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PubMed1989

Comparison of six commercial enzyme linked immunosorbent assays for detecting IgM antibodies against Toxoplasma gondii.

To evaluate the usefulness of different commercial enzyme linked immunosorbent assays (ELISAs) for the detection of IgM antibodies against Toxoplasma gondii the results of six of these assays for a panel of 81 sera were compared. The following tests were selected: Toxoplasma gondii IgM ELISA (Clark Laboratories), Toxoplasma IgM EIA (Labsystems), Toxo-M EIA (Abbott), Toxonostika M (Organon), Toxo M Enzyme Immunoassay (Hybritech) and Platelia Toxo IgM (Diagnostics Pasteur). An antibody capture ELISA developed at our laboratory was used as the reference test. An IgM immunoblotting assay was also performed. Four (Toxoplasma IgM EIA, Tox-M EIA, Toxonostika M, and Platelia Toxo IgM) of the commercial IgM ELISAs gave a high sensitivity and a high specificity. Toxo-M EIA, Toxonostika M, Toxoplasma IgM EIA and the Toxo M Enzyme Immunoassay were too insensitive, and the Toxoplasma gondii IgM ELISA was both insensitive and unspecific. No remarkable differences were observed between the results of indirect or antibody capture ELISAs, and between the results of ELISAs performed with polyclonal or monoclonal antibodies.

Animals↗

PubMed1993

Toxoplasma infection and systemic lupus erythematosus: analysis of the serological response by immunoblotting.

AIMS: To examine the serological response of patients with systemic lupus erythematosus (SLE) and toxoplasma infection and to compare the blot profiles with those from immunocompetent subjects of similar immune response. METHODS: Forty serum samples from patients with SLE were tested for toxoplasma antibodies using the dye and indirect haemagglutination tests. Specific IgM was measured by mu-capture enzyme linked immunosorbent assay (ELISA). The sera were immunoblotted using antigen strips prepared from the RH strain of Toxoplasma gondii. For comparison, control blots were prepared from pooled sera from immunocompetent subjects with serological evidence of acute (pool 1), or chronic (pool 2) toxoplasma infection, or with no evidence of infection (pool 3). RESULTS: Some of the blot profiles from the patients with SLE were compatible with the corresponding serology but others showed considerable variation, particularly among the IgM blots. The blots from sera with low dye test titres suggested that the latter could be false positive results. CONCLUSIONS: Toxoplasma infection may enhance the production of autoantibodies which, when combined with the high titres characteristic of SLE, might interfere in the dye test and other serological tests. Immunoblotting could prove useful in the immunocompromised for confirming the presence of specific toxoplasma antibodies and for the staging of infection in those with positive serology.

Animals↗

PubMed1995

Invasion of Toxoplasma gondii occurs by active penetration of the host cell.

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of vertebrate cells including macrophages. We have used a combination of video microscopy and fluorescence localization to examine the entry of Toxoplasma into macrophages and nonphagocytic host cells. Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. Invasion occurred rapidly and within 25-40 seconds the parasite penetrated into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting phagosome that formed over a period of 2-4 minutes. Phagocytosis involved both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins. In some cases, parasites that were first internalized by phagocytosis, were able to escape from the phagosome by a process analogous to invasion. These studies reveal that active penetration of the host cell by Toxoplasma is fundamentally different from phagocytosis or induced endocytic uptake. The novel ability to penetrate the host cell likely contributes to the capability of Toxoplasma-containing vacuoles to avoid endocytic processing.

Animals↗

PubMed1990

Tight junctional inhibition of entry of Toxoplasma gondii into MDCK cells.

Various conditions of cultures were performed to investigate the role of tight junctions formed between adjacent MDCK cells on the entry of Toxoplasma. When MDCK cells were cocultured with excess number of Toxoplasma at the seeding density of 1 x 10(5), 3 x 10(5), and 5 x 10(5) cells/ml for 4 days, the number of intracellular parasites decreased rapidly as the host cells reached saturation density, i.e., the formation of tight junctions. When the concentration of calcium in the media (1.8 mM in general) was shifted to 5 microM that resulted in the elimination of tight junction, the penetration of Toxoplasma increased about 2-fold (p less than 0.05) in the saturated culture, while that of non-saturated culture decreased by half. Trypsin-EDTA which was treated to conquer the tight junctions of saturated culture favored the entry of Toxoplasma about 2.5-fold (p less than 0.05) compared to the non-treated, while that of non-saturated culture decreased to about one fifth. It was suggested that the tight junctions of epithelial cells play a role as a barrier for the entry of Toxoplasma and Toxoplasma penetrate into host cells through membrane structure-specific, i.e., certain kind of receptors present on the basolateral rather than apical surface of MDCK cells.

