A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

J. Euk. Microbiol., 42(2), 1995, pp. 150-157 8 1995 by the Society of F’rotozoologistS

A Cell Culture System for Study of the Development of Toxoplasma gondii Brad yzoites

LOUIS M. WEISS,*.**.’ DENISE LAPLACE,** PETER M. TAKVORIAN,*** HERBERT B. TANOWITZ,*.** ANN CALI*** and MURRAY WITTNER*.**

*Department of Medicine. Division of Infectious Diseases, **Department of Pathology, Division of Parasitology, Albert Einstein College of Medicine. Bronx, New York 10461, and

***Department of Biological Sciences, Rutgers University, Newark, New Jersey 971 92

ABSTRACT. Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting With a 28 kDa antigen, was used to study bradyzoite development in vitro by imrnuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free- floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of y-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

Supplementary key words. Bradyzoite, cyst, Toxoplasma gondii, in vitro development.

XOPLASMA gondii, a ubiquitous apicomplexan parasite P of mammals and birds, is responsible for several distinct human diseases. After acute infection, the organism often remains latent in tissue in the form of the bradyzoites or cysts. In congenital T. gondii infection, relapsing chorioretinitis de- velops when cysts rupture and bradyzoites are transformed into tachyzoites. A host immune response to the extracellular parasite occurs subsequently, resulting in pathologic changes. In a patient with the human immunodeficiency virus (AIDS), T. gondii is a common cause of encephalitis. This is most likely due to the reactivation of latent encysted organisms (bradyzoites) into active replicating organisms (tachyzoites) and the failure of the immune system to adequately respond to the free parasites [7].

The mechanism of bradyzoite-tachyzoite interconversion is poorly understood. Studies have demonstrated that bradyzoites can develop in vitro and that development ofcyst-like structures can be demonstrated by transmission electron microscopy [ 10, 12-14, 18, 191. Morphologically it is difficult to distinguish bradyzoites from tachyzoites by light microscopy. Biologically such tissue culture derived cysts appear to be true bradyzoites as demonstrated by cat feeding experiments demonstrating a short prepatent period [9, 131. It is of interest that bradyzoites develop in cell cultures in the absence ofan exogenous immune response.

Western blot studies have demonstrated that tachyzoites and bradyzoites are antigenically distinct [5, 11, 15, 20-221. Mono- clonal antibodies specific to bradyzoites [3, 15, 20, 221 and tachyzoites [5] have been described. The development in vitro of bradyzoites and the conversion of bradyzoites isolated from mouse brains into tachyzoites has been demonstrated recently employing bradyzoite specific monoclonal antibodies [3, 191. In the present study, utilizing our previously developed bradyzoite- specific monoclonal antibodies, we describe a continuous cell culture system that allows for the in vitro study of bradyzoite development of T. gondii.

’ To whom correspondence should be addressed.

MATERIALS AND METHODS Parasite. Bradyzoites obtained from tissue cysts in murine

brain of the ME49 strain of T. gondii were used to inoculate cultures. Tissue cysts were harvested under sterile conditions from DMI mice at I to 2 months post infection homogenized in normal saline and purified by isopycnic centrifugation in 45% Percoll as previously described [4, 221. Purified cysts were sus- pended in sterile PBS and ruptured using a one minute trypsin (0.25% final concentration) incubation. The released bradyzoites were pelleted for 2 minutes at 12,000 g and resuspended in sterile PBS. 1,000 bradyzoites were added to a 50% confluent flask of human fibroblasts. Toxoplasma zoites were passaged weekly by inoculating an uninfected 50% confluent human fibroblast monolayer with 1,000-5,000 tissue culture derived T. gondii. Fresh medium was added weekly to flasks (25 cmz) observed longer than one week.

Cell line. A human fibroblast cell line, ATCC CRL 1475 (CCD-27SK), was used for culture. These were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 10 mM Hepes (pH 6.8, 7.2 or 8.2) and 1% Penicillin-Streptomycin (Gibco, Gaithersburg, MD) and incubated at 37O C and 7% CO,. Media were adjusted to pH 6.8, 7.2 or 8.2 and filter sterilized before being used. Cells were subcultured weekly using 0.25% trypsin- 0.03% EDTA at a ratio of 1 :4. Cells were used between passage 6 and 30.

