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Reactivation of chronic toxoplasmosis resulting in Toxoplasma encephalitis (TE) is a common event in acquired immune deficiency syndrome (AIDS) patients. Conversion from Toxoplasma gondii bradyzoites to tachyzoites is a prerequisite for reactivation. Until recently, the study of stage conversion in human tissue was not possible due to the lack of antibodies that recognize stage-specific epitopes after long-term formaldehyde fixation. Using the combination of a polyclonal anti-T. gondii antibody, the cyst-stage-specific monoclonal antibody CC2, and a tachyzoite-specific polyclonal antibody (anti-SAG1, recombinant), we tried to demonstrate parasite differentiation in the brain tissue of 10 AIDS patients with clinically suspected TE. Double labeling of the stage-specific antibodies enabled us to demonstrate interconversion between tachyzoites and bradyzoites for the first time in human tissue. The study confirmed that the transformation process is nonsynchronous and that the manifestation of TE depends on the degree and site of tissue destruction caused by invading tachyzoites. The original source of tachyzoites could never be located, but a few samples suggested that tachyzoites may invade by dissemination across the blood-brain barrier. Cyst rupture as the first event in the process of reactivation was not seen. We conclude that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical symptoms occur.

A murine model of pulmonary toxoplasmosis was examined morphologically and immunochemically using 3 strains of Toxoplasma gondii. BALB/c and NMRI mice were infected with tachyzoites of a virulent strain (RH) or with brain cysts of an avirulent (GAIL), or moderately virulent, strain (NED). Depending on the strain of T. gondii, the degree of infection, number of parasites, and replicative potential of T. gondii in lungs varied. In lungs of mice infected with the RH strain, the number of parasites increased in mice during the survival period. Only tachyzoites were found, as confirmed by stage-specific monoclonal antibodies. In lungs of mice infected with the GAIL strain or the NED strain, different stages of parasites were detected. Up to day 14 after infection, only tachyzoites were present, followed by bradyzoites between days 14 and 20 after infection. The number of cysts decreased and finally could not be detected in this organ. In general, the number of cysts that developed in the lungs was smaller than the number that developed in the brain.

Monoclonal antibody (mAb) TxE2, reactive with Toxoplasma gondii excretory products, detects an acidic 29 kDa protein (p29) which, in 2D gel electrophoresis, exhibits a migration pattern distinct from those of the toxoplasmic excretory proteins described so far. The sequence of seven peptides from tryptic digestion of isolated p29 allowed the design of primers to obtain the coding DNA sequence. The full-length gene was amplified from genomic DNA of T. gondii strain BK and the sequence was identical with that of the corresponding cDNA, providing evidence for an intron-free gene structure. A single mRNA transcript of 1.3 kb was detected by Northern blot analysis. The deduced 236 amino acid protein contains a putative N-terminal signal peptide, one site of potential N-linked glycosylation, and, close to the C-terminus, a further hydrophobic, putative transmembrane domain. With synthetic peptides spanning the sequence of p29, the epitope for mAb TxE2 was mapped adjacent to the putative signal sequence. The antigen, which represents almost 0.5% of T. gondii protein, is expressed in strains of all three intraspecies subgroups, and is associated with the parasite dense granules as demonstrated by immunoelectron microscopy. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. In bradyzoite-infected cells, p29 is present within the host cell cytoplasm as detected by immunofluorescence staining, and, furthermore, in the supernatant of cyst-bearing cell culture lacking extracellular parasites as shown by enzyme-linked immunosorbent assay (ELISA). Thus, p29 which is named dense granule protein (GRA)7 may indicate the presence of intracellular toxoplasma.

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo.

The phenomenon of released Toxoplasma gondii cyst antigens was studied immunohistochemically and cytochemically. It could be demonstrated that the preservation and localization of excreted/secreted antigens depended not only on the technique of preparation and fixation of the infected tissue but also on the preservation of the host cell. The soluble, bradyzoite-secreted amorphous cyst matrix material is released either after host cell destruction or permeabilization of the cyst wall. Ultrastructural studies could prove that the liberation of the cyst from its host cell does not affect the basic structure of the cyst wall but causes stretching of the invaginated limiting membrane.