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A comparative study of total saline extract (SE) and cyst vesicular fluid (VF) of Taenia solium metacestodes by ELISA and Western blotting assay (WB) tests was conducted to detect IgG in sera for diagnosis of human cysticercosis. Sera were obtained and analysed by ELISA in 1 : 20 and 1 : 100 dilutions from 208 individuals: 22 confirmed neurocysticercosis (NC) (group 1), 101 suspected NC (group 2), 55 with various intestinal parasitosis (group 3) and 30 healthy individuals (group 4). The WB test was carried out on SE and VF extracts with and without reducing agent, 2-beta-mercaptoethanol (2-ME) in 20 sera of each group. WB using extracts without 2-ME and ELISA at 1 : 100 dilution were compared in 20 sera from each group; sensitivity and specificity were calculated using samples from groups 1, 3 and 4. By ELISA, in the 1 : 100 sera dilution reactivity was reduced for both antigens without changes in the sensitivity of the test. By WB, antigens treated with 2-ME demonstrated low specificity. For SE and VF antigens, the proteins of 24, 39-42, 47-52, 56, 64-68, 126-155 kDa and 18, 24, 26-28, 32-36, 47-52, 75 kDa, respectively, were considered immunodominant markers, with high indices of specificity, suggesting a profile for NC patients. However, as the sensitivity was found to be low, it might still not be a definitive test for NC when used alone. These data suggest WB as an indicative test to determine exposure to T. solium. ELISA and WB together may supply reliable results for the diagnosis of human cysticercosis, since appropriate purified antigens are not available yet.

We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.

In the present investigation we studied some behavioral and immunological parameters of adult gastropod mollusk, Biomphalaria tenagophila, which have been reproducing for several generations under laboratory conditions. One group of gastropods was kept on a 14-h light/10-h dark cycle, corresponding to a regular circadian cycle, and another group was exposed to continuous light for 48 h. Animals were studied along (behavioral groups) or immediately after (immunological groups) 48 h of regular circadian cycle or continuous light conditions. Stopping/floating, dragging and sliding were the behavioral aspects considered (N = 20 for regular cycle; N = 20 for continuous illumination) and number of hemocytes/microl hemolymph was the immunological parameter studied (N = 15 for regular cycle, N = 14 for continuous illumination). Animals under continuous illumination were more active (sliding = 33 episodes, dragging = 48 episodes) and displayed a lower number of hemocytes (78.0 +/- 24. 27/microl) when compared with mollusks kept on a regular circadian cycle (sliding = 18 episodes, dragging = 27 episodes; hemocytes = 157.6 +/- 53.27/microl). The data are discussed in terms of neural circuits and neuroimmunological relations with the possible stressful effect of continuous illumination.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect IgG and IgM antibodies in human sera against a synthetic tripeptide derived from a hybrid peptide containing 3 specific epitopes from Trypanosoma cruzi. This assay was compared in Brazil with one using conventional antigen, the alkaline crude extract. Serum samples were divided into positive (40 samples) or negative (107 samples) for Chagas disease. Positive samples included 9 serum samples from patients with acute Chagas disease, while negative samples included 57 samples from patients suffering from viral diseases. The total percentages of IgG positive samples from patients with chronic Chagas disease for alkaline extract and synthetic tripeptide were 93.5% and 100%, respectively. All samples from patients with acute Chagas disease were confirmed positive for IgM antibodies by using both the tripeptide and the alkaline extract. However, the results for anti-T. cruzi IgM in the group of chronic Chagas disease patients demonstrated that 41.9% were positive for IgM with the alkaline extract, while the synthetic peptide showed a significantly lower number of positive samples (12.9%). The serum samples from healthy people showed similar results for both antigens. However, 40% of the serum samples from patients presenting with viral diseases were IgM positive for Chagas disease when assayed with conventional antigen; with the synthetic tripeptide as antigen, 100% of this group of samples were found to be negative. Thus, as the results of ELISA with synthetic tripeptide showed higher rates of sensitivity and specificity than ELISA with conventional antigen, the former should be included as a laboratory tool in the serodiagnosis of Chagas disease.

