Development of Schizont Stages of *Toxoplasma gondii* in Primary Cell Culture of Feline Enterocytes

*Renata M. de Muno, Marcos A. Moura, Letícia C. Medeiros, Pedro N. Caldas, Rafael M. Mariante and Helene S. Barbosa*

#### **Abstract**

Intestinal epithelial cell cultures are a potentially applicable model for investigating enteropathogens such as the protozoan *Toxoplasma gondii*, the etiological agent of toxoplasmosis. Felids such as domestic cats are the only known definitive hosts where the parasite undergoes sexual reproduction, which occurs in the enterocytes. Primary feline intestinal epithelial cell (FIEC) cultures were obtained from the fetal small gut of felines, and the epithelial nature of these cells was confirmed by the revelation of cytokeratin and intestinal alkaline phosphatase content by fluorescence microscopy, besides alignment, microvilli, and adherent intercellular junctions by ultrastructural analysis. FIECs infected with *T. gondii* bradyzoite forms showed that the parasite:cell ratio was determinant for establishing the lytic cycle and cystogenesis and the induction of schizont-like forms. Type C and D schizonts were identified by light and electron microscopies, which showed morphological characteristics like those previously described based on the analysis of cat intestines experimentally infected with *T. gondii*. These data indicate that FIECs simulate the microenvironment of the felid intestine, allowing the development of schizogony and classic endopolygeny. This cellular framework opens new perspectives for the *in vitro* investigation of biological and molecular aspects involved in the *T. gondii* enteric cycle.

**Keywords:** *Toxoplasma gondii*, enteric cycle, primary cell culture, feline enterocytes, schizont stages

#### **1. Introduction**

Intestinal cells act as barriers to prevent access to potentially harmful substances and the migration of the underlying cells in the lamina propria [1]. Several investigators have established culture methods for intestinal cells from different animal species that mimic normal intestinal development [2]. Culture techniques have been developed for cells from different sources, including adult [3, 4] and embryonic cells [5–8]. The introduction of growth factors or interaction of these systems with the extracellular matrix during recent decades has allowed the development of experimental approaches to study *in vitro* differentiation [9, 10]. These advances have afforded the

possible application of enterocyte cultures for *in vitro* studies (e.g., the interaction of these cells with enteroparasites [11] such as *Toxoplasma gondii* [8]).

*T. gondii*, the etiological agent of toxoplasmosis, is an obligatory intracellular parasite that causes one of the most common zoonoses in the world. Its transmission occurs by (i) oral infection via the ingestion of cysts, present in raw or poorly cooked meat; (ii) ingestion of oocysts, present in the feces of Felidae contaminating food, water, and soil [12, 13]; or (iii) vertical transmission via the transplacental route [14, 15]. Cats and other felines are the only definitive hosts capable of directly spreading *T. gondii* in the environment, since the enteroepithelial cycle, including the sexual stage of the parasite, occurs exclusively in those species [16]. The infection in felids is established after ingestion of cysts or oocysts in tissues whose walls are destroyed by proteolytic enzymes in the stomach and intestine, resulting in the release of bradyzoites or sporozoites that invade intestinal cells and initiate the enteroepithelial cycle of the parasite [17].

Five distinct enteroepithelial morphological stages or schizonts of *T. gondii* (Types: A, B, C, D, and E) are described in the felid gut, involving the processes of schizogony, gametogony, and sporogony, which result in the formation of immature oocysts [16–19]. Specific knowledge about the enteric cycle of *T. gondii* in Felidae is limited to morphological characterization *in vivo* [17, 20–25], which makes it difficult to monitor the kinetics of infection. Thus, the analysis of the temporal events established during this *in vivo* cycle is subjective since histological studies of the gut do not allow monitoring the actual sequence of differentiation events of the parasite's infectious stages. However, the practice of euthanasia of cats for scientific studies is restricted [26], which corroborates the need to introduce alternative research models to explore the enteroepithelial cycle of *T. gondii* in felids.

No cell models of the feline intestinal epithelium are commercially available to allow the study of the *T. gondii* enteroepithelial cycle *in vitro.* Several attempts to culture intestinal epithelial cells from adult animals or establish normal cell lines derived from normal enterocytes have not been very successful [27]. The lack of cellular models that allow the reproduction of the enteric cycle of *T. gondii* in felids motivated us to introduce primary cultures of cat enterocytes as an alternative for this study. Preliminary data from this interaction have been published by our group [8]. We now deepen this study by revealing the *T. gondii* development *in vitro* in feline enterocytes.

## **2. Experimental design**

#### **2.1 Feline enterocyte primary cell culture**

Feline enterocyte primary cell cultures (FIEC) were obtained from fetuses of a clinically healthy pregnant domestic cat (no gastrointestinal disease and serologically negative for *T. gondii*, feline immunodeficiency virus, and feline leukemia virus). All procedures were performed in accordance with the guidelines stipulated by the Brazilian College of Animal Experimentation (COBEA). This study was approved by the Fundação Oswaldo Cruz Committee of Ethics for the Use of Animals (license L042/2018 A1).

