Dynamics of <i>I</i><i>. suis</i> in intestinal porcine epithelial cells (IPEC-J2).

<p>Cells were infected with an infection dose of 1:10 (sporozoites: cells), medium with 5% FCS was used. The percentage of infected cells over time (A) and the number of zoites per group over time (B) are shown. A group was defined as the totality of merozoites derived from one dividing mother cell based on the arrangement of zoites in pairs or rosettes; dpi, days post infection.</p

Meronts of <i>I</i><i>. suis</i> in the <i>in vitro</i> culture in intestinal porcine epithelial cells (IPEC-J2).

<p>(A) PC image on dpi 9 and (B) DIC image at dpi 3 of bi-nucleated type I meronts; (C) PC and (D) DIC image of symmetric type II meronts with multiple nuclei; (E) PC image of asymmetric type II meronts on dpi 9; (F) rounded multinucleated subtype II meront on dpi 9. DIC, differential interference contrast; dpi, days post infection; HN, host cell nucleus; PC, phase contrast; bar = 10 µm.</p

Immunohistochemical staining of <i>I</i><i>. suis</i> stages with anti-<i>Tg</i>IMC3 and DAPI.

<p>Staining with anti-<i>Tg</i>IMC3 is shown in green, DAPI-staining is depicted as orange; (A) Type I meront, dpi 8; (B) merozoites, dpi 8; (C) merozoites and a weakly stained macrogamont indicated by arrow heads, dpi 8; (D), (E) presumptive early microgamonts, dpi 11; (F) mature microgamont with microgametes surrounding the residual body, dpi 11; dpi, days post infection; bar = 5 µm.</p

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

<div><p>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 <i>Toxoplasma gondii</i>. Although this parasite is well investigated there is no adequate <i>in vitro</i> model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of <i>Isospora</i><i>suis</i>, a sister taxon to <i>T. gondii</i> and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an <i>in vitro</i> model for neonatal porcine coccidiosis in cells representative for the <i>in vivo</i> 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>I</i><i>. suis</i>, 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 <i>T. gondii</i> (in our case, anti-<i>Tg</i>IMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of <i>I</i><i>. suis</i> 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.</p> </div

Development of <i>I</i><i>. suis</i> stages and cell condition over time dependent on infection dose (sporozoites: cells).

<p>Cells were cultivated in medium with 5% fetal calf serum. Group means from semi-quantitative evaluation of developmental stages (A–D) and of host cell condition (E) are shown; *indicates significant difference to at least one other infection dose in multiple comparisons with Bonferroni correction (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069797#tab1" target="_blank">Table 1)</a>; dpi, days post infection. No significant differences were detected for oocysts in multiple comparisons.</p

Sexual stages of <i>I</i><i>. suis</i> in intestinal porcine epithelial cells (IPEC-J2).

<p>(A) PC image of a mature microgamont with intracellular microgametes (MG) surrounding the residual body (RB) on dpi 14; (B) DIC image of a mature microgamont (MIG) and a macrogamete (MAG) in the neighbouring host cell on dpi 13, notice the pronounced granular structure of the MAG; (C) PC image of a free microgamete in motion showing the typical two flagella (FL) on dpi 8; (D) presumptive young macrogamonts within one parasitophorous vacuole, notice the distinct nucleoli and the granular structure of the cytoplasm typical for macrogamonts; (E) PC image of a mature macrogamete and a pair of type I merozoites (MZ) on dpi 7; (F) DIC image of a mature macrogamete on dpi 19. DIC, differential interference contrast; dpi, days post infection; HN, host cell nucleus; PC, phase contrast; bar = 10 µm.</p

Oocysts of <i>I</i><i>. suis</i> in the <i>in vitro</i> culture.

<p>(A) intracellular oocysts with a distinct parasitophorous vacuole (PV) and an already developed oocysts wall (OW) on dpi 13; (B) free unsporulated oocyst containing the sporont (SP); (C) sporulated oocyst 5 days after harvest from cell culture and incubation in tap water at room temperature with developed sporocyst walls (SPW) containing sporozoites; dpi, days post infection; bar = 10 µm.</p

Electron micrographs of a microgamont and microgametes of <i>I</i><i>. suis</i> in intestinal porcine epithelial cells (IPEC-J2).

<p>(A) section through a microgamont with budding microgametes on dpi 13; (B) transversal section through microgametes and flagella showing the 9x2 + 2 arrangement of microtubules within the flagella on dpi 12; (C) longitudinal section through the flagella and the body of a microgamete showing the typical large mitochondrion in the latter. dpi, days post infection; FL, flagella; HC, host cell; MI, mitochondrion; MV, host cell microvilli; N, nucleus; PG, polysaccharide granules; PL, plasmalemma; PV, parasitophorous vacuole; RM, residual cytoplasmatic mass; single bar = 1000 nm, double bar = 500 nm.</p

Sporozoites and merozoites of <i>I</i><i>. suis</i><i>in vitro</i> in intestinal porcine epithelial cells (IPEC-J2).

<p>(A) extracellular sporozoite before cell invasion on dpi 0; (B) intracellular sporozoite 24h after infection, note the anterior and posterior refractile body (arrow heads); (C), (D), and (E) type I merozoites on dpi 5; (F) smaller presumptive type II merozoites on dpi 7; (G) two large groups of subtype II merozoites (ST II) and a small group of type I merozoites (TI) on dpi 9. HN, host cell nucleus; dpi, days post infection; bar = 10 µm.</p

Size of intracellular <i>I</i><i>. suis</i> zoites in intestinal porcine epithelial cells (IPEC-J2).

<p>The correlation between length and group size (A) and the correlation between length and dpi for groups of 4 zoites (B) and 8 zoites (C) are shown. Groups of 4 zoites were first detected at 3 dpi, groups of 8 at 5 dpi. A group was defined as the totality of merozoites derived from one dividing mother zoite based on the arrangement of zoites in pairs or rosettes; dpi, days post infection.</p