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Secreted Antigens from Toxoplasma gondii Cysts
Zbl. Bakt. 284, 378-389 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Nonimmunological Factors Affecting the Release of Excreted/Secreted Antigens from Toxoplasma gondii Cysts * INGRID REITER-OWONA, MONIKA SAHM, and HANNS MARTIN SEITZ Institute for Medical Parasitology, University of Bonn, Germany
Received March 13, 1996 . Accepted March 27, 1996
Summary 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.
Introduction Metabolic products (exoantigens) of Toxoplasma gondii were localized by Werner et al. (21) around cryosectioned tissue cysts of chronically infected mice. A subsequent study of Hay et al. (9) failed to confirm the previous results. However, the constant stimulation of the immune response by excretion of exoantigens from the "dormant" T. gondii cysts has never been questionable since immunity for T. gondii is considered to depend on the presence of cysts in the host. The presence and immunogenicity of excreted/secreted antigens (EISA) in chronically infected hosts was shown indirectly by the demonstration of circulating antigens (8) in the serum and stage-specific antibodies (14, S, 20). More recently, a variety of cystlbradyzoite-specific monoclonal antibody has been raised (12, 18,2, 13,23). Antigens originating from granules inside the bradyzoites were located immunocytochemically by Torpier et al. (19) in the ground substance (GRA2) and on the limiting membrane (GRAS) ofthe cyst wall. The authors suggested an involvement of GRAS in the immune response resulting from T. gondii infection but could not demonstrate the antigen outside the cyst wall. Naturally processed T. gondii peptides were only isolated from tachyzoite-infected host cells (1) so far.
* Dedicated to Prof. Dr. H. Brandis on the occasion of his 80 th birthday.
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In order to analyse the observed release of E/SAs from T. gondii cysts, we examined this phenomenon under different preparative conditions, with emphasis on ultrastructural studies of the cyst wall. Materials and Methods Parasites
Two low-virulent strains (DX, TT 2975) of T.gondii were used. The DX strain was of swine origin. The TT 2975 strain was isolated from a newborn with congenital toxoplasmosis. 30 NMRI female mice (average weight 30 g) were infected intraperitoneally (i. p.) with approximately 5 cysts. Groups of mice were killed 3, 6 and 12 months after infection. The brains were immediately removed and further processed as described below. Preparation of tissue samples
Brain samples were processed either as whole tissue, as impression smears or homogenates. For standard paraffin embedding, the brain was fixed for 24 h in formaldehyde (4% in 0.1 M phosphate buffered saline, PBS, pH 7.4) at room temperature, washed in buffer, dehydrated and embedded in paraffin. For standard electron microscopy, brain tissue and homogenated brains were fixed with 2% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4 °e overnight and washed in the same buffer. Samples were then dehydrated in ethanol and embedded in Spurr resin. Embedded tissue blocks were sectioned with a diamond knife and stained with uranyl acetate and lead hydroxide. To prepare frozen sections, brain tissue was either frozen without previous fixation or after fixation in formaldehyde (2% in 0.1 M PBS, pH 7.4, 1 h). Fixed tissue samples were washed 3 times for 20 min in PBS. Before freezing, tissue samples were mounted on tissue Tek R , frozen in liquid nitrogen and stored at -60 °e in a deep freezer until sectioning. For sectioning, frozen samples were cut (7-9 fim) in a cryostat at -25°C to -30 0c. Sections from unfixed brains were thaw-mounted on glass slides and stored at -20°C for 24 h before fixation with cold acetone (10 min) or formaldehyde (2% in O.lM PBS, pH 7.4, 5, 10 or 30 min). Frozen sections from fixed tissue were thawmounted on slides precoated with a mixture of glycerol and egg white (1 Vt: 1 Vt, containing thymol) and dried for 1 hat 37°C. All slides were stored at -20°C until use for immunostaining. For preparation of impression smears, small samples of brain tissue were squeezed between two slides. Slides were then separated and each slide was dried for 30 min and fixed with cold acetone (10 min) or formaldehyde (2 % in 0.1 M phosphate buffer, pH 7.4, 5, 10 or 30 min). Other samples of brain tissue were homogenated in PBS (pH 7.4) with a pistil. The resulting homogenates were kept at 4°C and aliquots were removed immediately, after 15 min, after 3 h, and after 48 h. The material was dropped on slides, air-dried and fixed with cold acetone (10 min) or formaldehyde (2% in 0.1 M phosphate buffer, pH 7.4, 5, 10 or 30 min). Until use, all slides were stored at -20°C. Antibodies
Polyclonal immune rabbit serum (R-Pc) was prepared by oral infection with approx. 500 cysts of T. gondii strain DX, followed by strain Gail, and strain TT 2975, 2 months later. Serum was collected 6 months after the last application. The monoclonal antibody ee2 (mAb ee2) was kindly provided by Dr. U. Gross, Wiirzburg, Germany. Immunofluorescence assay (IFA)
For IFA, the slides were incubated with R-Pc for 60 min (1 :40 in PBS, pH 7.4), followed by washing and incubation with fluorescein-conjugated goat anti-rabbit IgG (H + L, Sigma,
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Heidelberg, Germany) diluted 1 :50 in PBS (pH 7.4), for 60 min at 37"C in a moist chamber. The slides were mounted in buffered glycerine with p-phenylenediamine (10). lmmunoperoxidase staining (PO)
For the PO with the indirect avidin-biotin method, the VectastainR-kit (Camon LaborService GmbH, Wicsbaden, Germany) was used and staining was performed according to the instructions given bv the manufacturer. Electron immullocytochemistry
