**3.3** *T. Gondii* **bradyzoite-FIEC interaction** *in vitro*

Previously, we described the behavior of bradyzoites during their interaction with FIECs [8]. Here, quantitative and qualitative analyses were performed with ratios

*Development of Schizont Stages of* Toxoplasma gondii *in Primary Cell Culture of Feline… DOI: http://dx.doi.org/10.5772/intechopen.105957*

#### **Figure 2.**

*Characterization of FIEC by fluorescence microscopy. (A) Culture of FIECs presenting epithelial morphology, revealed by double labeling: Actin filaments in green and cytokeratin in red. (b–d) Cytokeratin expression is located around the nucleus. (e–g) Actin filament organization revealed by phalloidin in FIECs shows the cell morphology and the major concentration at the focal adhesion points. (B–D) Intestinal alkaline phosphatase immunoreactivity in FIECs (red), shows activity that progressively increased in the cells as a function of the culture time. (B) 5 days, (C) 7 days, and (D) 9 days post-seeding. Actin filaments are shown in green. Bars: 20 μm.*

of 1:5, 1:10, and 1:20 (parasite: host cell). The number of infected cells was analyzed after 24 to 96 h of parasite and host cell interaction (**Figure 3**). The data indicated the influence of the parasite load on the number of infected enterocytes during the study period: 9% after 24 hours of interaction and 42.4% after 96 hours when the 1:5 ratio was used (**Table 1**). Ratios of 1:10 and 1:20 (parasite: host cell) resulted in a lower number of infected enterocytes when compared to the 1:5 ratio. The main difference between the 1:10 and 1:20 ratios was the occurrence of structures similar to cysts (cyst-like) or schizonts (schizont-like), as previously observed by our group [8]. Cyst-like structures were more common when the ratio of 1:10 was employed, while the ratio of 1:20 resulted in more schizonts-like structures (**Table 1**).

The analysis of the parasite-host cell interaction with the 1:5 ratio revealed that the parasites doubled during the first 24 h of infection, with rosette form indicating the occurrence of endodyogeny, as seen by Giemsa (**Figure 4A**) and immunofluorescence, revealing tachyzoites with anti-SAG antibodies (**Figure 4B**). The bradyzoitetachyzoite conversion occurred as shown by staining with the anti-SAG1-TRITC antibody (**Figure 4B**). After 96 hours of interaction, parasites were found in the extracellular environment, characterizing the lytic cycle of *T. gondii*, as seen by Giemsa (**Figure 4C**) and immunofluorescence (**Figure 4D**).

The establishment of *T. gondii* cystogenesis with the ratio of 1:10 was confirmed (**Figure 5**). Cyst-like intracellular structures in enterocytes were visible after 48 h

#### **Figure 3.**

*Absolute number of FIECs infected with T. gondii ME-49 strain bradyzoites. A total number of 400 cells were counted for each coverslip. The parasite-host cell ratios (MOI) were 1:5, 1:10 and 1:20. \*\*\*\* p < 0.0001 and \* p < 0.05 compared to 24 h at MOI 1:5. && p < 0.01 compared to 48 h at MOI 1:5. \$ p < 0.05 compared to 24 h at MOI 1:10. ## p < 0.01 compared to MOI 1:5 at 96 h.*


#### **Table 1.**

*Quantitative analyses of cyst-like and schizont-like forms during FIEC infection with ME49 T. gondii bradyzoites. Time comparison of IC for MOI 1:5: 96 h x 24 h \*\*\*\*, 96 h x 48 h \*\*, 72 h x 24 h \*. Time comparison of IC for MOI 1:10: 72 h x 24 h \*. MOI comparison of IC for 96 h: 1:20 x 1:5 and 1:10 x 1:5 \*\*. Time comparison of SL for MOI 1:20: 72 h x 24 h \*\*\*\*; 96 h x 24 h and 72 h x 48 h \*\*\*; 96 h x 72 h and 48 h x 24 h \*\*. Time comparison of CL for MOI 1:10: 96 h x 24 h \*\*; 96 h x 48 h \*. MOI comparison of SL for 48 h: 1:20 x 1:10 and 1:20 x 1:5 \*. MOI comparison of SL for 72 h: 1:20 x 1:10 and 1:20 x 1:5 \*\*\*\*. MOI comparison of SL for 96 h: 1:20 x 1:10 and 1:20 x 1:5 \*\*\*. MOI comparison of CL for 96 h: 1:20 x 1:10 \*; 1:10 x 1:5 \*\*. \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001, \*\*\*\* p < 0.0001.* *Development of Schizont Stages of* Toxoplasma gondii *in Primary Cell Culture of Feline… DOI: http://dx.doi.org/10.5772/intechopen.105957*