Animals↗

PubMed1995

Transmission of Toxoplasma gondii in Panama City, Panama: a five-year prospective cohort study of children, cats, rodents, birds, and soil.

A cohort of more than 500 children from Panama City, Panama was studied prospectively over five years for acquisition of antibody to Toxoplasma gondii. The direct agglutination test showed that 72 of 571 children seroconverted between one and six years of age, for a cumulative incidence of 12.6%. Children were examined by pediatricians quarterly, and illnesses that had occurred in the interval and their activities were noted on questionnaires. Thirty-eight variables were examined for their role as risk factors for seroconversion. There was a higher correlation between children's seroconversion and contact with dogs than with cats. Combinations of significant predictors without dogs explained only 67% of the seroconversions, but the same factors with dogs explained 90%. On the other hand, ingestion of raw or rare meat or eggs appeared to play no role in transmission. Cats were examined and 110 (45.6%) of 241 had Toxoplasma antibody on the first bleeding. Only two (0.5%) of 383 cat fecal specimens, when tested in mice, resulted in seroconversion. Ten (1.1%) of 924 soil samples resulted in seroconversion in mice that had been injected. Antibody to Toxoplasma was found in 52 (23.3%) of 226 rats (Rattus norvegicus) and two (0.035%) of 571 mice (Mus musculus). Two hundred sixteen birds of 16 different species were bled. Antibody to Toxoplasma was found in 13.4% of these birds, mostly in grackles, blue-gray tanagers, and doves. The rate of isolation of Toxoplasma was low: one of 23 in rats and three of 201 in birds. High relative risks (RRs) of transmission to children were predicted by contact histories with nursing dogs (RR = 5.8), weaned dogs (RR = 4.7), many flies (RR = 3.6), 6-12-month-old dogs (RR = 3.4), weaned cats (RR = 3.0), 6-12-month-old cats (RR = 2.7), nursing cats (RR = 2.5), much garbage (RR = 2.4), and many roaches (RR = 2.2). The high statistical correlation of dog contact with seroconversion in children suggests the possibility that dogs, by eating and rolling in cat feces, are instrumental in mechanically transmitting Toxoplasma infection. In addition, flies, and to a lesser extent, cockroaches, may have practically important roles in transmission.

Animals↗

PubMed1976

Prevalence of Toxoplasma infection in cattle slaughtered at an Ohio abattoir.

Specimens of heart, diaphragm and, in 159 instances, esophagus were taken from 352 cattle slaughtered in an Ohio abattoir. The total weight of the material was 32 kg, and this was fed to 12 specific-pathogen-free cats to test for Toxoplasma infectivity. None of the 12 cats acquired Toxoplasma infection, as evidenced by their failure to excrete oocysts or to develop antibodies, and by failure to isolate Toxoplasma from their tissues by inoculation into mice. In further tests, a 650-g sample of beef from 77 of the 352 cattle was digested in 1% trypsin, then inoculated intraperitoneally into 40 mice. Toxoplasma was not isolated from the mice. Serums from 186 of the 352 cattle were inactivated at 60 C for 1 hour, then examined by the Sabin-Feldman (dye) test for antibodies to Toxoplasma. None had titers of more than 1:8. Of the 186 serums, 77 were also examined for antibodies in the indirect hemagglutination test. One had a titer of 1:64, which was considered nonspecific. Thus, evidence for Toxoplasma infection was not found in 352 cattle.

Abattoirs↗

PubMed2002

[Study on the behavioral risk of toxoplasma infection in population working in the slaughterhouse].

OBJECTIVE: To explore the behavioral risk of toxoplasma infection in special population. METHODS: Serum samples from workers in slaughterhouse were collected and tested for the detection of cAg, toxo-IgG, toxo-IgM antibody to toxoplasma, using ELISA. Behavioral measurements were conducted through questionnaire, which was developed according to theory of reasoned action which consists of a measurement model that specifies the relation of measured to behavioral variables and a behavioral variable model to show the influence of behavior variables on each other. Factor analysis methods were used for estimating the parameter of the former and path analyses method for the latter. RESULTS: With data from 302 research subjects, the responding rate was 95.87%. The overall infection rate was of 19.20% including 15.89% having cAg, 5.63% with toxo-IgG, 2.32% with toxo-IgM, 1.32% with the third of cAg, toxo-IgG and toxo-IgM, 2.65% with the both cAg and toxo-IgG, 1.99% with the both cAg and IgM respectively. There was significant association between toxoplasma infections and contact with dystocia or live pigs, body fluid or harslet. There was no relation between toxoplasma infections and abortion. RESULTS: from miscarriage factor analyses showed that except evaluation of behavioral outcomes and behavioral attitudes, internal consistence reliability of others behavioral variables was good (Cronbach's alpha > 0.7). These behavior variables could be explained by their measure items. Path analyses suggested that two components had provided significant independent contributions to those preventive behaviors which significantly correlated to toxoplama infections. Half of the contribution leading to toxoplasma infections had come from behavioral factors. Belief was relatively more important in predicting the preventive behavior than subjective norm. CONCLUSION: Our findings supported the application of reasoned action theory in understanding the role of behavioral factors in toxoplasma infection.

