Progression of asexual to sexual stages of Cystoisospora suis in a host cell-free environment as a model for Coccidia

Abstract

Coccidia display a characteristic life cycle, where the parasites switch between asexual and sexual development, resulting in an environmental stage, the oocyst. The entero-pathogenic Cystoisospora suis, a coccidian parasite of swine and close relative to Toxoplasma gondii, undergoes development in one host-cycle. Despite the well-described intracellular development of Coccidia, the C. suis life cycle can progress in an in vitro, host cell-free system after initial intracellular development of merozoites. A novel host cell-free cultivation method was developed by transferring purified merozoites from cell culture supernatant (dpi 6) to culture medium and incubating them for 5 days to induce their progression to sexually differentiated stages. The development of sexual stages in the absence of host cells was verified by morphological studies, flow cytometry and the transcription analysis of three genes linked to sexual stages (HAP2, OWP and TyRP). The host cell-free culture permits the sexual development (and with this, the complete life cycle progression from sporozoites to oocysts) of C. suis in vitro and provides a new tool for detailed research on the development of C. suis and possibly other Coccidia. This will also be useful for the evaluation of novel drug or vaccine targets in these parasites.

Keywords: Cell-free culture; Isospora suis; gametes; gametogenesis; gamonts; qPCR.

Graphical abstract

Fig. 1.

Total amount of gamonts and oocysts of Cystoisospora suis in a host cell-free culture. Values represent the mean from five independent experiments. ****P < 0.0001.

Fig. 2.

Comparison of flow cytometry of an in vitro cell culture and the cell-free in vitro culture of parasite cells. (A) Microgamonts, macrogamonts and macrogametes (black) and host cells and few intact microgametes in the top right corner (blue), in vitro culture, 10 dpi. (B) Exclusively parasite stages (black) are found in the host cell-free culture 4 dpt.

Fig. 3.

Light microscopy of different stages of C. suis in host cell-free culture, captured with differential interference contrast (DIC) or brightfield microscopy. Scale bars: 20 μm unless indicated otherwise. (A) First-generation merozoite, 0 dpt, DIC. (B) Second-generation merozoite, 0 dpt, brightfield microscopy. (C) Microgamont with an opening on one end and moving microgametes in the centre, 3 dpt, DIC. (D) Macrogamont, 3 dpt, DIC. (E) Microgamete, 4 dpt, scale bar: 5 μm. DIC. (F) Macrogamete, 4 dpt, DIC.

Fig. 4.

Light microscopy of oocysts of C. suis in a host cell-free culture. (A) Unsporulated oocyst, 4 dpt. (B) Sporulated oocysts, 4 dpt. (C) Autofluorescent, unsporulated oocyst, 4 dpt. Scale bars: 20 μm.

Fig. 5.

Relative normalized expression levels of genes related to sexual development in a host cell-free culture evaluated by qRT-PCR 0, 3 and 4 dpt. (A) Microgamete-related gene HAP2. (B) Macrogamete related gene OWP1. (C) Macrogamete related gene TyRP. Values represent the mean ± standard error (s.e.) from five independent experiments. ****P < 0.0001.