#### **Figure 4.**

*Light microscopy of feline enterocytes infected with bradyzoites of T. gondii (1:5 parasite:host cell ratio). (A) Parasitophorous vacuoles show parasites in classic rosettes, indicating the interconversion from bradyzoites to tachyzoites. (B) Immunostaining for SAG1 (in red) reveals tachyzoites in FIECs and actin filaments (in green) with phalloidin. (D, E) The establishment of the lytic cycle was observed at 96 h provoked by tachyzoite proliferation based on Giemsa staining and immunolabeling with anti-SAG1 antibody. Bars: 20 μm.*

(**Table 1**) by differential interference contrast microscopy (**Figure 5A**), staining with Giemsa (**Figure 5B**), and confirmed by ultrastructural analysis showing cyst wall, bradyzoites containing various granules of amylopectin and electron-dense rhoptries (**Figure 5C**).

As described during the quantitative analyses, schizont-like forms of *T. gondii* were observed in FIECs with the 1:20 ratio (**Table 1**). Starting at 48 h, cultures stained with Giemsa presented large parasitophorous vacuoles containing multinucleated masses (**Figure 6**). These structures had wide variability of shape and size, resembling the schizonts found in the gut of felines during the process of schizogony. It was common to observe more than one of these multinucleated masses (**Figure 6A, E**) and cell division processes by endodyogeny and schizogony in the same cell (**Figure 6D, E**). These structures are characterized as C-type schizonts, as advocated by Speer & Dubey [25], in a process of multiple nuclear division, with migration from the nuclei to the periphery, as can be seen in **Figure 6A**–**D**. This process of schizogony gives rise to merozoites,

#### **Figure 5.**

*Cysts of T. gondii in FIECs infected with the 1:10 bradyzoites:host cell ratio. (A-B) Differential interference contrast (DIC) microscopy (A) and Giemsa staining (B) show cyst-like structures (arrowheads) after 72 hpi (C) Cyst of T. gondii revealed by TEM with the cyst wall (arrowhead), parasites containing amylopectin granules (Am), and electron-dense rhoptries (R). ER = endoplasmic reticulum; Mi = mitochondria. Bar: (a, B) 20 μm; (C) 0.5 μm.*

which are organized immediately below the schizont's membrane, as in a "budding," as suggested by **Figure 6F**. These will be better presented during the ultrastructural analyses. With the advancement of schizogony, the merozoites acquired a peripheral arrangement (**Figure 7A**–**D**) with the presence (**Figure 7B**–**D**) or not of a residual body (**Figure 7A**). The occurrence of different pathways of intracellular fate of the parasite was observed in a single cell: lytic cycle with formation of rosettes (**Figure 7B, D**) simultaneously to vacuoles containing C-type schizonts (**Figure 7A**–**D**).

Another type of schizont detected in enterocytes *in vitro,* according to the description of *in vivo* models, was a group of three or four parasites in a parallel arrangement, with or without residual body, identified as D-type schizonts. These structures were seen in PV isolated in the cytoplasm of cells, along with other PV-containing C-type schizonts of *T. gondii* (**Figure 8**).

Ultrastructural analysis of these infected cultures for periods ranging from 48 h to 9 days showed PV containing parasites with morphological characteristics similar to those of *T. gondii* enteroepithelial stages. Evidence of schizonts containing merozoites supports the suggestion that partial reproduction of the enteric cycle of *T. gondii* was

*Development of Schizont Stages of* Toxoplasma gondii *in Primary Cell Culture of Feline… DOI: http://dx.doi.org/10.5772/intechopen.105957*

#### **Figure 6.**

*Enterocytes infected with T. gondii bradyzoites of the ME-49 strain for 48 h. It is possible to observe several multinucleated masses of varied shapes and sizes in large vacuoles, corresponding to type C schizonts (arrowheads). (D, E) The same cell presents parasites in pairs (arrow in D) or rosettes (arrow in E) in process of cell division, corresponding to the lytic cycle, and vacuoles containing multinucleated masses (arrowheads). (F) Type C schizont showing merozoites (m) emerging from the multinucleated mass. Nu = enterocyte nucleus. Bars: 10 μm.*

#### **Figure 7.**

*Enterocytes infected with T. gondii bradyzoites of the strain ME-49 for 72 h. Note the presence of parasitophorous vacuoles that are morphologically smaller, along with peripherally arranged parasites inside the vacuoles with or without a large residual body (RB), as described for type C schizonts (arrowhead). Other vacuoles containing parasites in different stages of development can be seen in neighboring cells or in the same cell (arrows). Bars: 10 μm.*

#### **Figure 8.**

*Enterocytes infected with T. gondii bradyzoites of the strain ME-49 for 96 h. (A–C) Images suggestive of type D schizonts (TD) in groups of 3–4 parasites with or without residual bodies. (B–D) Several vacuoles containing parasites in different stages of cell division and development. Type C schizonts (TC) show parasites arranged peripherally inside the parasitophorous vacuoles. Bars: 10 μm.*

obtained *in vitro*, as will be detailed below. Electron micrographs showed enterocytes with multinucleated structures that correspond to the C-type schizont forms (**Figure 9**), similar to those detected here by light microscopy stained with Giemsa (**Figure 6**).