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

The potential of the RH strain of Toxoplasma gondii to invade trophoblast cells of the cricetid rodent Calomys callosus in a congenital infection in the initial third of pregnancy was investigated in this study using morphological and immunocytochemical approaches. The animals were intraperitoneally inoculated on the 1st day of pregnancy and the infection was observed on day 7. Various numbers of parasites could be observed inside the parasitophorous vacuoles in trophoblastic cells under light and electron microscopy. The trophoblast cells showed characteristics of healthy cells, and no alteration other than parasite vacuoles in their cytoplasm could be detected. Polyclonal or monoclonal anti-T. gondii antibodies (respectively, anti-T. gondii components and the major surface parasite antigen p30) labeled both the parasite surface and parasitophorous vacuole membranes, regardless of the number of parasites inside the compartment. In addition, p30-containing trails were detected in the extracellular matrix surrounding trophoblastic cells similar to those found with other parasites during locomotion and the invasion process. Our results show the ability of T. gondii to infect trophoblast cells during the early blastocyst-endometrial relationship and open new possibilities for more accurate study of the invasion process of this parasite and the role of the trophoblast as an embryo defense barrier.

An experimental model for acquired and congenital ocular toxoplasmosis as well as a model to induce experimental autoimmune uveitis (EAU) was investigated in Calomys callosus. Toxoplasma gondii, ME-49 strain, was used to infect males and pregnant- and not pregnant-females while S-antigen, a major glycoprotein of the retinal photoreceptor cell, was used to induce EAU. The ocular lesions elicited by T. gondii were characterized by the presence of cysts, free tachyzoites and inflammatory cells in the retina or related tissues. In the congenital form, 40% of the fetus presented ocular lesions, i.e., presence of cysts in the retina, vitreous, and extra-retinal tissues. In the acquired form, 75% of the females and 50% of the males presented unilateral ocular cysts both at 21 and 47 days post-infection. It was also demonstrated that S-antigen was not uveitogenic in the C. callosus model. No lesion was observed in the animals exclusively immunized with this retinal component, even when jacalin was used as additional adjuvant for polyclonal response to the retinal antigen. It can be concluded that C. callosus may constitute in a promising model for study both acquired and congenital ocular toxoplasmosis, particularly when it is important to make sure that a non autoimmune process is involved in the genesis of the ocular infection.

Stage conversion between bradyzoites and tachyzoites was investigated in C57BL/6 mice chronically infected with the ME-49 strain of Toxoplasma gondii. In order to promote bradyzoite-tachyzoite conversion, mice were treated in vivo with neutralizing doses of anti-gamma interferon (IFN-gamma) or anti-tumor necrosis factor alpha (TNF-alpha) antibodies. Expression of parasite-specific antigens SAG-1, SAG-2, and heat shock protein 70 (Hsp-70) was visualized in the central nervous system by immunocytochemistry and measured by photometric assay. The immunosuppressive effect of anti-IFN-gamma or anti-TNF-alpha treatment was immediate, leading to parasite stage conversion as indicated by the increased expression of tachyzoite-specific antigens (SAG-1 and SAG-2) and by rapid parasite replication. We also observed expression of high levels of Hsp-70 during a short period of conversion of bradyzoites to tachyzoites. Our data suggest that Hsp-70 may have an important role in the process of bradyzoite-tachyzoite conversion during the reactivation of chronic toxoplasmosis.

Calomys callosus, Rengger 1830 (Rodentia, Cricetidae), a wild rodent found in Central Brazil, was studied to investigate its susceptibility to Toxoplasma gondii experimental infection and its humoral immune response against this protozoa. The electrophoretic profile of the serum proteins of C. callosus showed that IgG, which shows no affinity to Protein A, has higher cross reactivity with rat IgG than with IgG from other rodents. The susceptibility assay was performed by inoculation groups of animals with various suspensions of T. gondii tachyzoites from 10(2) to 10(6) parasites. All animals died between 3 and 9 days after infection and the kinetics of antibody synthesis was determined. Basically, they recognized predominantly the immunodominant antigen SAG-1 (P30). The immunohistochemistry assays revealed that the liver was the most heavily infected organ, followed by the spleen, lungs, intestine, brain and kidneys. It can be concluded that C. callosus is an excellent experimental model for acute phase of Toxoplasma infection.