Cytokines. Interleukin 6 (IL-6) human recombinant (Gibco) was used at concentrations of 0.5, 1.0, 2.5, 5.0 and 10 ng/ml. Gamma interferon ( m y ) human recombinant (Gibco) was used at concentrations of 0.1,0.5, I , 5 and 10 ng/ml. Cytokines were added to cell cultures on day 3-5 by removing medium and replacing it with medium containing cytokines.

Antibodies. Mab 74.1.8, IgGZb, bradyzoite specific and reactive to a 28 kDa antigen (BAG-5) was used, at a 1:25 to 150 dilution for immunofluorescence. Mab DG52, IgG2a, tachyzoite specific and reactive to SAG-1 (p30) was used at a 1:500 to 1: 1,000 dilution for immunofluorescence [ 5 , 201. Mab 92- I OB, IgG 1, reactive to GRA- I found in both stages was used at a 1:25 to 1:50 dilution for immunofluorescence [23]. Secondary

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WEISS ET AL.-BRADYZOITE DEVELOPMENT IN VITRO 151

antibodies used at 1 :60 for immunofluorescence were fluorescein-antimouse IgG 1, fluorescein-antimouse IgGZa, rhodamine-antimouse IgG2b and rhodamine-antimouse immuno- globulins (Fc) (Southern Biotechnology Associates Inc., Bir- mingham, AL). For western blotting, HRP-antimouse immu- noglobulins (Fc) (Organon-Teknika-Cappel, West Chester, PA) were used at 1:250 dilution.

Western blotting. Samples of cell culture supernatant, containing free cysts, or T. gondii infected human fibroblast monolayers were split into equal samples which were then assayed for protein concentration (BioRad, Hercules, CA) and dissolved in 2 x gel sample buffer. Equal amounts of protein were loaded onto SDS-PAGE gels, electrophoresed and transferred to nitro- cellulose as previously described [24]. The amount of bradyzoite specific antigen was ascertained by western blotting as previously described using Mab 74.1.8 [22].

Immunofluorescence (IF). Tissue flasks were fixed for 30 minutes with 2% buffered formalin, washed 3 x with PBS per- meabilized with 0.2% Triton X- 100 for 20 minutes, washed 3 x with PBS, blocked with 1% bovine serum albumin in PBS overnight at 4” C and washed 3 x in PBS. Flasks were then incubated for 90 minutes at 37” C with the appropriate Mab, washed 3 x with PBS, incubated with the secondary antibody for 90 minutes at 37” C, washed three times with PBS and overlayed with PBS with 2.5% DABCO (Sigma, St. Louis, MO). Flasks were then examined with a Nikon Diaphot inverted U-V microscope (Ni- kon, Melville, NY) using rhodamine and fluorescein filters. Pho- tomicrographs were taken with Ektachrome T-320. For double labeling, flasks were then incubated with the second Mab as above followed by the monospecific secondary antibody and then examined with the Nikon Diaphot system. IF-positive organisms were counted in twenty-five 20 x microscope fields and total IF-positive counts in a flask were determined by multi- plying this count by the area of the flasklarea counted.

Electron microscopy. Cells containing organisms were cen- trifuged in 1.5 ml microfuge tubes to form a pellet. Cold (4” C) 2.5% (v/v) glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.2) was then added to each microfuge tube for 24 h. The fixed tissues were rinsed in buffer, post-fixed in buffered I % (w/v) OsO,, dehydrated in graded ethanols, placed in pro- pylene oxide, and embedded in Araldite 502. Thin sections (silver-gray) were placed on copper grids, stained with uranyl acetate and lead citrate, then examined with a Philips CM 10 transmission electron microscope operated at 80 kV. Chemicals for EM were from Polysciences Incorporated (Warington, PA).