Infected brain tissues were fixed for one hour at room temperature in 2 % formaldehyde and 0.1 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) and washed 3 times in the same buffer at 4 Qe on an ice-bath. The samples were dehydrated using serially graded ethanol solutions (at 4 Qq. From a 90% ethanol solution samples were embedded in Lowicryl K4M according to the instructions given by the manufacturer (Chemische Werke Lowi GmbH, Waldkraiburg, Germany). Polymerisation was performed under UV light (Philips lamp: TL 44 D25/09N) at room temperature overnight. The distance between samples and lamp was 40 em. For post-polymerisation, the samples were kept on the bench for 48 h. Ultrathin sectIons were cut with a diamond knife and collected on unsupported 400 mesh Ni-grids. Thin sections containing Toxoplasma tissue cysts were applied onto drops of R-Pc diluted 1 :40 in 0.1 M PBS, pH 7.4 containing 1 % bovine serum albumin (fraction V, Merck, Darmstadt, Germany). After washing with PBS (2 X 5 min), the grids were incubated with 12 nm colloidal gold-conjugated goat anti-rabbit IgG (whole molecule, Sigma Immuno Chemicals, Heidelherg, Germany), diluted 1: 40 in PBS with bovine serum albumin. The duration of each incubation was 60 min at 3rc. The mAb eC2 was used undiluted, grids were preincubated with normal goat serum (20 min, diluted I: 70 in 0.1 M PBS, pH 7.4) and incubated with 12 nm colloidal gold-conjugated goat anti-rat IgG (whole molecule, Sigma Immuno Chemicals, Heidelberg, Germany), diluted 1 :40 in PBS with bovine serum albumin. Grids were examined unstained or stained with 1 % uranyl acetate in methanol with a Zeiss EM 9 S elee'tron microscope. Results Immunolabelling on acetone-fixed brain tissue smears with R-Pc anti-T. gondii antibody resulted in a bright, intensive staining (PO, IFA) of the cyst wall, the cyst content and soluble, antigenic material outside the cyst wall overlying the brain tissue cells (Fig. 1a). Labelling of extracystic antigenic material was even more intensive on cryosections of infected brain tissue (not shown). When tissue smears from the same infected mouse brain were fixed with formaldehyde (2%, 10 min) and incubated with the same specific antibody, neither the exogenous antigenic material surrounding the cyst nor the cyst wall was stained (IFA, PO). Labelling of the intracystic antigenic material was reduced and the whole cyst had obviously shrunk (Fig. 1b). By using formaldehyde fixation in increasing concentrations (2-4%) and increasing duration on tissue smears of infected brain as well as on cryosections, it could be demonstrated that intra- and extracystic labelling by R-Pc gradually decreased and was finally abolished at a concentration of 4 % formaldehyde and 30 min of fixation. Prolonged fixation (12 h) of tissue at high formaldehyde concentration (4%) before deep freezing also led to a decrease of labelling. Following short-term fixation (30 min) of whole brain tissue samples, there was a decrease in labelling of the exoantigens of those
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Fig. 1. Immunolabelled Toxoplasma gondii cyst in an impression smear of infected mouse brain, strain DX, 175 d p.i., polyclonal Toxoplasma antibody (R-Pc) and indirect avidinbiotin-peroxidase method after a) acetone fixation and b) formaldehyde fixation (2% formaldehyde, 10 min). In the acetone-fixed tissue smears, cysts were surrounded by released antigenic material. The cyst content and the cyst wall (CW) were intensively stained. After formaldehyde fixation, the intensity of staining became reduced, and outside the unlabelled cyst wall, no immunoreaction was seen. .
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cysts which had been lying near the outer surface compared to those in the centre of the sample. The morphology of the T. gondii cysts and the brain tissue in frozen sections was - as expected - less preserved than after routine paraffin embedding (4% formaldehyde, 24 h). The treatment of paraffin sections with anti- T. gondii antibody (R-Pc) and PO staining revealed, that brain cysts from one host can show different staining patterns. A small zone of soluble antigenic material surrounding the cyst wall could only be suspected in some paraffin sections. To examine the way by which intra cystic antigen can diffuse through the lightmicroscopically intact cyst wall, immunocytochemical studies were performed. It was shown that the R-Pc used was reactive to three different components of the T. gondii brain cyst in K4M-embedded material: 1. dense granules of the bradyzoites, 2. cyst matrix material, 3. cyst walUmembrane (Fig. 2). In contrast to immunohistological results, no labelling was observed in the cytoplasm of the host cell associated to the cyst. Based on these results we presumed that the location of the cyst within the intact and later thoroughly fixed host cell would prevent the E/SAs from leaking out in detectable quantities. In a series of experiments, T. gondii cysts were mechanically isolated from the host cell by homogenizing the infected brain with a pistil. Under the light microscope, the development of a lucent area beneath the cyst wall was observed. Af-
Fig. 2. Toxoplasma gondii cyst in infected mouse brain (strain DX, 380 d p.i.) labelled with polyclonal rabbit antibody (R-Pc) followed by collodial gold-conjugated goat anti-rabbit IgG. The R-Pc showed considerable binding to the cyst wall (CW) including the vesicles. Gold particles were also localized on the electron-dense ground substance (GS) and on bradyzoites mainly within the dense grana (DG). The unlabelled host cell (HC) was only seen as a thin layer ad jeeting to the eyst wall.
Fig. 3a, b. Electron micrograph of a Toxoplasma gondii cyst in mouse brain infected with strain IT 2975 (87 d p.i.). The host cell (HC) was seen as a small cell-layer surrounding the cyst. The cyst wall (CW) showed deep and branching invaginations of the limiting membrane (ME). Bradyzoites (B) were embedded in an electron-dense ground substance (GS).