Abattoirs↗

PubMed2002

Seroprevalence of toxoplasma infection among primigravid women attending antenatal clinic at a secondary level hospital in North India.

A study was carried out among primigravid women attending a secondary level hospital in a district of North India. The objective of the study was to determine seroprevalence rate of Toxoplasma gondii infection using enzyme-linked immunosorbent assay (ELISA) test. Associations between toxoplasma seropositivity and selected variables (socio-demographic and biologically plausible variables) were also studied. Five hundred three women were enrolled from the antenatal clinic between August, 1996 and September, 1997. Of these, 210 women (41.75%) were seropositive for Toxoplasma gondii infection. These results indicate that a large number of the study subjects were vulnerable to toxoplasma infection. If these infections had occurred during the pregnancy, their foetuses would have been at high risk of infection. There was no statistically significant association between toxoplasma seropositivity and the variables studied except, in the case of type of meat usually consumed. There is a need to provide health education to pregnant women in order to prevent primary toxoplasma infection during pregnancy.

Adolescent↗

PubMed1989

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts.

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.

Acridine Orange↗

PubMed1987

Immune response to Toxoplasma gondii--analysis of suppressor T cells in a patient with symptomatic acute toxoplasmosis.

Unresponsiveness of antigen-dependent (Toxoplasma-specific and purified protein derivative of tuberculin [PPD]-specific) T-cell proliferative responses of peripheral blood leukocytes (PBL) was observed in a patient with symptomatic acute toxoplasmosis. The immunosuppression of T-cell responses was mediated by Leu 1+, Leu 2a+, and Leu 3a- suppressor T cells that were induced by Toxoplasma gondii antigen and suppressed both Toxoplasma-specific and PPD-specific PBL T-cell responses from a patient with chronic toxoplasmosis when PBL of these patients were mixed and cocultured in vitro. Participation of class II molecules of HLA in Toxoplasma-specific proliferative T-cell responses and activation of suppressor T cells was examined by using monoclonal antibodies specific for HLA-DR and HLA-DQ molecules. Anti-HLA-DQ monoclonal antibody released the suppressive activity, while anti-HLA-DR monoclonal antibody inhibited Toxoplasma-specific T-cell responses. Thus, the suppressive effect of PBL from a patient with acute toxoplasmosis on antigen-dependent PBL T-cell responses from a patient with chronic toxoplasmosis was mediated by HLA-DQ molecules. By contrast, Toxoplasma-specific T-cell responses were activated by HLA-DR molecules (presumably present on antigen-presenting cells).

Acute Disease↗

PubMed1988

Invasion of the guinea pig conjunctiva by Toxoplasma gondii.

Although interactions of Toxoplasma gondii with host cells have been studied extensively in vitro, relatively little is known about the initial interactions of Toxoplasma with mucosal surfaces in vivo. We therefore studied the onset of a Toxoplasma infection in guinea pig conjunctiva. Toxoplasma were inoculated onto the conjunctival epithelium. The tissue was fixed 15 min to 48 hr after inoculation and examined by electron microscopy. Guinea pigs similarly inoculated were maintained in the laboratory for 2 to 8 weeks and tested for antibody by the Toxoplasma dye test. We found that parasites invaded both epithelial and goblet cells within minutes of inoculation. Replication occurred within 4 hr of inoculation and took place mainly in epithelial cells. Within 48 hr, the organisms were found beneath the basal lamina of the epithelium. The host developed an inflammatory response consisting first largely of polymorphonuclear leukocytes and later largely of macrophages. The parasites also replicated in macrophages, showing their ability to evade host defenses in nonimmune animals. Inoculated guinea pigs kept in the laboratory for 8 weeks survived and developed elevated antibody titers against Toxoplasma. The guinea pig conjunctiva is a suitable tissue for studying the pathogenesis of toxoplasmosis.

Animals↗

PubMed1987

Population studies showing cross-reactivity of Toxoplasma gondii antibodies with antibodies to malignant cervical tissue antigens.

