Dissertation - University of Veterinary Medicine Vienna - 2022

Cystoisospora suis, an apicomplexan enteral parasite of pigs, causes severe economic losses in the livestock industry worldwide. It is characterized by a complex life cycle, during which asexual multiplication, with sporogony and merogony, is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Previous life cycle studies give an overview of the development in vivo; however, the detailed development of the sexual stages, i.e. fusion of macro- and microgametes and proteins involved in this is not yet known. However, this crucial step in the life cycle of C. suis may constitute a promising intervention target to interrupt parasite development and to prevent further formation of transmissible stages. This thesis aimed to provide a detailed morphological characterisation of all stages of the sexual development of C. suis, including fertilization, and to identify and characterise stage-specific proteins linked to sexual development by various cell cultivation, molecular and imaging techniques. First and foremost, we could demonstrate that the detailed life cycle of C. suis can be produced in vitro and all life stages can be found at distinct timeslots. Early sexual stages of C. suis are first observed on day 8 of cultivation (doc) and look like sunny-side-up eggs, are immobile and have a size of 11.6×15.6 μm. Late sexual stages comprise of two types of gamonts, micro- and macrogamonts, which are similar in size and morphology. Mature micro- and macrogametes are mainly found at 9-11 doc and are often observed in close proximity to each other to support fusion. Mature microgametes consist of an uninucleate body (3-5 μm), with two flagella of 10.8-12.3 μm length on opposite sides, which makes them actively motile. Macrogametes are immobile, spherical with a smooth surface and have a diameter of 11.5-13.0 μm. After fusion a zygote is formed which is the stage preceding the infectious, transmissible sporozoites. From the proteins of the macrogamete an oocyst wall forms, and the environmentally resistant oocysts can be found in vitro between 10-13 doc. The present work also identified genes linked to the developmental cycle of C. suis by RNASeq analysis and showed the expression changes of selected genes by quantitative real time PCR during the course of development. Genes related to the sexual stages (micro- or macrogametes) showed an upregulation from 6 doc onwards with a peak on the days when gamonts were also present in vitro (10-13 doc). For microgamonts and macrogametes an increased expression of DLC1 and HAP2, proteins related to fertilization, was shown. The occurrence of macrogamonts and macrogametes was correlated with elevated OWP1 and TyRP expression, proteins necessary for oocyst wall formation, both of which increased steadily during cultivation. Moreover, a new host cell free culture system was developed during this project. For this, merozoites collected from in vitro IPEC monolayer cultures infected with C. suis were transferred to a host cell-free environment on the 6th doc developed further into sexual stages and thus continued their life cycle in the same time slots as in the cell culture system. Also, the gene expression levels of sexual stage specific genes were comparable in the host cell-free culture system to the already established cell in vitro system. This novel system provides a new tool for detailed research on the development of C. suis and possibly other Coccidia and will also be useful for evaluation of novel drug or vaccine targets in these parasites. Furthermore, the gene transcribing for the TyRP in C. suis could be identified, and the native protein was bound by a recombinant anti-TyRP-antibody in host cell-free culture. Inhibition of TyRP stunned the formation of the oocyst wall, rendering the parasite non-infectious. Hence, during the course of this project, a possible candidate for the interruption of the C. suis life cycle could be described. Overall, this work sheds light on the sexual development of an important intestinal parasite of pigs, and paves the ways to more applied research for the development of tools and methods to effectively interrupt the parasite’s development as the basis of novel control options.

Dissertation - Veterinärmedizinische Universität Wien - 2022

application/pdf

75 Blätter