming strain Toxoplasma HanR. In mice infected by Hammondia hammondi low antibodies titres were found that showed a cross-reaction with Toxoplasma. The enzyme immunoassay for evidence of Toxoplasma antigens demonstrated a negative reaction in these animals.

Animals↗

Distribution of antibodies against Coxsackie B viruses, arboviruses and Toxoplasma gondii among patients with endomyocardial fibrosis (EMF) compared with normal subjects from EMF endemic and non-endemic zones of Nigeria.

The sera of eight endomyocardial fibrosis (EMF) subjects, 11 siblings of one of them and 16 normal children matched with the EMF patients for age, sex and socio-economic status from Ogunmakin and Shao/Oloru communities (eight each), situated in EMF-endemic and non-endemic areas of Nigeria respectively, were examined for the presence of antibodies against Coxsackie viruses B1-6, 16 arboviruses and Toxoplasma gondii. Sera from 36 other randomly selected normal children from Ogunmakin and 26 other randomly selected children from Shao/Oloru were also tested for the presence of antibodies against Toxoplasma gondii and the 16 arboviruses. None of the eight EMF subjects nor the 11 siblings of one of them had antibodies against any of the Coxsackie viruses B1-6 in their sera. Two of the 16 matched control subjects, one from each community, had positive antibodies, at equivocal titres against Coxsackie B1 (Ogunmakin) and B4 (Shao/Oloru). There was no significant difference in the distribution of antibody titres to the arboviruses between the EMF patients and matched controls. Normal children from the Shao/Oloru community had higher percentage antibody reactions and higher titres to the arboviruses compared with the children from Ogunmakin. All the eight EMF patients had high antibody titres against Toxoplasma gondii. Seven (87.5%) of the matched controls from Ogunmakin were sero-positive for Toxoplasma gondii compared with three (37.5%) of the matched controls from Shao/Oloru. Of the 36 normal children from Ogunmakin, 32 (88.9%) were sero-positive compared with 11 (42.3%) of the 26 normal children from Shao/Oloru. Four (36.4%) of the 11 siblings of one of the EMF patients had weak sero-positivity. It is therefore concluded that further studies are needed to clarify the role, if any, of Toxoplasma gondii in EMF.

Adolescent↗

Impaired in vitro lymphocyte response to toxoplasma antigen in HIV1 infected patients.

The containment of Toxoplasma gondii infection is largely dependent of T cell mediated immunity. In this study, in vitro lymphocyte responsiveness to T. gondii antigen was examined in 59 HIV1 infected individuals and in 58 HIV non-infected controls. Of the 45 patients with serological evidence of past Toxoplasma infection, a significant proliferative response was found in only 18, whereas responses were present in 48 out of 51 controls with anti-Toxoplasma antibodies. In the 27 non-responder patients, the lack of proliferative response to T. gondii antigen was correlated with the loss of CD4+ cells, and the impairment of proliferative responses to other microbial antigens, whereas responsiveness to phytohaemagglutinin and concanavalin A were not significantly diminished. Results are consistent with impairment of cell mediated immunity to T. gondii in patients at risk for reactivation of chronic Toxoplasma infection. Of note, in the one year clinical following, 2 of the 27 non-responder patients developed toxoplasmic encephalitis compared to 0 of 18 with a Toxoplasma specific proliferative response.

Acquired Immunodeficiency Syndrome↗