Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.
A method is described for the excystation and collection of infective sporozoites of Eimeria separata. The procedure uses conditions that resemble the in vivo environment. The first treatment of the oocysts in a 0.4% pepsin/HCl solution alters the oocyst wall, which becomes thinner. The second treatment in a 0.4% trypsin/0.75% taurocholate solution breaks the oocyst wall and sporocysts are released. A third incubation of the oocyst-sporocyst mixture in trypsin-free medium with 0.75% taurocholate and an additive of MgCl2 followed by a final incubation in RPMI medium supplemented with 1% fetal calf serum yields a sporozoite excystation rate of up to 90%.
Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca2+ and H+ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca2+] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca2+ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling.
To study the pathophysiology of diarrhoea in coccidial infections, Na+ and Cl- fluxes (J(Na), J(Cl)), short circuit current (I(sc)) and tissue conductance (g(t)) were determined in stripped gut epithelia of Eimeria separata infected rats employing the Ussing chamber technique. E. separata invades enterocytes of the caecum and proximal colon. Na+ absorption was generally reduced in infected tissues, Cl- absorption only in the caecum. I(sc) values were increased in the caecum and reduced in the proximal colon. Tissue conductance was not affected. Values tended to normal with time after infection. Theophylline caused markedly increased I(sc) and g(t) values in the caecum epithelia of infected rats. In the epithelia of the distal colon, i.e. the non-infested part of the large intestine, g(t) values remained unaffected but I(sc) was fourfold increased. This I(sc) increase was strongly sensitive to amiloride, suggesting a compensatory activation of Na+ channels in the distal colon of infected rats. Accordingly, serum levels of aldosterone, which activates Na+ channels in the distal colon, were increased to eightfold levels in infected animals. Thus compensatory Na+ absorption was under endocrine control.
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