Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

Abstract

Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isospora suis , a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I . suis , including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I . suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.

Characterization of Cystoisospora suis sexual stages in vitro

“…Piglets were raised with the sow in the animal facilities of the Institute of Parasitology, University of Veterinary Medicine Vienna, Austria. Infection of piglets, oocyst collection, oocyst isolation, oocyst purification and excystation were performed as described previously [7]. In deviation to the original protocol, after NaOCl treatment and washing, the oocysts were vortexed three times for 45 s with Precellys ® glass beads (Peqlab, Erlangen, Germany) in 2% sodium taurocholate hydrate (Sigma-Aldrich, St. Louis, USA) in DMEM/Ham's F-12 culture medium (Gibco, Thermo Fisher Scientific, Waltham, USA).…”

“…Previous studies have shown that the development of C. suis in vitro is comparable with the life-cycle in vivo; however, the development of sexual stages in vitro is delayed [7]. This makes it possible to observe, harvest and examine sexual stages in the short time frame in which they occur.…”

Cystoisospora suis – A Model of Mammalian Cystoisosporosis

“…Like other Cystoisospora species, C. suis entirely develops in one host ( 26 , 27 ) (Figure 1 ). Directly after ingestion, sporulated oocysts undergo excystation and sporozoites invade the small intestine epithelium ( 12 , 28 ) to reproduce within a parasitophorus vacuole ( 29 , 30 ).…”

“…An experimental model mimicking the field situation ( 10 ) in conventional piglets gave deeper insight into neonatal porcine cystoisosporosis. This was further strengthened by the establishment of an in vitro culture system supporting the entire lifecycle of C. suis in intestinal porcine epithelial cells ( 26 ). Moreover, gnotobiotic piglets are available as infection models for specific applications ( 3 , 21 ).…”

Vaccination against parasites – status quo and the way forward

“…New techniques in biotechnology and bioinformatics have enabled rapid and cost-effective screening of genomes and transcriptomes of parasites for vaccine and drug target candidates (as reviewed in [4, 12], and others) including A. suum [13] , T. suis [14] , T. gondii (www.toxodb.org/) and C. suis (Palmieri, submitted). For C. suis a genome size of 83 Megabases encoding >8300 genes is estimated, and a recently developed pipeline for the search of vaccine candidates in apicomplexan parasites (Vacceed; [10]) has detected 562 candidates in C. suis which now need to be evaluated further in silico, in vitro (using a cell culture system supporting the complete life cycle of C. suis ; [50]) and in vivo .…”