The excreted/secreted antigens (ESA) of Toxoplasma gondii are thought to play an important role in the pathogenesis and immune escape of the parasite and the host's immunity to infection with it. In the present study, ESA from peritoneal exudates of infected mice were precipitated with ammonium sulphate (a 40% saturated solution giving the best yield of antigenic components). They were then analysed by immunoblot assay and dot-ELISA using serum samples from 25 patients with toxoplasmosis (19 in the acute phase and six chronic). Three groups of antigens, with molecular masses of 35, 30-32 and 26 kDa, reacted strongly with IgG antibodies, and antigens from two of these groups (30-32 and 26 kDa) also reacted with IgM and IgA.

We evaluated the titers of anti-T. gondii antibodies by various serological tests in 40 serum samples from dogs exhibiting clinical signs of infectious diseases. Indirect immunofluorescence (IgG-IFI), indirect haemagglutination (IHA and M-Toxo) and immunoenzymatic (ELISA and PA-ELISA) tests were carried out. Titers > or = 64 were considered as positive. Anti-Toxoplasma antibodies were found in 9 (22.5%), 14 (35%), 14 (35%) and 12 (30%) samples, respectively for IHA, IgG-IFI, ELISA and PA-ELISA. The results showed that 57% were negative in all tests and 43% of the dogs presented antibodies to T. gondii; from these, 20% were positive in all three tests with high titers of antibodies and 23% were positive in only one or two tests with low titers of antibodies and mainly related to the IFI and ELISA tests. We observed 5 (12.5%) and 1 (2.5%) reactive samples, respectively, by M-Toxo and IHA with or without 2-mercapthoethanol, in the attempt to detect specific IgM. We can conclude that serodiagnosis of toxoplasmosis in dog have to be based on the combination of serological tests (IFI and ELISA) and with emphasis at the determination of the titers and the classes of the specific antibodies.

Serum samples from 107 cervids were examined for Toxoplasma gondii antibodies using indirect hemagglutination (IHA), indirect immunofluorescence (IFA), enzyme linked immunosorbent assay (ELISA) and Dot-ELISA. Samples were obtained from 66 marsh deer (Blastocerus dichotomus) in the State of São Paulo (Brazil) and from 41 pampas deer (Ozotocerus bezoarticus) in the State of Goiás (Brazil). Antibodies to T. gondii were found in 23 (22%) of the deer, with 18 and 5 positive samples, respectively, for B. dichotomus and O. bezoarticus. The highest prevalence of T. gondii antibodies were young adults (32%), following by adults (27%) and fawns (13%). Only one serum sample (8%) from a newborn fawn was positive in the serological tests. The convenience of the Dot-ELISA test is obvious when compared with other serological tests for both laboratory or field surveys, mainly due to its features of practicability and reagent stability.

Paired human saliva and serum samples from 60 individuals were tested for specific IgA and IgG antibodies to Toxoplasma gondii. The study in both fluids was carried out by indirect immunoenzymatic assay (ELISA). Saline antigenic extract of T. gondii was used to coat plastic surfaces upon which the samples were then incubated; monospecific conjugates of anti-IgA and anti-IgG-peroxidase were then incubated with the samples after a washing procedure to separate the unbound antibodies. The enzymatic activity was measured and the results expressed in terms of ELISA index. Toxoplasma-specific IgG antibodies were detected in 43 of the serum samples (71.7%) and in 12 of the saliva samples (20.0%) whereas Toxoplasma-specific IgA antibodies were detected in 18 of the serum samples (30.0%) and in 12 of the saliva samples (20.0%). No association was observed when the Toxoplasma-specific IgG reactive and non-reactive serum samples were compared with the reactive and non-reactive saliva samples for this class of immunoglobulin. On the other hand, a significant association was observed when the Toxoplasma-specific IgA reactive and non-reactive serum samples were compared with the reactive and non-reactive saliva for this type of antibody. In conclusion, our results show that the detection of salivary IgA reflects the serum level of this isotype but salivary IgG does not. Moreover, the isolated detection of salivary IgG may not contribute to epidemiological studies of chronic toxoplasmic infections.