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ter prolonged incubation at room temperature (30 min) the originally tightly packed bradyzoites gradually became loosened. Magnification at ultrastructural level revealed, that the lucent area beneath the cyst wall was due to a distinct space between the cyst wall and the amorphous electrondense material surrounding the clustered bradyzoites. Electron micrographs taken after 3 h of incubation documented that the formerly undulated limiting membrane (Fig. 3b) had stretched to become nearly linear without any sign of disruption (Fig.4a). The previously electron-dense, amorphous material between the bradyzoites (Fig. 3) became electron lucent and the formerly closely associated bradyzoites were now loosely packed (Fig. 4). When examined after 48 h of incubation (4 0q, the structure of the cyst wall had changed from a thick layer with many branching invaginations of the limiting membrane to a wall with an intact straight limiting membrane supported by a layer of amorphous material. The mAb CCl was found to bind strongly to the cyst wall (Fig. 5). Gold particles were not observed between parasites, suggesting a different origin than the amorphous cyst wall and cyst matrix material labelled by the R-Pc. Immunoreaction with the R-Pc and PO/Igold-staining of the liberated cysts and examination by light and electron microscopy revealed that the intensity of intracystic labelling decreased when the incubation of free cysts was prolonged. After 48 h of incubation (4 0q, there remained only a faint reaction in the interior of the cysts. The cyst wall was not labelled. Results recorded were the same for both Toxoplasma strains examined and independent from the time that had passed after infection (examined at 3,6, 12 months). At the electronmicroscopic level, the DX strain differed from other isolates insofar as brain cysts in tissue and in homogenated material contained more submembrane and cyst matrix vesicles than did cysts from other isolates. Discussion The cystic stage of T. gondii cannot be easily maintained in brain cells (17). This is one reason why we know very little about the release of bradyzoite excreted/secreted antigens (EISA) and their transport to and through the cyst wall. Although it has been suggested that bradyzoite E/SAs playa major role in the maintenance of immunity (4), the processing and presentation of the antigens by the host cell as well as the function of E/SAs in the mechanism of reactivation of latent infection are not yet known. The major aim of our study was to analyze the release of parasite antigens into the surrounding of T. gondii tissue cysts of the brain. Our labelling experiments using a polyclonal antibody (R-Pc) have clearly demonstrated that the preservation and localization of the amorphous material that comprises the cyst matrix in vivo depends mainly on the technique of preparation and fixation of the infected tissue. When the infected mouse brains were processed for immunohistology, the minority of the cysts detected revealed a faintly stained cyst wall, whereas in the majority of them, the cyst wall was not reactive but the content of the cysts was intensively stained. Antigenic substances - so called exoantigens - of T. gondii were described by Werner et al. (21) in the vicinity of older brain cysts in acetone-fixed, frozen sections. We also found a specific and very bright labelling of exoantigens after cryopreservation and acetone-fixation not only around older cysts. However, electronmicroscopy revealed that the morphology of the unfixed frozen tissue was poorly preserved and
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Fig.4a, b. Electron micrograph of a Toxoplasma gondii cyst (strain DX), after 3 h of incubation in homogenized mouse brain (175 d p.i.). The cyst wall (eW) has stretched. The deep and branching invaginations of the limiting membrane (ME) have disappeared and bradyzoites (B) were loosely packed in a lucent ground substance. Only rest material of the host cell (HC) was found. 25
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Fig. Sa, b. Immunogold labelling of mAb CC2: gold labels intensively the wall of Toxo~ plasma gondii tissue cysts. a) A similar labelling pattern existed for the cyst wall of liberated cysts, here labelled after 3 h of incubation in homogenized brain tissue. b) CW = cyst wall, HC = host cell, B = bradyzoites. Fixation: 2 % formaldehyde, 1 h.
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therefore the concentration of soluble matrix antigen outside the cyst wall has been probably due to a permeabilization of the cyst wall rather than to an active transmembrane transport of parasitic antigens to the host cell. The same is true of tissue smears where dehydration, which is obviously faster outside than inside the intact cyst wall, causes the diffusion of soluble cyst antigens. When Hay et al. (9) failed to confirm the presence of exoantigens in mouse brain tissue, they were not aware of the influence exerted by the pretreatment of their tissue samples, namely the whole body perfusion and postfixation with 10% formaldehyde. In the present study the gradual decrease in immunogenicity of matrix-derived antigens after formaldehyde fixation is demonstrated. We could also observe that aldehyde fixation did preserve the highly invaginated structure of the limiting membrane including the underlying cyst wall material and the structure of the host cell. As a consequence, the matrix antigens were preserved within the cyst at higher concentration and labelled even after fixation with 10% formaldehyde. The observation of Bonhomme et al. (3) that intracystic staining may result primarily from matrix antigen and not from bradyzoites is supported by our immunocytochemical (R-Pc) results. The polyclonal antibody used in our experiments was derived from a rabbit orally infected with avirulent Toxoplasma strains and thus antibodies were directed only against natural antigens present in the host. The R-Pc did recognize the cyst wall after acetone-fixation of tissue smears, in a minority of cysts in paraffin sections, and when whole tissue was Lowicryl-embedded. The nature of the cyst wall is poorly understood and there are only few reports on its formation in vivo and the membrane-associated transport of molecules. The thick fibrillar structure of the cyst wall demonstrated by electron microscopy (6) was thought to prevent any transfer of material to the host cell. Immunostaining of paraffin sections with anti-Toxoplasma antibodies can produce a faint staining of the wall but obviously not of the host cell. Sims et al. (15) concluded that the immunolabelling of the cyst wall in paraffin sections may represent parasitic antigen diffusion out of the cyst. Immunocytochemical labelling studies with monoclonal antibodies suggest that the cyst wall is enriched in bradyzoite-secreted antigens (22, 13, 19). In brain tissue, our polyclonal antibody equally labelled cyst matrix, cyst wall material and dense granules suggesting the same origin as bradyzoite-secreted antigens. The cyst wall material which is underlying the limiting membrane consists primarily of amorphous, electron-dense material and is supposed to give a rigid form to the cyst structure (11). We could demonstrate that the amorphous cyst wall material remains associated to the limiting membrane even when the membrane stretches until its branching invaginations disappear and most of the amorphous cyst matrix material is released through the intact membrane. Interestingly, the recognition by mAb CC2 of the amorphous material was restricted to the cyst wall material underlying the limiting membrane of the cyst wall. This finding suggests the presence of structure proteins of different, unknown origin, which are not released when the cyst is free. In situ labelling studies with the polyclonal antibody did not reveal any difference between the cyst wall and cyst matrix-derived amorphous material as already described by Parmeley et al. (13) who used a monoclonal antibody which recognized a 65-kDa antigen. The rapid diffusion of cyst matrix material through the cyst wall which we observed when cysts had been mechanically liberated from the tissue suggest that the host cell plays a central role in the release of parasite antigens, as already mentioned by Ferguson and Hutchison (7). Apparently, only if combined, the cyst wall and the host cell are able to prevent the rapid diffusion of T gondii excreted/secreted antigens. In a se-
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ries of experiments, we could never detect Toxoplasma-antigen in the host cell at electronmicroscopic level as already described by Sims et al. (16). This might be due to the intensity of the fixation method (formaldehyde). Some other reason could be the low amount of antigen (EISA) released under normal in vivo conditions and/or a bad conservation of excreted/secreted antigens in the host cell. In summary, the most convincing event which in vivo will cause the EISAs to be released in high quantities into the vicinity of the cyst and thus be presented to the host immune response will be the death or destruction of the host cell. In contrast to the hypothesis of Jones et al. (11), we could prove that the preservation of the host-derived limiting membrane does not prevent the exposure of T. gondii antigens to the host or the culture medium. It is quite conceivable that the results of in vitro tests are influenced by the preservation of the host cell and the structure of the cyst wall. Immunological assays, the demonstration of cyst specific antigens, and the testing of compounds against T. gondii have to be considered under these aspects. Also our observations offer a new hypothesis to the process of reactivation of T. gondii infection in the immunocompromised patient. The above mentioned death of the host cell or destruction of the host cell will result in a release of a considerable amount of intracystic Toxoplasma antigen, leading to cellular infiltration followed by disintegration of the cyst and finally release of bradyzoites in vivo. Acknowledgements. We gratefully acknowledge the assistance of Dr. G. Chioralia. The study was supported by grant No. 01 KI 9456 from the German Federal Ministry for Research and Technology (Bundesministerium fiir Forschung und Technologie).
