Characterization of Cystoisospora suis sexual stages in vitro

Abstract

Background: The porcine coccidium Cystoisospora suis is characterized by a complex life cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development.Methods: Sexual stages of C. suis (micro- and macrogametes) developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Dynein light chain type 1 and male gamete fusion factor HAP2 expression is presumably restricted to microgametes. Tyrosin-rich proteins are a part of the wall-forming bodies and oocyst wall proteins. The RAD51/Dmc1 protein plays a role during the cell division process, and Nima-related protein kinases regulate the cell cycle and are associated with male or female Plasmodium gametocytes. Transcription levels were compared for parasites in culture supernatants at 6–9 doc (day of cultivation) and intracellular parasites at 6–15 doc.Results: Sexual stage of C. suis could be found during 8–11 doc in vitro. Microgametes had a spherical body (3.0–5.0 µm) and two flagella (10.8–12.3 µm). Macrogametes were spherical with a diameter of 11.5–13.0 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. Conclusions: The present work linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in vitro. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of development of C. suis (and coccidian parasites in general) which could be exploited as novel targets for control.