Small intestine samples corresponding to the jejunum-ileum region (~5 cm) were collected aseptically. The samples were dissected, and the fragments were gathered in ice-cold sterile phosphate-buffered saline (PBS) with a 10% antibiotic solution (Sigma-Aldrich-St. Louis, MO, United States). This tissue was opened longitudinally,

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

washed three times with PBS, and maintained in this solution with 10% antibiotics for 20 min at room temperature. Fragments were divided into small pieces (1 cm3 ) and washed into PBS. The fragments were placed in nonenzymatic dissociation buffer (pH 7.2) containing 1 mM EDTA (Sigma-Aldrich-St. Louis, MO, United States), 1 mM EGTA (Sigma-Aldrich-St. Louis, MO, United States), 0.5 mM dithiothreitol (Sigma-Aldrich-St. Louis, MO, United States), and 10% antibiotic solution for 20 min under stirring at room temperature [2–6]. The cell aggregates were plated in DMEM/Hams medium Dulbecco's Modified Eagle's Medium/Ham's Nutrient F12 (1:1) containing 1% antibiotic solution, 1 mM glutamine, 5% fetal bovine serum (Life Technologies, São Paulo, SP, Brazil), 20 ng/ml epidermal growth factor (Sigma-Aldrich-St. Louis, MO, United States) [4, 7], 0.1% human insulin (Humulin N - Lilly, Indianapolis, IN, United States), 100 nM hydrocortisone (Sigma-Aldrich-St. Louis, MO, United States), 1% nonessential amino acids 100x (Life Technologies, São Paulo, SP, Brazil), and 1 μg/ml 3,3′, 5-triiodo-L-thyronine sodium salt (Sigma-Aldrich-St. Louis, MO, United States) [10]. The cultures were maintained at 37°C in a 5% CO2 atmosphere, and the medium was renewed every two days.

Confluent FIECs were treated for 10 min at 37°C with dissociation solution (PBS with 0.01% EDTA and 0.25% trypsin). After dissociation, the cells were placed in culture medium at 4°C with 10% fetal bovine serum to inhibit the action of trypsin, centrifuged for 7 min at 650 × *g* at 4°C, and grown in 24-well plates on coverslips (105 cells/well) or on 35 mm3 plastic disks (5.0 x 105 cells/disk). Cell cultures were daily analyzed by light microscopy (Zeiss Imager A2 microscope) after fixation in Bouin's solution and Giemsa staining to assess morphology and proliferation. Images were captured with Soft Axion Vision 40 v.4.8.2.0 and an Axion cam MRc color camera.

#### **2.2 Characterization of FIEC by immunolabeling**

Several monoclonal antibodies were applied to characterize the FIECs: anti-pancytokeratin clone PCK-26 (Sigma-Aldrich-St. Louis, MO, United States); anti-vimentin clone VIM-13.2 (Sigma-Aldrich-St. Louis, MO, United States); anti-intestinal alkaline phosphatase clone AP-59 (Sigma-Aldrich-St. Louis, MO, United States); and anti-desmin clone DE-U-10 (Sigma-Aldrich-St. Louis, MO, United States). The cells were fixed for 10 min at 4°C with 4% paraformaldehyde in PBS, washed three times for 10 min in PBS, and then incubated for 30 min in 50 mM ammonium chloride to block free aldehyde radicals. Afterward, the cells were permeabilized for 20 min in a PBS solution containing 0.05% Triton X-100 (Roche, Rio de Janeiro, RJ, Brazil) and 4% BSA (Sigma-Aldrich-St. Louis, MO, United States) to block nonspecific binding. For the indirect immunofluorescence assays, the cells were incubated for 2 h at 37°C with the following primary antibodies: anti-vimentin (1:200); anti-cytokeratin (1:100); anti-intestinal alkaline phosphatase; and anti-desmin (1:100). After this incubation, the cells were washed 3 times for 10 min in PBS containing 4% BSA and incubated for 1 h at 37°C with the secondary antibody at a dilution of 1:1000 (mouse anti-IgG conjugated with FITC or TRITC) (Sigma-Aldrich-St. Louis, MO, United States). To reveal actin filaments, the cells were incubated for 1 h at 37°C with 4 μg/mL phalloidin-FITC in PBS (Sigma-Aldrich-St. Louis, MO, United States). Next, the cultures were washed 3 times for 10 min in PBS and incubated for 5 min with 0.1 μg/ mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich-St. Louis, MO, United States) diluted in PBS. After wash, the coverslips were mounted on slides with a solution of 2.5% DABCO (1,4-diazabicyclo-[2,2,2]-octane-triethylenediamine)

(Sigma-Aldrich-St. Louis, MO, United States) in PBS containing 50% glycerol, pH 7.2. Controls were performed by omission of the primary antibody. The samples were examined with a confocal laser-scanning microscope (CLSM Axiovert 510, META, Zeiss) with a 543 helium laser (LP560 filter), 488 argon/krypton laser (Ar/Kr) (filter LP515), and a 405 Diode laser (LP 420 filter).