References
1. Aosai, F., T. H. Yang, M. Ueda, and A. Yano: Isolation of naturally processed peptides from a Toxoplasma gondii-infected human B lymphoma cell line that are recognized by cytotoxic T lymphocytes. J. Parasitol. 80 (1994) 260-266 2. Bohne, w., J. Heesemann, and U. Gross: Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages. Infect. Immun. 61 (1993) 1141-1145 3. Bonhomme, A., F. Boulanger, L. M. Bharadwaj, D. Puygauthier-Toubas, P. Bonhomme, M. Plout, and J. M. Pinon: Toxoplasma gondii: immunocytochemistry of four immunodominant antigens with monoclonal antibodies. Exp. Parasito!' 71 (1990) 439-451 4. Darcy, F., H. Charif, D. Deslie, R.J. Pierce, M. F. Cesbron-Delauw, A. Decoster, and A. Capron: Identification and biochemical characterization of antigens of tachyzoites and bradyzoites of Toxoplasma gondii with cross-reactive epitopes. Parasito!' Res. 76 (1990) 473-478 5. Decoster, A., F. Darcy, and A. Capron: Recognition of Toxoplasma gondii excreted and secreted antigens by human sera from acquired and congenital toxoplasmosis: identification of markers of acute and chronic infection. Clin. Exp. Immuno!. 73 (1988) 376-382 6. Ferguson, D.]. and W. M. Hutchison: An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasito!' Res. 73 (1987) 483-491 7. Ferguson, D.]. and W. M. Hutchison: The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch. Patho!' Anat. 411 W. M. (1987) 39-43 8. Hassi, A. and H. Aspock: Detection and characterization of circulating antigens in acute experimental infections of mice with four different strains of Toxoplasma gondii. Zentralbl. Bakteriol. 272 (1990) 526-534
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9. Hay,]., D. I. Graham, G. N. Dutton, and S. Logan: The immunocytochemical demonstration of Toxoplasma gondii in the brains of congenitally infected mice. Z. Parasitenkd. 72 (1986) 609-615 10.Johnson, G. D. and A. Nogueira: A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Meth. 43 (1981) 349-350 11. Jones, T. c., K. A. Bienz, and P. Erb: In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gamma interferon. Infect. Immun. 51 (1986) 147-156 12. Omata, Y., M. Igarshi, M. I. Ramos, and T. Nakabayashi: Toxoplasma gondii: Antigenic differences between endozoites and cystozoites defined by monoclonal antibodies. Parasitol. Res. 75 (1989) 189-193 13. Parmeley, St. F., S. H. Yang, G. Harth, L. D. Sibley, A. Sucharczuk, and J. S. Remington: Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in matrix of tissue cysts. Mol. Biochem. Parasitol. 66 (1994) 283-296 14. Partanen, P., J. Turunen, R. A. Passivuo, and P. o. Leiniki: Immunoblot analysis of Toxoplasma gondii antigens by human immunoglobulins G, M and A antibodies at different stages of infection. J. Clin. Microbiol. 20 (1984) 133-135 15. Sims, T. A.,]. Hay, and I. C. Talbot: Host-parasite relationship in the brains of mice with congenital toxoplasmosis. J. Pathol. 156 (1988) 255-261 16. Sims, T. A.,]. Hay, and I. C. Talbot: Ultrastructural immunocytochemistry of the intact tissue cyst of Toxoplasma in the brains of mice with congenital toxoplasmosis. Ann. Trop. Med. Parasitol. 84 (1990) 141-147 17. Smith,]. E. and E. K. Lewis: The growth and development of Toxoplasma gondii tissue cysts in vitro. In: NATO ASI series. ]. E. Smith, ed. Springer Verlag, Berlin - Heidelberg (1992) 18. Tamaroo, S., B. Fortier, M. Soerte, C. Ansel, D. Camus, and]. F. Dubremetz: Characterization of bradyzoite-specific antigens of Toxoplasma gondii. Infect. Immun. 59 (1991) 3750-3753 19. Torpier, G., H. Charif, F. Dracy,]. Liu, M. L. Darde, and A. Capron: Toxoplasma gondii: differential location of antigens secreted from encysted bradyzoites. Exp. Parasitol. 77 (1993) 13-22 20. Verhofstede, c., P. van Geldeer, and M. Rabaey: The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting. Parasitol. Res. 74 (1988) 997-1004 21. Werner, H., K. N. Masihi, and U. Senk: Latent Toxoplasma-infection as a possible risk factor for eNS-disorders. Zentralbl. Bakteriol. Orig. A 250 (1981) 368-375 22. Woodison, G. and J. E. Smith: Identification of the dominant cyst antigens of Toxoplasma gondii. Parasitology 100 (1992) 389-392 23. Zhang, Y. W. and]. E. Smith: Toxoplasma gondii: Identification and characterization of a cyst molecule. Exp. Parastiol. 80 (1995) 228-233 Dr. med. vet. Ingrid Reiter-Owona, Institut fiir Medizinische Parasitologie, SigmundFreud-StrafSe 25, D-53105 Bonn, Germany
Nonimmunological Factors Affecting the Release of Excreted/Secreted Antigens from Toxoplasma gondii Cysts * INGRID REITER-OWONA, MONIKA SAHM, and HANNS MARTIN SEITZ Institute for Medical Parasitology, University of Bonn, Germany
Received March 13, 1996 . Accepted March 27, 1996
Summary 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.