The initial attachment of Toxoplasma tachyzoites to target host cells is an important event in the life cycle of the parasite and hence critical in the pathogenesis of this infection. The efficiency of Toxoplasma attachment to synchronized populations of Chinese hamster ovary cells and bovine kidney cells was investigated by using a glutaraldehyde-fixed host cell assay system. For both cell lines, parasite attachment increased as the synchronized host cells proceeded from the G1 phase to the mid-S phase and then decreased as the cells entered the G2-M boundary. Postulating that these differences in attachment reflect the upregulation of a specific receptor, polyclonal antibodies were generated against whole MDBK antigen at 0 and 4 h into the S phase. Both antisera were shown to inhibit parasite attachment to both synchronous and asynchronous host cell populations. However, the attachment blockade observed with the 4-h antiserum was significantly greater than that with the 0-h antiserum, completely abolishing the cell cycle-dependent increase in attachment found in control samples. These findings suggest that Toxoplasma tachyzoites bind specifically to a host cell receptor which is upregulated in the mid-S phase of the cell cycle.

Levels of total and specific anti-Trypanosoma cruzi immunoglobulin E (IgE) were determined by immunoenzymatic assay among 101 samples of pericardial fluid from patients who had died in one trypanosomiasis endemic area in central Brazil. These samples were divided into 6 groups. Group I, 17 samples from patients with the cardiac form of Chagas disease; group II, 11 samples from patients with the digestive form of Chagas disease, presenting megaoesophagus and/or megacolon; group III, 41 samples from patients with the indeterminate form of Chagas disease; group IV, 4 samples from patients with both cardiac and digestive forms of Chagas disease; group V, 5 samples from patients who suddenly died and were seropositive for T. cruzi antibodies; group VI, 23 samples, used as a control group, which came from patients seronegative for T. cruzi antibodies. Significantly high levels of total IgE were observed in groups I, II, III, IV and V when compared with group VI (mean concentrations 708-1157 iu/mL compared with 394 iu/mL). In groups I-V, 32 samples (41%) had specific anti-T. cruzi IgE antibodies. The individual percentage positivity rates in these groups were 64.7% (group I), 45.4% (group II), 34.1% (group III), nil (group IV), and 40.0% (group V). A significant correlation between total IgE and specific anti-T. cruzi IgE was observed only in the samples from patients with the cardiac form of Chagas disease (group I).

During its intracellular life cycle within the infected host cell, Toxoplasma gondii is able to undergo rapid asexual replication. Neither the mechanism by which the parasite initiates this process nor the requirements for maintaining it are understood. We produced a monoclonal antibody, 1B8, that identifies a parasite antigen of approximate M(r) 97 kDa as determined by SDS-PAGE. The epitope recognized by mAb 1B8 appears as a collection of vesicular structures scattered throughout the cell cytoplasm. When RH strain parasites are incubated with mAb 1B8 in the absence of serum complement, parasite growth is inhibited by > 90% as determined by radioisotope incorporation. Both attachment and invasion assays show that neither of these parasite-host cell interactions are inhibited by the mAb. However, a marked reduction in the number of intracellular rosettes was observed following mAb treatment of the parasites. Viable extracellular parasites are able to endocytose mAb 1B8. Once within the parasite cytosol the antibody recognizes the vesicular structures similar to those observed with fixed parasites. Immunofluorescence assays with Besnoitia jellisoni and Plasmodium falciparum show that the epitope recognized by mAb 1B8 is conserved among Coccidiae but not the kinetoplastid Leishmania.

