Acta Tropica 95 (2005) 74–78 Short communication Extracellular like-gregarine stages of Cryptosporidium parvum M.J. Rosales ∗ , G. Per´ ez Cord ´ on, M. S´ anchez Moreno, C. Mar´ ın S´ anchez, C. Mascar ´ o. Departamento de Parasitolog´ ıa, Facultad de Ciencias, Instituto de Biotecnolog´ ıa, Universidad de Granada, Espa˜ na Received 24 August 2004; received in revised form 18 January 2005; accepted 8 March 2005 Abstract The present study confirms the existence of extracellular stages of Cryptosporidium parvum during in vitro culture on MDCK, HCT 8 and Vero cells as well as alveolar macrophages, by optic, Nomarski and transmission electron microscopy images. Extracellular trophozoite/gamont, stages in syzygy, zygotes and spores with eight sporozoites were seen in the supernatant of the cultures. The first ultrastructural images of extracellular stages of C. parvum are shown in this study. The morphology of these stages, which have characteristics similar to those of some gregarines, support the contention that Cryptosporidium has closer affinity with gregarines. It also supports the necessity of reconsidering the life cycle of Cryptosporidium and the classification within the coccidia. © 2005 Elsevier B.V. All rights reserved. Keywords: Cryptosporidium; Gregarines; Extracellular 1. Introduction Cryptosporidium parvum was originally classified as a coccidian based on its possession of similar life cycle features (Levine, 1988). However, Cryptosporid- ium demonstrates several peculiarities that separate it from any other coccidian: the location of Cryp- tosporidium within the host cell where the endogenous developmental stages were confined to the apical surfaces of epithelial cells (intracellular but extracyto- plasmic); the presence of feeder organelle; the oocyst ∗ Corresponding author. E-mail address: mjrosale@goliat.ugr.es (M.J. Rosales). which lacks morphological structures such as sporo- cyst, micropyle and polar granules and the insensitivity of Cryptosporidium to all anticoccidial agents tested. These characteristics of Cryptosporidium have been confirmed further by the results of molecular stud- ies, which consistently group Cryptosporidium as a clade separate from the coccidian taxa (Morrison and Ellis, 1997; Carreno et al., 1998). Bull et al. (1998) demonstrated that an immunofluorescent antibody test based on anti-Cryptosporidium Mab cross-reacted with oocysts of Monocystis species. Recent studies by Carreno et al. (1999), based on SSrRNA sequence, and Leader et al. (2003), based on SSU rDNA and - tubulin sequences showed that the gregarines and Cryp- 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.03.009