Introduction Metabolic products (exoantigens) of Toxoplasma gondii were localized by Werner et al. (21) around cryosectioned tissue cysts of chronically infected mice. A subsequent study of Hay et al. (9) failed to confirm the previous results. However, the constant stimulation of the immune response by excretion of exoantigens from the "dormant" T. gondii cysts has never been questionable since immunity for T. gondii is considered to depend on the presence of cysts in the host. The presence and immunogenicity of excreted/secreted antigens (EISA) in chronically infected hosts was shown indirectly by the demonstration of circulating antigens (8) in the serum and stage-specific antibodies (14, S, 20). More recently, a variety of cystlbradyzoite-specific monoclonal antibody has been raised (12, 18,2, 13,23). Antigens originating from granules inside the bradyzoites were located immunocytochemically by Torpier et al. (19) in the ground substance (GRA2) and on the limiting membrane (GRAS) ofthe cyst wall. The authors suggested an involvement of GRAS in the immune response resulting from T. gondii infection but could not demonstrate the antigen outside the cyst wall. Naturally processed T. gondii peptides were only isolated from tachyzoite-infected host cells (1) so far.
* Dedicated to Prof. Dr. H. Brandis on the occasion of his 80 th birthday.
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In order to analyse the observed release of E/SAs from T. gondii cysts, we examined this phenomenon under different preparative conditions, with emphasis on ultrastructural studies of the cyst wall. Materials and Methods Parasites
Two low-virulent strains (DX, TT 2975) of T.gondii were used. The DX strain was of swine origin. The TT 2975 strain was isolated from a newborn with congenital toxoplasmosis. 30 NMRI female mice (average weight 30 g) were infected intraperitoneally (i. p.) with approximately 5 cysts. Groups of mice were killed 3, 6 and 12 months after infection. The brains were immediately removed and further processed as described below. Preparation of tissue samples
Brain samples were processed either as whole tissue, as impression smears or homogenates. For standard paraffin embedding, the brain was fixed for 24 h in formaldehyde (4% in 0.1 M phosphate buffered saline, PBS, pH 7.4) at room temperature, washed in buffer, dehydrated and embedded in paraffin. For standard electron microscopy, brain tissue and homogenated brains were fixed with 2% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4 °e overnight and washed in the same buffer. Samples were then dehydrated in ethanol and embedded in Spurr resin. Embedded tissue blocks were sectioned with a diamond knife and stained with uranyl acetate and lead hydroxide. To prepare frozen sections, brain tissue was either frozen without previous fixation or after fixation in formaldehyde (2% in 0.1 M PBS, pH 7.4, 1 h). Fixed tissue samples were washed 3 times for 20 min in PBS. Before freezing, tissue samples were mounted on tissue Tek R , frozen in liquid nitrogen and stored at -60 °e in a deep freezer until sectioning. For sectioning, frozen samples were cut (7-9 fim) in a cryostat at -25°C to -30 0c. Sections from unfixed brains were thaw-mounted on glass slides and stored at -20°C for 24 h before fixation with cold acetone (10 min) or formaldehyde (2% in O.lM PBS, pH 7.4, 5, 10 or 30 min). Frozen sections from fixed tissue were thawmounted on slides precoated with a mixture of glycerol and egg white (1 Vt: 1 Vt, containing thymol) and dried for 1 hat 37°C. All slides were stored at -20°C until use for immunostaining. For preparation of impression smears, small samples of brain tissue were squeezed between two slides. Slides were then separated and each slide was dried for 30 min and fixed with cold acetone (10 min) or formaldehyde (2 % in 0.1 M phosphate buffer, pH 7.4, 5, 10 or 30 min). Other samples of brain tissue were homogenated in PBS (pH 7.4) with a pistil. The resulting homogenates were kept at 4°C and aliquots were removed immediately, after 15 min, after 3 h, and after 48 h. The material was dropped on slides, air-dried and fixed with cold acetone (10 min) or formaldehyde (2% in 0.1 M phosphate buffer, pH 7.4, 5, 10 or 30 min). Until use, all slides were stored at -20°C. Antibodies
Polyclonal immune rabbit serum (R-Pc) was prepared by oral infection with approx. 500 cysts of T. gondii strain DX, followed by strain Gail, and strain TT 2975, 2 months later. Serum was collected 6 months after the last application. The monoclonal antibody ee2 (mAb ee2) was kindly provided by Dr. U. Gross, Wiirzburg, Germany. Immunofluorescence assay (IFA)
For IFA, the slides were incubated with R-Pc for 60 min (1 :40 in PBS, pH 7.4), followed by washing and incubation with fluorescein-conjugated goat anti-rabbit IgG (H + L, Sigma,
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Heidelberg, Germany) diluted 1 :50 in PBS (pH 7.4), for 60 min at 37"C in a moist chamber. The slides were mounted in buffered glycerine with p-phenylenediamine (10). lmmunoperoxidase staining (PO)
For the PO with the indirect avidin-biotin method, the VectastainR-kit (Camon LaborService GmbH, Wicsbaden, Germany) was used and staining was performed according to the instructions given bv the manufacturer. Electron immullocytochemistry