Immunogold electron microscopy. Cells containing organisms and cell-free parasites were pelleted as described above. The pellets were then fixed in cold (4” C) fixative containing 0.5% glutaraldehyde (v/v), 2% (v/v) freshly made paraformaldehyde in a 0.1 M cacodylate buffer (pH 7.2) for 1 h. The pellets were rinsed several times in buffer and slowly dehydrated through graded ethanols to 70% alcohol. The partially dehydrated pellets were then transferred to several changes of LR White embedding resin, infiltrated and then embedded in gelatin capsules. The resin filled gelatin capsules were polymerized for 24 h with ultraviolet light at room temperature. After polymerization, the tissue blocks were sectioned, placed on nickel grids and dried. The grids containing sections were then incubated overnight at 4” C in “blocking” buffer containing 0.1 M phosphate-buffered saline (PBS), 5% v/v normal goat serum (Jackson Immuno- Research Laboratories, West Grove, PA), and 1% w/v bovine serum albumin (BSA) to block any nonspecific antigenic activ- ity. The grids were then rinsed several times in “rinsing” buffer containing 0.1 M PBS, and 1% w/v BSA. After rinsing, the grids were incubated for 2 h at room temperature, in the primary monoclonal antibodies 73.18, 74.1.8 or 92.10B respectively.

The Mab were diluted 1 5 0 with “rinsing” buffer. After incubation with the Mab, the grids were once again rinsed several times with “rinsing” buffer. Subsequently, the grids were then incubated for 2 h with goat anti-mouse IgG (H + L) secondary antibody conjugated to 12 nm colloidal gold particles (Jackson Immuno Research Laboratories, West Grove, PA) diluted 1 :20 with “rinsing” buffer. Control sections were treated exactly as above with the omission ofthe primary Mab from the incubating solution. After incubation with the secondary antibody (colloidal gold) and several rinses, the grids were stained with aque- ous saturated uranyl acetate and then examined with a Philips CM 10 transmission electron microscope operated at 80 kV. After initial examination and identification of the gold particle distribution, some of the grids were stained with lead citrate for increased contrast. All aspects of standard and immunogold electron microscopy were conducted at the Rutgers University Electron Microscopy facility.

RESULTS Three days after infection of human fibroblasts with ME49

T. gondii tissue cyst development was clearly evident in vitro. Development was similar when either freshly isolated bradyzoites or T. gondii from the first 20 cell passages after isolation from mice (DMI) were used to inoculate human fibroblasts. Tissue cyst-like structures containing rounded loosely packed parasites were evident in the early bradyzoite parasitophorous vacuole (Fig. IA, B). Subsequently, by day 4, we noted the development of thick-walled phase lucent structures many of which were free floating (Fig. IC, D). Often, the cyst-like vacuole contained an odd number of parasites [ 181 rather than the usual multiples of 2 seen in the tachyzoite rosettes which were also present in the cell cultures (Fig. IA, B). This odd number of organisms may be a consequence of asynchronous and/or slow replication of bradyzoites in these vacuoles. A typical T25 culture flask contained from 2,000 to 8,000 tissue cysts in the host cells at day 4.

Immunofluorescence unambiguously demonstrated that the organisms present in the tissue cyst-like structures, observed by light microscopy, expressed BAG-5 but not SAG- 1, confirming that bradyzoite-specific but not tachyzoite-specific antigen was being expressed by these organisms (Fig. 1 E, F, G). IF staining was similar to that seen in bradyzoites isolated from mouse brain (Fig. IH). By western blot, BAG-5 expression was also demonstrable (Fig. 2) and increased from day 3 to day 6. This was consistent with the IF observations. Immediately after infection of human fibroblasts with freshly isolated bradyzoites from a mouse, transitional forms expressing both BAG-5 and SAG- 1 were observed by IF [ 191.

Within the same fibroblast, separate parasitophorous vacuoles could be seen that expressed either bradyzoite or tachyzoite antigens (Fig. 11, J, K). Evidently, the host cell was not the only determining factor in the development of a bradyzoite versus a tachyzoite vacuole. In this regard, on occasion, both bradyzoite and tachyzoite forms were seen within a single parasitophorous vacuole so that a mixed population was present (Fig. 1 L). This may have represented a transition of bradyzoite to tachyzoite or tachyzoite to bradyzoite. In such vacuoles the tachyzoites predominated, presumably due to their greater replication rate. On occasion, extracellular organisms that were BAG-5 positive (free bradyzoites) could be identified often surrounded by numerous SAG-1 positive organisms. We believe it is likely that bradyzoite differentiation is a relatively uncommon event that occurs as part of normal division (i.e. 1 in a 1,000 cells). As a consequence of the bradyzoite’s slower rate of replication tachyzoites predominate in tissue culture. This is supported by data demonstrating that when tachyzoites are removed from

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cultures by frequent media changes bradyzoites can be seen to increase in these cultures over time [ 141.