The ultrastructural morphological characteristics of these multinucleated masses showed a varying number of nuclei in each of these structures, with diverse sizes and shapes, higher incidence of rounded shapes, presence of voluminous dense granules, and lipid bodies (**Figure 9**). These forms also presented a well-developed tubulovesicular membrane network (TMN) in the vacuolar matrix, best seen in **Figure 9A, C**, and **E**. From these multinucleated masses in a certain stage of development of the endopolygeny or schizogony, the merozoites constructed within these masses began to migrate to the periphery, like "budding" on the surface of these structures, being easily recognized by the presence of the emerging conoid, and these stages were identified as type C schizonts (**Figure 9**). **Figure 10** is suggestive of the formation of D-type schizonts, as ascertained by Giemsa-stained light microscopy (**Figure 8A**–**C**).

*Development of Schizont Stages of* Toxoplasma gondii *in Primary Cell Culture of Feline… DOI: http://dx.doi.org/10.5772/intechopen.105957*

#### **Figure 9.**

*Enterocytes infected with T. gondii bradyzoites of the strain ME-49 for 96 h. Ultrastructure of enterocytes containing type C schizonts. Large vacuoles containing multinucleate mass with the presence of dense granules (DG) and lipids (Li). Tubulovesicular membrane network (TMN) is present in the vacuolar space. Merozoites are seen emerging from the multinucleated masses (arrows). N = nucleus; R = rhoptries. Bars: (A, D, and E) 3 μm; (B and C) 1 μm; (F) 2 μm.*

#### **Figure 10.**

*Enterocytes infected with T. gondii bradyzoites from strain ME-49 for 144 h. Ultrastructure of D-type schizontcontaining enterocytes. N = nucleus. Bars: (A) 2 μm; (B) 5 μm.*

#### **4. Discussion**

The universal distribution of toxoplasmosis and the important role of felines in the transmission of *T. gondii* stimulate research to understand its enteroepithelial cycle better. In this context, the present study employed primary cultures of feline intestinal epithelial cells as a model to investigate the *T. gondii*-host cell interaction.

Some methodological aspects employed in the present study deserve special attention. The use of bradyzoites as a source of infection is justified because it represents one of the natural routes of transmission of *T. gondii* (through the consumption of raw meat by carnivorous animals or humans), based on the hypothesis that the parasite's enteric cycle is most efficient when cats consume tissue cysts (carnivores) [32]. Ferguson, through schematic diagrams, showed all possible routes of development and parasite conversion stages that could occur among the various forms of infection during the life cycle of *T. gondii* [24]. He demonstrated that the only infectious stage capable of direct conversion into merozoites was bradyzoites. These data support the choice of bradyzoites as a source of infection in the present study, making possible the successful differentiation of bradyzoites into merozoites *in vitro*, evidenced by light microscopy and transmission electron microscopy.

*In vitro* experiments have shown that some parasites initially replicate quickly as tachyzoites to amplify the infection, independent of the infective form (tachyzoites, bradyzoites, or sporozoites) [33]. The conversion of bradyzoites into tachyzoites is a natural process that occurs beginning at 15 h in cell cultures without the addition of immunomodulatory substances [34]. We confirmed this process in FIECs infected with ME49 strain bradyzoites at the 1:5 (parasite: host cell) ratio and, to a smaller extent, 1:10 and 1:20 ratios. It was possible to analyze the intracellular fate of *T. gondii* in the three routes: lytic cycle, cystogenesis, and schizogony in these ratios for periods ranging from 3 to 9 days of interaction.

Our results revealed that decreasing the parasite ratio to 1:10 (bradyzoite: host cell) caused the spontaneous formation of well-defined intracellular cysts in enterocytes after 72 h without any modulation (physical, chemical, or immunological) of the cell culture. Like other researchers, we consider that cystogenesis is a spontaneous event dependent on the strain of *T. gondii*. For example, low-virulent strains (type II) such as ME49 have a natural ability to form cysts in mammalian cells [35–40]. Therefore, we believe that the cell type might be one of the factors that determine the intracellular parasite fate and that intrinsic cellular factors could promote the differentiation stage of *T. gondii* without the need for extrinsic stress factors [41–43].