Infected brain tissues were fixed for one hour at room temperature in 2 % formaldehyde and 0.1 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) and washed 3 times in the same buffer at 4 Qe on an ice-bath. The samples were dehydrated using serially graded ethanol solutions (at 4 Qq. From a 90% ethanol solution samples were embedded in Lowicryl K4M according to the instructions given by the manufacturer (Chemische Werke Lowi GmbH, Waldkraiburg, Germany). Polymerisation was performed under UV light (Philips lamp: TL 44 D25/09N) at room temperature overnight. The distance between samples and lamp was 40 em. For post-polymerisation, the samples were kept on the bench for 48 h. Ultrathin sectIons were cut with a diamond knife and collected on unsupported 400 mesh Ni-grids. Thin sections containing Toxoplasma tissue cysts were applied onto drops of R-Pc diluted 1 :40 in 0.1 M PBS, pH 7.4 containing 1 % bovine serum albumin (fraction V, Merck, Darmstadt, Germany). After washing with PBS (2 X 5 min), the grids were incubated with 12 nm colloidal gold-conjugated goat anti-rabbit IgG (whole molecule, Sigma Immuno Chemicals, Heidelherg, Germany), diluted 1: 40 in PBS with bovine serum albumin. The duration of each incubation was 60 min at 3rc. The mAb eC2 was used undiluted, grids were preincubated with normal goat serum (20 min, diluted I: 70 in 0.1 M PBS, pH 7.4) and incubated with 12 nm colloidal gold-conjugated goat anti-rat IgG (whole molecule, Sigma Immuno Chemicals, Heidelberg, Germany), diluted 1 :40 in PBS with bovine serum albumin. Grids were examined unstained or stained with 1 % uranyl acetate in methanol with a Zeiss EM 9 S elee'tron microscope. Results Immunolabelling on acetone-fixed brain tissue smears with R-Pc anti-T. gondii antibody resulted in a bright, intensive staining (PO, IFA) of the cyst wall, the cyst content and soluble, antigenic material outside the cyst wall overlying the brain tissue cells (Fig. 1a). Labelling of extracystic antigenic material was even more intensive on cryosections of infected brain tissue (not shown). When tissue smears from the same infected mouse brain were fixed with formaldehyde (2%, 10 min) and incubated with the same specific antibody, neither the exogenous antigenic material surrounding the cyst nor the cyst wall was stained (IFA, PO). Labelling of the intracystic antigenic material was reduced and the whole cyst had obviously shrunk (Fig. 1b). By using formaldehyde fixation in increasing concentrations (2-4%) and increasing duration on tissue smears of infected brain as well as on cryosections, it could be demonstrated that intra- and extracystic labelling by R-Pc gradually decreased and was finally abolished at a concentration of 4 % formaldehyde and 30 min of fixation. Prolonged fixation (12 h) of tissue at high formaldehyde concentration (4%) before deep freezing also led to a decrease of labelling. Following short-term fixation (30 min) of whole brain tissue samples, there was a decrease in labelling of the exoantigens of those
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Fig. 1. Immunolabelled Toxoplasma gondii cyst in an impression smear of infected mouse brain, strain DX, 175 d p.i., polyclonal Toxoplasma antibody (R-Pc) and indirect avidinbiotin-peroxidase method after a) acetone fixation and b) formaldehyde fixation (2% formaldehyde, 10 min). In the acetone-fixed tissue smears, cysts were surrounded by released antigenic material. The cyst content and the cyst wall (CW) were intensively stained. After formaldehyde fixation, the intensity of staining became reduced, and outside the unlabelled cyst wall, no immunoreaction was seen. .
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cysts which had been lying near the outer surface compared to those in the centre of the sample. The morphology of the T. gondii cysts and the brain tissue in frozen sections was - as expected - less preserved than after routine paraffin embedding (4% formaldehyde, 24 h). The treatment of paraffin sections with anti- T. gondii antibody (R-Pc) and PO staining revealed, that brain cysts from one host can show different staining patterns. A small zone of soluble antigenic material surrounding the cyst wall could only be suspected in some paraffin sections. To examine the way by which intra cystic antigen can diffuse through the lightmicroscopically intact cyst wall, immunocytochemical studies were performed. It was shown that the R-Pc used was reactive to three different components of the T. gondii brain cyst in K4M-embedded material: 1. dense granules of the bradyzoites, 2. cyst matrix material, 3. cyst walUmembrane (Fig. 2). In contrast to immunohistological results, no labelling was observed in the cytoplasm of the host cell associated to the cyst. Based on these results we presumed that the location of the cyst within the intact and later thoroughly fixed host cell would prevent the E/SAs from leaking out in detectable quantities. In a series of experiments, T. gondii cysts were mechanically isolated from the host cell by homogenizing the infected brain with a pistil. Under the light microscope, the development of a lucent area beneath the cyst wall was observed. Af-
Fig. 2. Toxoplasma gondii cyst in infected mouse brain (strain DX, 380 d p.i.) labelled with polyclonal rabbit antibody (R-Pc) followed by collodial gold-conjugated goat anti-rabbit IgG. The R-Pc showed considerable binding to the cyst wall (CW) including the vesicles. Gold particles were also localized on the electron-dense ground substance (GS) and on bradyzoites mainly within the dense grana (DG). The unlabelled host cell (HC) was only seen as a thin layer ad jeeting to the eyst wall.
Fig. 3a, b. Electron micrograph of a Toxoplasma gondii cyst in mouse brain infected with strain IT 2975 (87 d p.i.). The host cell (HC) was seen as a small cell-layer surrounding the cyst. The cyst wall (CW) showed deep and branching invaginations of the limiting membrane (ME). Bradyzoites (B) were embedded in an electron-dense ground substance (GS).
