Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites. Methods 13

and formation of the parasitophorous vacuole mem-Apical organelles contain molecules that are of critical im- brane, and dense granules are important for remod-portance for the interaction of all apicomplexan parasites eling the vacuole after invasion (for example, see with their target host cells. Thus, there is considerable inter- Refs. 1±5). Sequencing of genes for organelle pro-est in characterizing and understanding the function of mole- teins has shown several domains and motifs con-cules that reside in these organelles. Large numbers of sur- served among genera, especially in microneme pro-face-sterilized oocysts of Eimeria tenella, an apicomplexan teins, supporting the idea that apical proteins have coccidian of the chicken, can be routinely obtained from the similar functions across the phylum. Studies to elu-animal host, and invasive sporozoites, which contain abun- cidate the structure and function of the apical organ-dant apical organelles, can be rapidly prepared from these elles will be critical for understanding how apicom-oocysts in the laboratory. Thus, E. tenella is proving to be an plexan parasites enter cells and establish parasitism amenable parasite for subcellular fractionation techniques and also for identifying novel strategies that could that allow the direct isolation and characterization of apical be used to block or control these events. organelles. In this paper, a series of protocols is described for the large-scale culture of E. tenella parasites, the prepara-tion of invasive sporozoites, the isolation of apical organelles, and the use of in vitro culture for localization and functional RATIONAL