<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

Cellular Cargo Delivery: Toward Assisted Fertilization by Sperm-Carrying Micromotors

We present artificially motorized sperm cellsa novel type of hybrid micromotor, where customized microhelices serve as motors for transporting sperm cells with motion deficiencies to help them carry out their natural function. Our results indicate that metal-coated polymer microhelices are suitable for this task due to potent, controllable, and nonharmful 3D motion behavior. We manage to capture, transport, and release single immotile live sperm cells in fluidic channels that allow mimicking physiological conditions. Important steps toward fertilization are addressed by employing proper means of sperm selection and oocyte culturing. Despite the fact that there still remain some challenges on the way to achieve successful fertilization with artificially motorized sperms, we believe that the potential of this novel approach toward assisted reproduction can be already put into perspective with the present work

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

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

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

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

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

Sperm-Hybrid Micromotor for Targeted Drug Delivery

A sperm-driven micromotor is presented as a targeted drug delivery system, which is appealing to potentially treat diseases in the female reproductive tract. This system is demonstrated to be an efficient drug delivery vehicle by first loading a motile sperm cell with an anticancer drug (doxorubicin hydrochloride), guiding it magnetically, to an <i>in vitro</i> cultured tumor spheroid, and finally freeing the sperm cell to deliver the drug locally. The sperm release mechanism is designed to liberate the sperm when the biohybrid micromotor hits the tumor walls, allowing it to swim into the tumor and deliver the drug through the sperm–cancer cell membrane fusion. In our experiments, the sperm cells exhibited a high drug encapsulation capability and drug carrying stability, conveniently minimizing  toxic side effects and unwanted drug accumulation in healthy tissues. Overall, sperm cells are excellent candidates to operate in physiological environments, as they neither express pathogenic proteins nor proliferate to form undesirable colonies, unlike other cells or microorganisms. This sperm-hybrid micromotor is a biocompatible platform with potential application in gynecological healthcare, treating or detecting cancer or other diseases in the female reproductive system

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