Antibody inhibition studies have established that ethanol-soluble extracts prepared from a pool of malignant cervical tissues contained substances capable of reacting with antibodies specific for Toxoplasma gondii and malignant tissue factors. Extracts from normal cervical tissue did not possess Toxoplasma or malignant tissue antigens. Antibodies to malignant cervical tissue extract could be detected singly or in combination with Toxoplasma antibodies. The overall incidence of antibodies to malignant tissue extract in over 2,000 samples of blood tested was 4.4%. Of 98 apparently healthy donors with antibodies to malignant tissue extract, 50% also possessed Toxoplasma antibodies. The combined incidence of anti-Toxoplasma activity was 16.5% of all samples tested. Methods for the isolation and determination of IgG antibodies to malignant tissue and Toxoplasma antigen extracts are described, and the possible significance of the findings discussed.

Animals↗

PubMed1983

Continual cultivation of Toxoplasma gondii on HeLa cells.

Continual cultivation of Toxoplasma gondii in monolayers of HeLa cells has been performed for 6 years. The toxoplasmas were then used for serial cultivation experiments. While searching for optimal conditions for toxoplasma inoculation it was found that infected HeLa cells which were scraped off the glass were a more suitable inoculum than those released by trypsinization. The optimal dose was one zoite per 12 host cells, medium level height at adsorption 2 mm and adsorption time 90 min at 37 degrees C. The concentration of sodium bicarbonate ranging from 8.7 to 26.1 mM had no effect on the multiplication of toxoplasmas. In serial passages in Roux bottles, when the medium was changed every 2-3 days, the average yield of toxoplasmas was 6.2 X 10(6) per ml of medium (after the second change of medium, i.e., 7 days after inoculation). The toxoplasmas obtained in serial passages were suitable for purification studies, for preparation of small amounts of antigens and as an inoculum for suspension cultures.

Animals↗

PubMed1983

Effects of neonatal spleen cell products on the multiplication of toxoplasma in rat peritoneal macrophages.

We studied the resistance of newborn rats to infection with T. gondii. Neonates of chronically infected rats were significantly more resistant to infection with Toxoplasma than were the newborn of normal, uninfected rats. Neonatal athymic rats (nu/nu) from immune mothers were susceptible to infection, whereas normal littermates (nu/+) were very resistant. Rat peritoneal macrophage monolayers did not exhibit enhanced toxoplasmacidal activity after the addition of immune serum to the culture medium. However, inhibition of Toxoplasma multiplication was observed when the parasites were exposed to immune serum just before the infection of macrophages. The effects of spleen cell products from neonates of chronically infected mothers on the multiplication of Toxoplasma in rat peritoneal macrophages were examined. A significant inhibition of Toxoplasma multiplication occurred in macrophages incubated with the supernatants of spleen cell cultures obtained from immune newborn rats, with added lysate antigen. The ability to be induced to make an inhibitory supernatant disappeared by the third week after birth. Also examined were supernatants of spleen cells sensitized in vitro by incubation with a Toxoplasma lysate antigen; when these were added to macrophage monolayers, inhibition of Toxoplasma multiplication was observed only if the antigen was accompanied by supernatant of immune spleen cells.

Animals↗

PubMed1982

[Studies on the correlation of toxoplasma antibody between mothers and their infants in toxoplasmosis in pregnancy].

The author examined immunoglobulin and toxoplasma antibody titer using a routine serologic test in 55 pregnant women with positive antibody titer and their infants (including one twin). After evaluation of the immunological correlationship between mothers and infants, the toxoplasma antibody titer half life in infants and its decay curve were derived from IgG toxoplasma antibody titer in infants. The IgG toxoplasma antibody titer at delivery well correlated with the titer transition in infants thereafter, and the infants were divided into the following three groups for comparison with mothers' titer at delivery. Group A (mothers' titer = infants' titer), group B (mothers' titer greater than infants' titer), group C (mothers' titer less than infants' titer). The antibody titer in group A and C decreased to negative at one year of age, the value in group B, however, at 6 months after delivery. Toxoplasma antibody titer (X) and antibody half life (T) from delivery to one month, from one month to 3 months and from 3 months to 6 months respectively, were derived as follows: (formula; see text) When the antibody titer of an infant is higher than the decay curve, toxoplasma infection is strongly indicated.

Antibodies↗

PubMed1995

[The prevalence of Toxoplasma infection among pregnant women and their newborn infants in Chengdu].