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ter prolonged incubation at room temperature (30 min) the originally tightly packed bradyzoites gradually became loosened. Magnification at ultrastructural level revealed, that the lucent area beneath the cyst wall was due to a distinct space between the cyst wall and the amorphous electrondense material surrounding the clustered bradyzoites. Electron micrographs taken after 3 h of incubation documented that the formerly undulated limiting membrane (Fig. 3b) had stretched to become nearly linear without any sign of disruption (Fig.4a). The previously electron-dense, amorphous material between the bradyzoites (Fig. 3) became electron lucent and the formerly closely associated bradyzoites were now loosely packed (Fig. 4). When examined after 48 h of incubation (4 0q, the structure of the cyst wall had changed from a thick layer with many branching invaginations of the limiting membrane to a wall with an intact straight limiting membrane supported by a layer of amorphous material. The mAb CCl was found to bind strongly to the cyst wall (Fig. 5). Gold particles were not observed between parasites, suggesting a different origin than the amorphous cyst wall and cyst matrix material labelled by the R-Pc. Immunoreaction with the R-Pc and PO/Igold-staining of the liberated cysts and examination by light and electron microscopy revealed that the intensity of intracystic labelling decreased when the incubation of free cysts was prolonged. After 48 h of incubation (4 0q, there remained only a faint reaction in the interior of the cysts. The cyst wall was not labelled. Results recorded were the same for both Toxoplasma strains examined and independent from the time that had passed after infection (examined at 3,6, 12 months). At the electronmicroscopic level, the DX strain differed from other isolates insofar as brain cysts in tissue and in homogenated material contained more submembrane and cyst matrix vesicles than did cysts from other isolates. Discussion The cystic stage of T. gondii cannot be easily maintained in brain cells (17). This is one reason why we know very little about the release of bradyzoite excreted/secreted antigens (EISA) and their transport to and through the cyst wall. Although it has been suggested that bradyzoite E/SAs playa major role in the maintenance of immunity (4), the processing and presentation of the antigens by the host cell as well as the function of E/SAs in the mechanism of reactivation of latent infection are not yet known. The major aim of our study was to analyze the release of parasite antigens into the surrounding of T. gondii tissue cysts of the brain. Our labelling experiments using a polyclonal antibody (R-Pc) have clearly demonstrated that the preservation and localization of the amorphous material that comprises the cyst matrix in vivo depends mainly on the technique of preparation and fixation of the infected tissue. When the infected mouse brains were processed for immunohistology, the minority of the cysts detected revealed a faintly stained cyst wall, whereas in the majority of them, the cyst wall was not reactive but the content of the cysts was intensively stained. Antigenic substances - so called exoantigens - of T. gondii were described by Werner et al. (21) in the vicinity of older brain cysts in acetone-fixed, frozen sections. We also found a specific and very bright labelling of exoantigens after cryopreservation and acetone-fixation not only around older cysts. However, electronmicroscopy revealed that the morphology of the unfixed frozen tissue was poorly preserved and
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Fig.4a, b. Electron micrograph of a Toxoplasma gondii cyst (strain DX), after 3 h of incubation in homogenized mouse brain (175 d p.i.). The cyst wall (eW) has stretched. The deep and branching invaginations of the limiting membrane (ME) have disappeared and bradyzoites (B) were loosely packed in a lucent ground substance. Only rest material of the host cell (HC) was found. 25
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Fig. Sa, b. Immunogold labelling of mAb CC2: gold labels intensively the wall of Toxo~ plasma gondii tissue cysts. a) A similar labelling pattern existed for the cyst wall of liberated cysts, here labelled after 3 h of incubation in homogenized brain tissue. b) CW = cyst wall, HC = host cell, B = bradyzoites. Fixation: 2 % formaldehyde, 1 h.
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therefore the concentration of soluble matrix antigen outside the cyst wall has been probably due to a permeabilization of the cyst wall rather than to an active transmembrane transport of parasitic antigens to the host cell. The same is true of tissue smears where dehydration, which is obviously faster outside than inside the intact cyst wall, causes the diffusion of soluble cyst antigens. When Hay et al. (9) failed to confirm the presence of exoantigens in mouse brain tissue, they were not aware of the influence exerted by the pretreatment of their tissue samples, namely the whole body perfusion and postfixation with 10% formaldehyde. In the present study the gradual decrease in immunogenicity of matrix-derived antigens after formaldehyde fixation is demonstrated. We could also observe that aldehyde fixation did preserve the highly invaginated structure of the limiting membrane including the underlying cyst wall material and the structure of the host cell. As a consequence, the matrix antigens were preserved within the cyst at higher concentration and labelled even after fixation with 10% formaldehyde. The observation of Bonhomme et al. (3) that intracystic staining may result primarily from matrix antigen and not from bradyzoites is supported by our immunocytochemical (R-Pc) results. The polyclonal antibody used in our experiments was derived from a rabbit orally infected with avirulent Toxoplasma strains and thus antibodies were directed only against natural antigens present in the host. The R-Pc did recognize the cyst wall after acetone-fixation of tissue smears, in a minority of cysts in paraffin sections, and when whole tissue was Lowicryl-embedded. The nature of the cyst wall is poorly understood and there are only few reports on its formation in vivo and the membrane-associated transport of molecules. The thick fibrillar structure of the cyst wall demonstrated by electron microscopy (6) was thought to prevent any transfer of material to the host cell. Immunostaining of paraffin sections with anti-Toxoplasma antibodies can produce a faint staining of the wall but obviously not of the host cell. Sims et al. (15) concluded that the immunolabelling of the cyst wall in paraffin sections may represent parasitic antigen diffusion out of the cyst. Immunocytochemical labelling studies with monoclonal antibodies suggest that the cyst wall is enriched in bradyzoite-secreted antigens (22, 13, 19). In brain tissue, our polyclonal antibody equally labelled cyst matrix, cyst wall material and dense granules suggesting the same origin as bradyzoite-secreted antigens. The cyst wall material which is underlying the limiting membrane consists primarily of amorphous, electron-dense material and is supposed to give a rigid form to the cyst structure (11). We could demonstrate that the amorphous cyst wall material remains associated to the limiting membrane even when the membrane stretches until its branching invaginations disappear and most of the amorphous cyst matrix material is released through the intact membrane. Interestingly, the recognition by mAb CC2 of the amorphous material was restricted to the cyst wall material underlying the limiting membrane of the cyst wall. This finding suggests the presence of structure proteins of different, unknown origin, which are not released when the cyst is free. In situ labelling studies with the polyclonal antibody did not reveal any difference between the cyst wall and cyst matrix-derived amorphous material as already described by Parmeley et al. (13) who used a monoclonal antibody which recognized a 65-kDa antigen. The rapid diffusion of cyst matrix material through the cyst wall which we observed when cysts had been mechanically liberated from the tissue suggest that the host cell plays a central role in the release of parasite antigens, as already mentioned by Ferguson and Hutchison (7). Apparently, only if combined, the cyst wall and the host cell are able to prevent the rapid diffusion of T gondii excreted/secreted antigens. In a se-
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ries of experiments, we could never detect Toxoplasma-antigen in the host cell at electronmicroscopic level as already described by Sims et al. (16). This might be due to the intensity of the fixation method (formaldehyde). Some other reason could be the low amount of antigen (EISA) released under normal in vivo conditions and/or a bad conservation of excreted/secreted antigens in the host cell. In summary, the most convincing event which in vivo will cause the EISAs to be released in high quantities into the vicinity of the cyst and thus be presented to the host immune response will be the death or destruction of the host cell. In contrast to the hypothesis of Jones et al. (11), we could prove that the preservation of the host-derived limiting membrane does not prevent the exposure of T. gondii antigens to the host or the culture medium. It is quite conceivable that the results of in vitro tests are influenced by the preservation of the host cell and the structure of the cyst wall. Immunological assays, the demonstration of cyst specific antigens, and the testing of compounds against T. gondii have to be considered under these aspects. Also our observations offer a new hypothesis to the process of reactivation of T. gondii infection in the immunocompromised patient. The above mentioned death of the host cell or destruction of the host cell will result in a release of a considerable amount of intracystic Toxoplasma antigen, leading to cellular infiltration followed by disintegration of the cyst and finally release of bradyzoites in vivo. Acknowledgements. We gratefully acknowledge the assistance of Dr. G. Chioralia. The study was supported by grant No. 01 KI 9456 from the German Federal Ministry for Research and Technology (Bundesministerium fiir Forschung und Technologie).
