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ABSTRACTThis work evaluated the kinetics of events that occur in the placenta of Calomys callosusafter Toxoplasma gondiiinfection. Animals on the first day of pregnancy (dop) and virgin nonpregnant females were perorally infected with 20 cysts of T. gondiistrain ME49. After 100 days of infection, the virgin animals were mated and received an additional 20 cysts on the first dop. The placentas and the embryos from both acutely and chronically infected animals were analyzed up to day 20 of pregnancy by morphological and immunocytochemical assays. Noninfected and infected animals exhibited placenta with normal morphology. From the seventh dop and infection onwards, liver and spleen cells of the infected animals contained several parasitophorous vacuoles. On the 13th day, the maternal blood present at the placental blood spaces contained T. gondii-infected leukocytes. Infected placental cells were only seen on the 15th dop, being the trophoblast giant cells, the first cell type to contain signs of the parasite internalization, followed by labyrinth zone cells 24 h later and spongiotrophoblast cells only after the 19th dop. Fetal liver and brain were infected by T. gondiiconcomitantly to the labyrinth cell infection. No signals of infection were observed on placentas and embryos from chronically infected animals. Therefore, considering the sequence of events leading to the infection of the various organs, it could be hypothesized that the placenta is infected later on during pregnancy, which may be related to the defense roles played by this structure. However, trophoblast giant cells are unable to completely stop the progression of T. gondiiinfection towards the fetal tissues. C. callosuswas demonstrated to be a suitable experimental model to study the dynamics of congenital toxoplasmosis.

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.

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.

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.

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.