In up to passage 16, tachyzoite replication was usually slow enough to permit clear visualization of tissue cyst-like structures in the monolayer; however, on subsequent passages tachyzoite replication often destroyed the monolayer by day 6. Neverthe- less, even in such ruptured monolayers, tissue cyst-like structures were observed floating in the medium and on western blotting expression of the bradyzoite specific antigen recognized by Mab 74.1.8 (BAG-5) was evident. The addition of INFy to the culture (0.1 ng/ml) on day 3 post-inoculation appeared to decrease the overgrowth of the cultures by tachyzoites and al- lowed visualization of tissue cyst formation through 28 days of observation in early passage cultures (< 10 passages). In long term cultures and on passaging of cultures there were often bursts of tachyzoite replication alternating with periods of qui- escence when tissue cyst development was easily seen. In addition, such INFy treated cultures could usually be maintained for up to 35 passages without the development of rapid tachyzoite overgrowth.

We had previously observed that stress conditions on the host cell monolayer, such as elevated C02 , appeared to increase tissue cyst formation in vitro therefore we investigated the effect of both pH alterations and IL-6 (as an inducer of an acute phase response [ l , 81) in our system. Alteration of the medium to either pH 6.8 or 8.2 on day 1 of infection increased the number of tissue cysts and decreased the rupture of the cells by tachyzoites.

Increasing the concentration of INFy from 0.1 to 10 ng/ml in the presence or absence of IL-6 (0.5 ng/ml) did not affect the number of tissue cysts when added at 3 days post-infection in pH 6.8, 7.2 or 8.2 media. This was demonstrated by western blotting and IF for BAG-5. The addition of high concentrations of INFy before infection resulted in a decrease in the number of infected fibroblasts and thus the number of tissue cysts and tachyzoites seen. There was an increase in BAG-5 expression on western blotting in tissue culture at pH 7.2 with an increase in IL-6 concentration; this was not evident at pH 6.8 or 8.2. In the presence of 0.1 ng/ml of INFy in pH 8.2 or 6.8 media, increased IL-6 concentration from 0.5 to 10 ng/ml decreased the number of free tissue cysts in the medium by half (from 600 to 300 per flask).

Transmission electron microscopy (TEM) demonstrated that the free-floating cyst-like structures and the BAG-5 positive T. gondii vacuoles (Fig. 3A, B and C) in the monolayers displayed the characteristics of tissue cysts (bradyzoites): a thick cyst wall,

Fig. 2. Western blot demonstrating BAG-5 expression in vitro. Lane 1, ME49 tachyzoites from long term (> 60 passages) in vitro culture in L,E, cells 123). Lane 2, ME49 bradyzoites from chronically infected DMl mice [22]. Lane 3, uninfected human fibroblasts. Lane 4, bradyzoites developing in human fibroblasts, day 4 of culture as described in materials and methods. Lane 5 , supernatant from infected fibroblasts containing tissue cyst-like structures. As seen in this western blot using Mab 74.1.8 (BAG-5) specific to bradyzoites, expression of this antigen is present in both the infected supernatant and the human fibroblast monolayer. The antigen is the same as that seen in bradyzoites from mouse brain and is not present in uninfected fibroblasts or tachyzoites.

electron lucent vacuoles, solid appearing rhoptries, numerous micronemes and a posterior situated nucleus [6]. Similar to bradyzoites isolated from brain but not tachyzoites, Mab 74.1.8 stained the electron lucent structures seen only in bradyzoites (Fig. 3D, E). Mab 92.10b reacted with GRA- 1 and stained the dense granules in both tachyzoites and bradyzoites (Fig. 3F, G).

DISCUSSION We have demonstrated by IF, TEM and western blotting the

in vitro development of bradyzoites together with tachyzoites in continuous cell culture of human fibroblasts. We have also seen the development of bradyzoites in L,E, rat myoblasts and cat intestinal cells (fibroblast like) (unpubl. data) but the im- proved morphology and the ability to examine the effects of the available cytokines makes the human fibroblast a better cell model for studying the interactions between host and parasite. Unlike the models described by Smith [ 181 and Soete [ 191 these cultures are many passages removed from the initial isolation of bradyzoites purified directly from mouse brain.

