1163 J. Parasitol., 90(5), 2004, pp. 1163–1165  American Society of Parasitologists 2004 Alternative Mechanism of Eimeria bovis Sporozoites to Invade Cells In Vitro by Breaching the Plasma Membrane J. H. Behrendt, W. Clauss, H. Zahner*, and C. Hermosilla*†, Institute of Animal Physiology, Justus Liebig University Giessen, Wartweg 95, 35392 Giessen, Germany; *Institute for Parasitology, Justus Liebig University Giessen, Rudolf-Buchheim-Strasse 2, 35392 Giessen, Germany; †To whom correspondence should be addressed. e-mail: carlos.r.hermosilla@vetmed.uni-giessen.de FIGURE 1. Cell wound assay. A–B. ‘‘Positive’’ control. Scratch mark in a BSLEC monolayer, applied in the presence of FITC-dextran. (A, phase contrast picture, arrows indicate dextran positive cells; B, fluorescence picture.). C–D. Pattern after infection of a BSLEC monolayer with Eimeria bovis sporozoites in the presence of FITC-dextran. (C, phase contrast picture, arrows indicate dextran positive cells; D, fluorescence picture.) ABSTRACT: In vitro Eimeria bovis sporozoites invade a wide range of cell types, and in the case of bovine cells, they may develop to first- generation schizonts. Often, however, they subsequently leave their host cell to invade a new one, which seems contrary to the classical way of infecting a cell by forming a parasitophorous vacuole. Using a standard, ‘‘cell wound assay,’’ we show that E. bovis can invade bovine endo- thelial cells by breaching the plasma membrane and may again leave the surviving cell. Eimeria bovis sporozoites also infected VERO and HT29 cells but obviously without damaging the plasma membrane. The same held true when bovine endothelial cells were exposed to tachy- zoites of Toxoplasma gondii and Neospora caninum. According to a literature report dealing with Plasmodium yoelii sporozoites, breaching the membrane of certain host cells may be a common phenomenon for coccidian sporozoites but may not be for merozoites. Eimeria bovis is a common and pathogenic coccidium of cattle, which may cause severe hemorrhagic enteritis (Daugschies et al., 1998). Its endogenous development shows a number of peculiarities. Sporo- zoites are liberated in the host’s gut and must invade endothelial cells of the central lymph capillaries in the villi of the ileum, where they replicate, forming multinucleated macroschizonts, which contain hun- dreds of thousands of first-generation merozoites. Second-generation schizonts and gamonts then develop quickly in epithelial cells of the large intestine (Hammond et al., 1944). It is unclear how the sporozoites reach their destination. If migrating directly, they would need to traverse the ileum epithelial cell layer. Because invasion of cells by coccidia is a complex process involving the release of particular parasite products and an invagination of the host cell plasma membrane to form a par- asitophorous vacuole (PV) around the invading stage (Dubremetz et al., 1998; Entzeroth et al., 1998), one might speculate that sporozoites take a paracellular route on their way to the predetermined host cell. How- ever, while monitoring the invasion of cells by E. bovis sporozoites in vitro, we have often observed parasites entering and leaving cells quick- ly and repeatedly, without harming them severely, as has been described by others (Fayer and Hammond, 1967). A similar observation was re- cently reported by Mota et al. (2001) for sporozoites of Plasmodium yoelii, which similarly traverse hepatocytes in vitro. The latter authors suggested that these sporozoites may use an alternative mode of inva- sion by breaching the host cell membrane without forming a PV. This breaching of the plasma membrane would be followed by a rapid mem- brane repair, i.e., cells usually survive it. To determine whether E. bovis sporozoites also share the ability to simply traverse cells, we performed several experiments using a variety of host cell types and compared the results with those obtained in the course of host cell invasion by Toxoplasma gondii and Neospora can- inum tachyzoites. The E. bovis strain was isolated in 1988 in the field in northern Germany and maintained by passages in calves (Fiege et al., 1992). Sporozoites were isolated from oocysts according to Hermosilla et al. (2002). Toxoplasma gondii (RH strain; Sabin, 1941) tachyzoites were harvested from the peritoneal cavity of BALB/c mice 48 hr after intra- peritoneal injection of the parasites and were washed several times with phosphate-buffered saline (PBS), then centrifuged at 400 g for 10 min. Neospora caninum (strain NC-1; Dubey et al., 1988) was maintained in VERO cells. Tachyzoites were washed off the cultures and prepared as above. The experiments used bovine aortic endothelial cells (BAEC), bovine spleen lymphatic endothelial cells (BSLEC), African green monkey kid- ney cells (VERO), and human colon adenocarcinoma cells (HT29). BAEC were isolated from freshly resected aortas by collagenase diges- tion (20 min in 0.2% collagenase type II [Worthington Biochemical Corp., Lakewood, New Jersey] in Puck’s saline A [PSA, GIBCO, Eg- genstein, Germany] salt solution at 37 C and 5% CO 2 ). They were washed, resuspended in endothelial cell growth medium (ECGM; Prom-