Previous observations have demonstrated that monoclonal and polyclonal antibodies directed at SAG-1, the major surface protein of Toxoplasma gondii, decreased the number of T. gondii that infected fibroblast monolayers. Direct evaluation of parasite-host cell attachment using glutaraldehyde-fixed human fibroblasts and live tachyzoites was performed to determine whether SAG-1 was a ligand for the host cell receptor. The interaction between the fixed cells and T. gondii was specific and saturable as determined by a radioisotope competitive binding assay. Moreover, the specificity of this interaction was confirmed by comparison to another member of the Apicomplexa, Besnoitia jellisoni. Treatment of fresh extracellular T. gondii with rabbit polyclonal anti-SAG-1 serum inhibited parasite attachment to host cells by 71%. A monoclonal antibody (6A8) directed at SAG-1 was able to inhibit parasite binding to fixed host cells by 65%. Other mAb's directed at SAG-1 failed to inhibit parasite attachment in this assay. Fab derived from 6A8 mAb showed dose-dependent inhibition of parasite attachment. At an Fab concentration of 25 micrograms/ml, 47% inhibition was observed. Attachment assays using mutant parasites with defective SAG-1 (PTgA and PTgC) showed significantly reduced binding (26 and 39%) when compared to wild-type (SAG-1+) parentals. Taken together, these observations suggest that SAG-1 is an important parasite ligand that binds to the host cell in the process of T. gondii invasion.

Recent studies indicate that Toxoplasma gondii attachment is mediated via a parasite ligand-host cell receptor interaction. Lloyd Kosper and Jose Mineo here survey factors involved in the attachment to and penetration and invasion of host cells by T. gondii.

Using a modified enzyme-linked immunosorbent assay that included dissociation of antigen antibody complexes with 6M urea solution, we analyzed the avidity of Toxoplasma-specific IgG in aqueous humor and serum samples from 24 patients with toxoplasmic chorioretinitis. As a control, we studied aqueous humor and serum samples from 14 cataract patients without history of uveitis and serum samples from 10 patients with recent primary systemic toxoplasmic infection without ocular lesions. IgG avidity was markedly lower in aqueous humor samples from patients with toxoplasmic chorioretinitis than in serum samples, despite those samples presenting higher levels of Toxoplasma-specific IgG than in serum samples. The detection of the low-avidity Toxoplasma-specific antibodies can offer a valuable aid to make a specific etiologic diagnosis and perhaps contribute to understand the pathogenic mechanisms of ocular toxoplasmosis.

Monoclonal and polyclonal, monospecific antibodies to the major surface antigen of Toxoplasma gondii (SAG-1, P30) inhibit infection of human fibroblasts and murine enterocytes. Fab prepared from polyclonal, monospecific antibody to P30 also have this inhibitory effect on invasion, which indicates that this antibody directly blocks parasite infection of host cells rather agglutinating the parasite. Antibodies to another surface protein (P22) did not alter in vitro infection. If the inhibitory effect of antibody to P30 was due to steric hindrance or complexing of surface epitopes contiguous to P30, antibodies to other surface epitopes would also be inhibitory and they are not. Urea treatment of antibody (which permits discrimination of high and low avidity antibody) did not alter the effect of anti-P30 antibody. This observation indicates that the effect of the antibody to P30 was not an artifact of differences in the avidity of the antibody to P22 and P30. Heat inactivated antisera from mice infected with either RH or PTg strain T. gondii (P30+) inhibit infection of fibroblasts when challenged with autologous wild-type parasites by 87 and 40%, respectively. In contrast, these antisera have little inhibitory effect (13 and 19%, respectively) against infection of human fibroblasts by a P30-deficient mutant (PTgB). Antisera raised to the P30-deficient mutant had no significant effect on infection of cells by wild-type strains that have surface P30. The neoglycoprotein, BSA-glucosamide, competitively blocks infection of human fibroblasts by P30+ tachyzoites with surface P30 in higher level than those without surface P30. This observation indicates that there is likely to be a glycosylated host cell receptor to which T. gondii's major surface Ag SAG-1 (P30) binds. Mice infected perorally develop intestinal IgA antibody to the major 30-kDa epitope of T. gondii. Thus, the major surface epitope of T. gondii, SAG-1 (P30), has an important, functional role in infection of host cells by T. gondii and elicits an intestinal antibody response after peroral infection.

