Microgametes of the porcine coccidium Cystoisospora suis

Unicellular parasites of the order Coccidia are important pathogens of humans and animals. As a representative of this group, Cystoisospora suis parasitizes in the small intestine of piglets and can cause severe gastrointestinal diseases. These parasites undergo a complex development, during which multiplication by fission is followed by sexual replication. During this process two different stages develop, the small, motile microgametes and the large, immotile macrogametes. The development and fusion of these stages is essential for the fulfilment of the developmental cycle of the parasite; however, little is known about these final steps. By means of bioimaging, molecular and protein biochemical analyses the maturation and fusion of the gametes will be investigated more closely in order to detect and characterize parasite proteins restricted to this developmental phase. In order to demonstrate and investigate these proteins on a cellular level a cell culture system is available which supports the complete development of Cystoisospora suis in cells of the small intestine which permits the observation and analysis of the gametes and their fusion. This biological process is also mandatory for related organisms, so that its detailed description does not only hold promise for novel control strategies against Cystoisospora suis but also against other parasites such as Toxoplasma gondii, an important pathogen of humans. For the detailed analysis of the parasite stages they will be grown in cell culture, isolated, separated from their host cell and characterized by RNA analysis of gene expression and protein analysis via bioinformatic methods and mass spectrometry as well as by imaging techniques (confocal laser dissection microscopy). The focus will be on the microgametes. As soon as microgamete-specific proteins are defined, these will be functionally characterized in more detail by immunological methods intended to inhibit fertilization of the macrogamete and consequently further development of the parasites in cell culture. This project is an important step in the research of the cellular and developmental biology of the Coccidia and will allow for the definition of specific new targets for infection prophylaxis of Coccidia, especially Cystoisospora suis of pigs.

Porcine cystoisosporosis, a gastrointestinal disease of suckling piglets caused by the single-celled parasite Cystoisospora suis, affects pig breeding farms worldwide. Despite its global distribution, little is known about details of the parasite's life cycle that could provide clues for intervention targets for drug application or vaccination. In this project we developed a cell culture format to propagate all stages of the parasite in the laboratory and subsequently analyzed specifically the sexually differentiated stages (gamonts and gametes) which provide a so-called developmental bottleneck for the parasites in that the population size during this development is at a minimum. They consist of macrogametes, "female" stages that are large and immotile, and flagellated, highly motile "male" stages, the microgametes. These stages are typical for parasites from the protozoan phylum Apicomplexa and provide the basis of its genetic diversity due to sexual reproduction; however, they are poorly characterized since they are only transiently present for short time periods and in limited numbers. We could show that the gametes of C. suis fuse to form environmentally resistant stages, the oocysts, in the laboratory, and that this formation could be interrupted by antibodies directed against proteins present specifically in pre-sexual and sexual stages of the parasite. To test whether vaccination of pigs with these antigens could truncate the parasite's development in the pig host as well, we vaccinated pregnant sows and experimentally infected their offspring. Antibodies present in the sows' milk were transferred to their piglets immediately after birth and were able to protect the piglets against the outcome of experimental C. suis infection, i.e. parasite shedding and weight loss. This project lead us from unravelling the -omics of the developmental biology of C. suis to the application of basic research to the generation of a veterinary anti-parasite vaccine. In addition, we could demonstrate that not only cellular but also excreted proteins as well as lipids, enclosed in vesicles exuded from the parasite's surface, have a stage-specific composition and may serve for communication with the host as well as intra-specifically, e.g. between macro-and microgametes, to facilitate their interaction. The results of this project have given rise to several peer-reviewed publications in international journals and to a patent on anti-C. suis vaccination of pigs and sparked new research questions on the biology of C. suis and its closest relative, Toxoplasma gondii, the most prevalent parasite of humans worldwide. Unlike C. suis, T. gondii cannot propagated in the laboratory throughout its development, so C. suis can complement T. gondii research as an alternative organism that can be studied under laboratory conditions requiring only a limited number of animal experiments.