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. 2022 Oct 13:9:1033380.
doi: 10.3389/fvets.2022.1033380. eCollection 2022.

Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture

Mariana M Rezende-Gondim  1 , Aristeu V da Silva  2 , Jitender P Dubey  3 , Gereon R M Schares  4 , Luís F P Gondim  1
Affiliations

Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture

Mariana M Rezende-Gondim et al. Front Vet Sci. .

Abstract

Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.

Keywords: Hammondia hammondi; Hammondia heydorni; Toxoplasma gondii; immunomagnetic; monoclonal antibody; tissue cyst wall.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Immunomagnetic separation of Toxoplasma gondii tissue cysts (TC). (A) In the direct capture, magnetic beads coated with goat anti-mouse IgG are firstly incubated with the monoclonal antibody (mAb) K8/15-15. Then, the cell suspension containing TC are incubated with the beads. (B) In the indirect method, the cell suspension containing tissue cysts are firstly incubated with the mAb, followed by incubation with the magnetic beads. (A,B) The beads bound to TC are magnetically captured by the magnetic support (magnetic cell separator).
Figure 2
Figure 2
Two in vitro produced tissue cysts of Toxoplasma gondii bound to magnetic beads containing the monoclonal antibody K8/15-15. (A) One of the cysts has about the size of the magnetic bead, which has 4.5 μm in diameter. (B) Bradyzoites inside the captured cysts were labeled by immunofluorescence using a rabbit serum to the bradyzoite antigen BAG-1.
Figure 3
Figure 3
A tissue cyst of Toxoplasma gondii derived from mouse brain bound to magnetic beads containing the monoclonal antibody K8/15-15. (A) The beads cover most of the tissue cyst surface. (B) The same tissue cyst shown in A was mechanically ruptured by pushing the coverslip against the glass slide; note that some beads were detached from the cyst wall and numerous bradyzoites (black arrow) were released from the tissue cyst.
Figure 4
Figure 4
Cysts of Hammondia heydorni produced in cell culture and captured by magnetic beads containing the monoclonal antibody K8/15-15. (A) The captured cyst observed in bright field. (B) Immunofluorescence using a rabbit serum to the bradyzoite antigen BAG-1.
Figure 5
Figure 5
Direct and indirect immunomagnetic separation methods for in vitro produced cysts of Toxoplasma gondii were each tested at two reaction temperatures (4 and 24°C). (A) Test 4 (indirect capture at 24°C) presented the best performance, as the cyst/host cell ratio had a maximum increase of 3.78 times. (B) Test 3 (indirect capture at 4°C) showed the highest non-specific binding, contrasting with test 4, that showed the lowest non-specific binding.
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