Areas where mucocutaneous leishmaniasis, kala-azar and Chagas' disease are prevalent often overlap in Latin America. Due to a variable degree of cross-reactivity between the three etiological agents and antibodies synthesized during the course of the disease, a test able to distinguish between them is greatly needed. Saline-extracted antigen from live Trypanosoma cruzi epimastigotes behaved towards Chagas' disease sera in immunoenzymatic assays (ELISA) with the same specificity and sensitivity as the antigen used routinely for such tests. In tests using mucocutaneous leishmaniasis or kala-azar sera, the live T. cruzi antigen showed a much lower geometric mean titer (GMT) than the standard T. cruzi antigen. In ELISA tests with L. braziliensis antigen a higher GMT was seen with kala-azar sera than with those from cases of mucocutaneous leishmaniasis. Also, sera from Vale do Ribeira leishmaniasis showed higher GMT with an L. donovani antigen than with a homologous one. Such discrepancies were not seen in immunofluorescence tests employing the same sera and antigens.

Enzyme-linked immunosorbent assay (ELISA) for toxoplasmosis was evaluated in serum samples presenting defined toxoplasmosis serological patterns, as determined by results in immunoglobulin (Ig)G and IgM immunofluorescence (IgG-IF, IgM-IF), hemagglutination, and complement fixation tests. ELISA was carried out with alkaline phosphatase-labeled anti-IgG and anti-IgM antibodies. Serum titer was expressed as the serum dilution end point determined by mere observation of color development in the test. A straight agreement was found between IgG-ELISA and IgG-IF titers, both in group A sera of ancient infections (patterns II and III) and group B sera of recent infections (pattern I). A similar agreement was found between IgG-ELISA and hemagglutination titers in group A sera, for which coincident IgG-IF and hemagglutination titers are also frequent. However, in group B sera, in spite of the same toxoplasma extract being used to sensitize both plastic surfaces and erythrocytes, IgG-ELISA titers were much higher than hemagglutination titers, in a way similar to that observed for IgG-IF titers. IgM-ELISA was positive in every group B serum, with higher titers than corresponding IgM-IF titers. Occasional low-titered positive IgM-ELISA results were seen for group A sera, sometimes due to IgM-antiglobulin antibodies. An easy test to perform, ELISA seems to be an adequate substitute for toxoplasmosis IF tests for routine purposes.

Toxoplasma gondii tachyzoites were purified from mouse peritoneal exudates using lectins to remove the host cells. Best results were obtained with phytohemagglutinin P at a concentration of 0.01% (W/V), which provided a 99.7% pure Toxoplasma suspension. The process was reproducible and easy to perform. Toxoplasmas so obtained were infective and served as sources of high quality antigens for immunofluorescence, hemagglutination, and complement fixation tests.

A polysaccharide fraction from Toxoplasma gondii was adsorbed to polystyrene plates, and the enzyme-linked immunosorbent assay was performed (poly-ELISA) with peroxidase-labeled anti-immunoglobulin G and anti-immunoglobulin M antibodies. A comparison was made with a T. gondii total protein extract ELISA (protein ELISA) in serum samples presenting different toxoplasmosis serological patterns, as indicated by a battery of tests for toxoplasmosis. Very low titers and negative results were seen for immunoglobulin G poly-ELISA both for serum samples corresponding to ancient or transitional-period infections (serological patterns II and III) and for samples of recent or acute toxoplasmosis (pattern I). On the contrary, immunoglobulin M poly-ELISA furnished high titer results for pattern I sera, and a very close agreement of titers was seen between immunoglobulin M protein ELISA and immunoglobulin M poly-ELISA. When the polysaccharide fraction was added to pattern I sera, a complete blocking of immunoglobulin M antibody reactivity resulted only for poly-ELISA. In the same way, the total protein extract could completely block only reactivity for protein ELISA. In both cases, a limited decrease in titers was observed for respective heterologous assays.