References
1. Aosai, F., T. H. Yang, M. Ueda, and A. Yano: Isolation of naturally processed peptides from a Toxoplasma gondii-infected human B lymphoma cell line that are recognized by cytotoxic T lymphocytes. J. Parasitol. 80 (1994) 260-266 2. Bohne, w., J. Heesemann, and U. Gross: Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages. Infect. Immun. 61 (1993) 1141-1145 3. Bonhomme, A., F. Boulanger, L. M. Bharadwaj, D. Puygauthier-Toubas, P. Bonhomme, M. Plout, and J. M. Pinon: Toxoplasma gondii: immunocytochemistry of four immunodominant antigens with monoclonal antibodies. Exp. Parasito!' 71 (1990) 439-451 4. Darcy, F., H. Charif, D. Deslie, R.J. Pierce, M. F. Cesbron-Delauw, A. Decoster, and A. Capron: Identification and biochemical characterization of antigens of tachyzoites and bradyzoites of Toxoplasma gondii with cross-reactive epitopes. Parasito!' Res. 76 (1990) 473-478 5. Decoster, A., F. Darcy, and A. Capron: Recognition of Toxoplasma gondii excreted and secreted antigens by human sera from acquired and congenital toxoplasmosis: identification of markers of acute and chronic infection. Clin. Exp. Immuno!. 73 (1988) 376-382 6. Ferguson, D.]. and W. M. Hutchison: An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasito!' Res. 73 (1987) 483-491 7. Ferguson, D.]. and W. M. Hutchison: The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch. Patho!' Anat. 411 W. M. (1987) 39-43 8. Hassi, A. and H. Aspock: Detection and characterization of circulating antigens in acute experimental infections of mice with four different strains of Toxoplasma gondii. Zentralbl. Bakteriol. 272 (1990) 526-534
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9. Hay,]., D. I. Graham, G. N. Dutton, and S. Logan: The immunocytochemical demonstration of Toxoplasma gondii in the brains of congenitally infected mice. Z. Parasitenkd. 72 (1986) 609-615 10.Johnson, G. D. and A. Nogueira: A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Meth. 43 (1981) 349-350 11. Jones, T. c., K. A. Bienz, and P. Erb: In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gamma interferon. Infect. Immun. 51 (1986) 147-156 12. Omata, Y., M. Igarshi, M. I. Ramos, and T. Nakabayashi: Toxoplasma gondii: Antigenic differences between endozoites and cystozoites defined by monoclonal antibodies. Parasitol. Res. 75 (1989) 189-193 13. Parmeley, St. F., S. H. Yang, G. Harth, L. D. Sibley, A. Sucharczuk, and J. S. Remington: Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in matrix of tissue cysts. Mol. Biochem. Parasitol. 66 (1994) 283-296 14. Partanen, P., J. Turunen, R. A. Passivuo, and P. o. Leiniki: Immunoblot analysis of Toxoplasma gondii antigens by human immunoglobulins G, M and A antibodies at different stages of infection. J. Clin. Microbiol. 20 (1984) 133-135 15. Sims, T. A.,]. Hay, and I. C. Talbot: Host-parasite relationship in the brains of mice with congenital toxoplasmosis. J. Pathol. 156 (1988) 255-261 16. Sims, T. A.,]. Hay, and I. C. Talbot: Ultrastructural immunocytochemistry of the intact tissue cyst of Toxoplasma in the brains of mice with congenital toxoplasmosis. Ann. Trop. Med. Parasitol. 84 (1990) 141-147 17. Smith,]. E. and E. K. Lewis: The growth and development of Toxoplasma gondii tissue cysts in vitro. In: NATO ASI series. ]. E. Smith, ed. Springer Verlag, Berlin - Heidelberg (1992) 18. Tamaroo, S., B. Fortier, M. Soerte, C. Ansel, D. Camus, and]. F. Dubremetz: Characterization of bradyzoite-specific antigens of Toxoplasma gondii. Infect. Immun. 59 (1991) 3750-3753 19. Torpier, G., H. Charif, F. Dracy,]. Liu, M. L. Darde, and A. Capron: Toxoplasma gondii: differential location of antigens secreted from encysted bradyzoites. Exp. Parasitol. 77 (1993) 13-22 20. Verhofstede, c., P. van Geldeer, and M. Rabaey: The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting. Parasitol. Res. 74 (1988) 997-1004 21. Werner, H., K. N. Masihi, and U. Senk: Latent Toxoplasma-infection as a possible risk factor for eNS-disorders. Zentralbl. Bakteriol. Orig. A 250 (1981) 368-375 22. Woodison, G. and J. E. Smith: Identification of the dominant cyst antigens of Toxoplasma gondii. Parasitology 100 (1992) 389-392 23. Zhang, Y. W. and]. E. Smith: Toxoplasma gondii: Identification and characterization of a cyst molecule. Exp. Parastiol. 80 (1995) 228-233 Dr. med. vet. Ingrid Reiter-Owona, Institut fiir Medizinische Parasitologie, SigmundFreud-StrafSe 25, D-53105 Bonn, Germany