t

Fig. 1. Development of bradyzoites in human fibroblasts. A. Phase contrast micrograph (60 x) of early bradyzoite development (B), confirmed by reaction with 74.1.8, in two separate vacuoles in a human fibroblast cell on day 3 of culture. Early bradyzoite vacuoles often display an odd number of rounded organisms located at the edge of the vacuole (demonstrated in the vacuole on the right) and can display a cruciform orientation (as demonstrated on the left). B. Phase contrast micrograph (20 X ) of typical tachyzoite (T) rosettes on the left and a cyst-like structure (B) on the right. C. Phase contrast micrograph of a free floating cyst ( 6 0 ~ ) in cell culture. A phase lucent cyst wall (CW) is evident in this picture. The cysts that form in culture are often flattened as a consequence ofdevelopment in the tissue flask adherent monolayers. D. Phase contrast micrograph (40 x ) of a mature cyst, confirmed by reaction with 74.1.8, in human fibroblasts. Such cysts often separate from the monolayer to become free floating structures as demonstrated in C. E. Immunofluorescence micrograph (60 x ) demonstrating BAG-5 expression in bradyzoites developing in vitro. F. Low power ( 2 0 ~ ) IF of bradyzoite development in vitro. At least nine BAG-5 positive cysts can be seen. G. Immunofluorescence micrograph (40 x ) demonstrating BAG-5 expression in vitro. H. lmmunofluorescence micrograph of bradyzoites isolated from mouse brain with 74.1.8 (BAG-5). Cytoplasmic staining around the nucleus is present. This is similar to that seen with the in vitro bradyzoites, see 1E. I. Immunofluorescence micrograph using the bradyzoite specific Mab 74.1.8 (BAG-5). Bradyzoite staining is indicated by B and tachyzoites by T. The intensity of staining is low in this early vacuole but is clearly more than that of the tachyzoites. J. Immunofluorescence micrograph using Mab DG52 (SAG-1). As can be seen with this tachyzoite specific antibody the tachyzoites (T) are stained and the bradyzoites (B) are not stained. K. Immunofluorescence micrograph using Mab 74.1.8 demonstrating bradyzoite and tachyzoite vacuoles within a single cell. L. Immunofluo- rescence micrograph using Mab 74. I .8 demonstrating bradyzoites and tachyzoites within the same vacuole. When stained with Mab DG52 (SAG- 1) the dark areas of this vacuole were brightly fluorescent confirming that these cells were tachyzoites.

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Similar to that described by Soete et al. [ 191, parasites that exhibited staining of both SAG-1 and BRA-5 were observed when freshly isolated bradyzoites from mouse brain transformed to tachyzoites during the initial passage; but in subsequent passages these intermediate organisms were rarely seen. In addition, in our culture system we observed organisms in separate parasitophorous vacuoles in the same host cell expressing either bradyzoite or tachyzoite specific antigens. This suggests that the “state” or condition of the host cell may not be the only factor in determining if the organism that invades the cell will form a bradyzoite or tachyzoite vacuole. In addition, rare extracellular organisms were seen expressing bradyzoite antigen.

Smith [ 181 distinguished bradyzoite from tachyzoite vacuoles by light microscopy, noting that bradyzoite vacuoles had rounded (or pip-shaped) organisms [ 181 that were loosely packed and that these vacuoles often contained an odd number of organisms (due to asynchronous replication). In addition, as development progressed a tissue cyst wall could be seen on phase or confocal microscopy. This was confirmed by our observations on day 3 of infection and suggested that the relationship between bradyzoites and the host cell differed from that of tachyzoites early in the development of the parasitophorous vacuole. This suggests that differentiation to bradyzoites and development of a unique parasitophorous vacuole is probably determined either at the time of invasion or perhaps organisms are already com- mitted to differentiation to either a tachyzoite or bradyzoite before invasion. The formation of a bradyzoite vacuole does not however preclude subsequent differentiation to bradyzoites (or the converse) as rare mature vacuoles were seen containing both stages of T. gondii.