Here, the occurrence of cystogenesis in feline enterocytes was well characterized ultrastructurally. Our group has already demonstrated in epithelial cells that infection of the feline renal epithelial line CRFK with bradyzoites of strain ME49 (the same strain used in the present study) was more efficient in the establishment of cystogenesis compared to the mouse intestinal epithelial cell line IEC-6 [44]. Our data, combined with the fact that FIEC differentiates in culture, reinforces the concept that cystogenesis *in vitro* is contingent upon several factors, including the strain, parasite load, and cell type (or the interaction of these factors), in addition to the stage of differentiation of the host cells [44–47].

The experimental conditions applied in our experiments using bradyzoites of a low-virulent strain of *T. gondii*, such as ME49, and feline enterocytes allowed us to obtain infective stages corresponding to the morphological characteristics of schizont forms of the parasite, very similar to those characterized *in vivo* [24, 25]. The reproducibility of the sexual cell cycle of *Isospora suis* in intestinal swine epithelial cells was obtained before [48], and it was pointed out the significant influence of the infective dose on the development of intracellular merozoites. The researchers obtained a high density of merozoites when the 1:10 ratio (parasite: host cell) was used and it even allowed the production of oocysts *in vitro* with low parasite loads (1:100 or 1:200). These data are corroborated in part by the results of our group [8] that FIEC

#### *Development of Schizont Stages of* Toxoplasma gondii *in Primary Cell Culture of Feline… DOI: http://dx.doi.org/10.5772/intechopen.105957*

infection with bradyzoites of the ME49 strain using different parasite:host cell ratios was decisive for the intracellular fate of the parasites in enterocytes, in particular, to obtain schizonts. Several experiments were carried out to induce higher production of schizonts in feline enterocytes to, perhaps, obtain sexual forms of *T. gondii.* In this study, we used parasitic load variation, but we were faced with the difficulty of observing a sufficient number of infected cells to allow ultrastructural analysis. We, therefore, chose to infect with the 1:20 load to explore this interaction better. Another strategy also employed was the reduction in the concentration of fetal bovine serum to 1% in the culture medium, as suggested before for *Isospora* [48], but in our system, there was no apparent influence on further induction of schizogony.

Our analysis by optical microscopy of Giemsa-stained monolayers was elucidating because the images of the intracellular stages had a close morphological correlation with the description of schizont stages traditionally reported in the gut of experimentally infected cats [17, 24–26, 49]. The development of schizonts and gametogony in feline enterocytes has been established from the infection of cats with bradyzoites, giving rise to the various stages of *T. gondii* schizonts [49]. It has been postulated that bradyzoites, when penetrating enterocytes, trigger the production of five distinct enteroepithelial stages or schizonts, which are conventionally called types A, B, C, D, and E [25].

The presence of large numbers of multinucleated masses in our enterocyte cultures was the first indication that the *T. gondii* enteroepithelial cycle was established *in vitro*. These structures would correspond to C-type schizonts in the multiple multiplication process, characterized as endopolygeny and/or schizogony by optical microscopic analysis and confirmed by observing the ultrastructure, as proposed by Speer and colleagues [25, 28, 50]. These C-type schizonts are characterized by TMN, mitochondria, electron-dense rhoptries, a large volume of lipids, amylopectin granules, and nuclei displaced to the periphery, as shown in **Figure 9**, in agreement with Speer and Dubey's descriptions [25]. The comparative analysis of the intracellular structures evidenced in enterocytes *in vitro* (**Figure 6A** and **9A**) revealed high similarity with those described by Ferguson [26], who characterized the enteroepithelial stages of histological sections of the intestines of cats. Later stages of the developing schizont type C in enterocyte cultures (**Figure 7**) are similar to those seen in sections of intestinal tissue from infected cats [25], confirming that we were able to partly reproduce the enteroepithelial cycle of *T. gondii in vitro*. Representative images of the schizogony in the process of "budding" of merozoites were observed by electron microscopy (**Figure 9E, F**). When compared to the corresponding ones from this type of schizont described in the original article by Ferguson [26], these images indicate the morphological similarities between these structures. Light and electron microscopic analysis enabled the identification of D-type schizonts, which are also characterized by multinucleated masses that give rise to merozoites from asymmetric nuclear division, which generates organisms with various morphological aspects [17]. Comparing the enterocyte culture images of **Figure 8A** and **10A** with those of histological sections of infected cat intestine [25] shows similarities, indicating that D-type schizonts were produced *in vitro*.

Thus, under our experimental conditions, cultures of FIECs infected with bradyzoites revealed structures very similar to the schizonts of types C and D according to the classification established by Dubey and Frenkel [17] and Speer and Dubey [25] in histological sections of the small intestine of cats orally infected with *T. gondii* cysts.