Purpose of the study was to determine the prevalence of toxoplasma infection among the pregnant women and their newborn infants in Chengdu and to identify risk factors of acquiring toxoplasma infection. The Maternal and Child Health Hospital was selected by random cluster sampling method in the study. Each pregnant women admitted to the above hospital consecutively and her surviving newborn at birth were included in this survey. History, physical examination and blood specimens were obtained from 1,211 pairs of mother-newborns. ELISA was used to detect toxoplasma IgG and IgM antibodies. Results revealed that sera prevalence of toxoplasma IgG antibodies and IgM antibodies of pregnant women were 39.14% and 4.21% respectively. Seraprevalence of toxoplasma IgM antibodies of newborn infants was 1.07%. Congenital malformation of newborn infants may be associated with congenital toxoplasma infection (P < 0.05, OR = 6.32).

Animals↗

PubMed1993

Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice.

This study was performed to determine the T-cellular immune responses following Toxoplasma gondii oral infection and to assess further toxoplasma antigens on their ability to stimulate in vitro mucosal and systemic T-cell immunity. Parasite-specific cellular immune responses in Peyer's patches (PP), in mesenteric lymph nodes (MLN) and in spleen (SPL) were investigated using a lymphoblastic transformation test following oral infection of mice with strain 76K cysts of T. gondii. An early toxoplasma sonicate-induced mucosal T-cell proliferation occurred in MLN and PP with a peak responsiveness on day 6 post-infection (PI) and rapidly reached background levels on day 7 PI in PP and on day 8 PI in mesenteric lymph nodes. A later splenic cellular blastogenesis was observed from day 28 PI and persisted throughout the experiment (day 91). At the time of T-cell proliferation, FACS analyses revealed a decrease in the relative percentages of CD4+ and CD8+ T cells with a predominance of CD8+ lymphocytes which leads to an inversion of the CD4/CD8 ratios. We found that CBA/J is a high responder mouse strain in the induction of mesenteric and splenic T-lymphocyte blastogenesis compared to the intermediate responder BALB/c and low responder C57BL/6. Toxoplasma gondii antigens SAG1 (30,000 MW) and GRA4 (40,000-41,000 MW), which are known to induce locally IgA antibodies, are shown to stimulate primed mucosal T lymphocytes from CBA/J and BALB/c mice whereas no proliferation was demonstrated with C57BL/6 T cells. 229-242 peptide, derived from the deduced amino acid sequence of GRA4, only induces detectable proliferation of primed-CBA/J T lymphocytes. Following oral experimental infection, the in vitro mesenteric response to a toxoplasma sonicate is dominated by a Th2-type cytokine pattern whereas a predominant Th1 cytokine response is observed in the spleen. Finally, in vitro stimulation of mesenteric T cells with the three defined toxoplasma antigens resulted in secretion of interleukin-5 (IL-5) and IL-6 (except for SAG1) and interferon-gamma (IFN-gamma) whereas no detectable IL-2 or IL-4 was observed.

Animals↗

PubMed1997

The importance of detecting circulating Toxoplasma antigens in human cases.

This work was performed on 100 suspected toxoplasmosis cases including 75 females with complicated obstetric histories, 15 children presented with hydrocephalus, retino-chorditis and lymphadenitis and 10 children suffering from leukaemia. Also 40 age-matched controls were included in this study. Serum samples from all patients and controls were examined to detect anti-Toxoplasma IgG and IgM using indirect ELISA. Antiserum against Toxoplasma avirulent strain was prepared in New-Zealand white rabbit, then it was used after purification for detecting circulating Toxoplasma antigens in the sera of these studied groups using a double antibody sanwich ELISA technique. Positive anti-Toxoplasms IgG was detected in 45% of the female group while 19% were IgM positive. According to IgM/IgG ratio, 9 cases were considered as acutely infected-females and 25 as chronically infected ones. The mean optical density of both IgG and IgM among acutely and chronically infected females were found significantly higher than of the control groups. Ten non-leukaemic children were IgG and IgM positive cases and were considered as acutely infected patients. On the other hand, all children with leukaemia were negative for anti-Toxoplasma IgG and IgM. Eleven out of 19 acutely infected cases (58%) had positive Toxoplasma antigens, whereas, only one case (4%) with chronic infection had positive antigen level. The mean optical density of circulating antigen level was significantly higher among acutely infected females and children than chronically infected and control groups. No antigen level difference was found between chronically infected patients and control groups. Six children from leukaemic group (60%) had positive circulating Toxoplasma antigen levels with significantly higher mean optical density than control groups (P < 0.001). As a conclusion the results demonstrate that the detection of circulating antigens can aid in the diagnosis of infection by T. gondii in humans, specially in immuno-compromised patients whose serologic response can be impaired.

Adult↗