Previous studies have demonstrated that bradyzoites lack SAG-1 and 2 and have unique antigens, BAG 1-5 [5, 1 1 , 15, 20-221. In addition, tachyzoites and bradyzoites share common antigenic components, i.e. SAG-3, SAG-4 and GRA 1-5 [21]. This study demonstrated the presence of GRA- 1 in cysts isolated from mice and in tachyzoites and bradyzoites in culture. The relative contribution, if any, of these common and unique antigens to the development of the different parasitophorous vac-

Fig. 3. Electron micrographs of ME49 bradyzoite development in vitro. A. Free floating in vitro tissue cyst of ME49. A cyst wall (Cw) can be clearly seen as well as lucent vacuoles in the bradyzoites. Mag- nification 15 K. Bar = 1 pm. B. Early ME49 tissue cyst in vitro demonstrating an electron dense ground substance (asterisk) between the bradyzoites (B). Magnification 18 K. Bar = 1 pm. C. ME49 in vitro tissue cyst in human fibroblast monolayer. The cyst wall (CW) dem- onstrates the ruffled appearance of the limiting membrane of the cyst (parasitophorous vacuole) and the precipitation of underlying material. The bradyzoites are in an electron lucent ground substance (asterisk) and are loosely packed. A posterior placed nucleus (Nu) and electron lucent vacuoles (V) are evident. Magnification 22 K. Bar = 1 pm. D. Immunoelectron micrograph using immunogold (arrows) of cultured bradyzoites reacting with Mab 74. I .8. This Mab reacts with the electron lucent vacuoles of the bradyzoite. Similar reaction was seen in bradyzoites isolated from mouse brain. Magnification 33.5 K. Bar = 0.5 pm. E. Immunoelectron micrograph using immunogold (arrows) of bradyzoites developing in vitro with Mab 74. I .8. Magnification 46.5 K. Bar = 0.5 pm. F. Immunoelectron micrograph using immunogold (arrows) of bradyzoites in vitro with Mab 92.10B (GRA- 1 reactive). Dense granules (arrow) are labeled. Magnification 15 K. Bar = I pm. G. lmmu- noelectron micrograph using immunogold (arrows) of bradyzoite isolated from mouse brain with Mab 92.10B (GRA-I reactive). Dense granules are labeled. Magnification 26.5 K. Bar = 1 pm.

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uoles seen in bradyzoites (tissue cysts) and tachyzoites remains to be determined.

Some vacuoles contained both tachyzoites and bradyzoites. In these vacuoles tachyzoites were the predominant form; bradyzoites were often situated at one edge of the vacuole. Since it is unlikely that these vacuoles were subsequently invaded by a second organism or that two organisms invaded simulta- neously interconversion between stages must have occurred il- lustrating that interconversion is a dynamic process. Thus the formation of a cyst-like parasitophoms vacuole does not preclude future differentiation in that vacuole to a tachyzoite. It is possible that such transitions may occur in tissue cysts in vivo and may be the prelude t o rupture and reactivation of latent infection [7]. In this regard, it is interesting to note that tachyzoite-like organisms have been described in association with degenerating bradyzoites in mature tissue cysts on TEM from ME49 infected mice [16].

We also examined the influence of INFy and IL6 on the development of bradyzoites in vitro. While it is clear that development occurred in the absence o f exogenous immune modulators it was thought likely that immune modulators may be capable of shifting the dynamics of differentiation. Thus, INFy was effective a t decreasing tachyzoite replication, especially in tryptophan free medium [ 171, and was useful in allowing pro- longed observation of cultures by preventing tachyzoite overgrowth. INFy had no effect on bradyzoite development. The addition of IL-6 appeared to increase the number of T. gondii tachyzoites in the culture and a t low concentrations (0.5-1 .O ngl ml) t o increase the number of free tissue cysts present. This was most evident in p H 7.2 medium. As IL-6 has been reported to induce an acute phase response in many cell types we had reason to believe it might increase bradyzoite development in vitro as we had previously observed that culture conditions unfavorable to host cells led to bradyzoite development in vitro (unpubl. observ.). To this end, bradyzoite differentiation appeared to be more frequent and consistent when using pH 8.2 or 6.8 medium. Both of these conditions also are believed t o induce stress re- sponses in host cell monolayers. Recently Bohne [2] has described that in T. gondii infected murine macrophages a n increase in bradyzoite antigen expression was associated with a decreased replication of tachyzoites induced by nitric oxide, pyrimethamine or aphidicolin. Further studies on the relationship between host cell stress and bradyzoite differentiation is in progress.

We have described an in vitro cultivation system which allows for the study of bradyzoite differentiation in vitro. This system should prove useful for the further examination of conditions and substances that affect the interconversion of bradyzoites and tachyzoites.

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Received 6- 14-94; accepted 9-30- 94.

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A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

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