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Early and late stages of C. parvum merogonic development from 2 h post-cultivation. (a) Meront with new budding merozoites, showing micronemes, thickening of the membranes underneath the pellicle (arrow) and an artifact (asterisk). (b–e) Extra-cellular meronts with budding merozoites displaying conoids (C), rhoptries (arrowheads), micronemes (Mn) and dense granules (Dg). (f, g) Nearly fully developed merozoites (2 and 12 h) budding from the surface with electron-dense collars at the neck of attachment (arrows). Note the vacuolization of the meront's residuum and the extension of its outer pellicle as a parasitophorous vacuole-like structure (arrowhead). (h) Two merozoites within the residual body. Inset: Five longitudinal sub-pellicle microtubules. Bar = 2 m (g), 1 m (d, f), 500 nm (b, c, e, h) and 200 nm (a).
Contexts in source publication
Context 1
... of the meront's cytoplasm into numerous com- partments and thickening of the membranes underneath its outer pellicle was identified (Figs 2b, 4a, b). This new mem- brane ultimately became the inner membrane complex of the newly completed merozoite. As development progressed, continued elevation of the membrane occurred, forming finger-like buds ( Fig. 4c-e); the merozoite primordia were recognizable by their micronemes and electron-dense rhop- try anlagens at the anterior ends ( Fig. 5a-c). As development proceeded, the rhoptries extended into the posterior parts, and the inward fold of the outer membrane continued to encase the entire merozoite (Fig. 4d, e). At more advanced stages, ...
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... occurred, forming finger-like buds ( Fig. 4c-e); the merozoite primordia were recognizable by their micronemes and electron-dense rhop- try anlagens at the anterior ends ( Fig. 5a-c). As development proceeded, the rhoptries extended into the posterior parts, and the inward fold of the outer membrane continued to encase the entire merozoite (Fig. 4d, e). At more advanced stages, daughter merozoites had completely developed, apart from a posterior attachment to the residuum, and electron-dense col- lar thickenings were observed at the points where separation will occur (Fig. 4f, g). Finally, the attachment was broken, releasing free extra-cellular merozoites (Fig. 6). An exten- sion of ...
Context 3
... into the posterior parts, and the inward fold of the outer membrane continued to encase the entire merozoite (Fig. 4d, e). At more advanced stages, daughter merozoites had completely developed, apart from a posterior attachment to the residuum, and electron-dense col- lar thickenings were observed at the points where separation will occur (Fig. 4f, g). Finally, the attachment was broken, releasing free extra-cellular merozoites (Fig. 6). An exten- sion of the outer pellicle of the meront as a parasitophorous vacuole-like membrane was detected (Fig. 4g). Notably, developing meronts showing dividing nuclei were also observed inside the oocysts in association with cytoplasmic budding ...
Context 4
... from a posterior attachment to the residuum, and electron-dense col- lar thickenings were observed at the points where separation will occur (Fig. 4f, g). Finally, the attachment was broken, releasing free extra-cellular merozoites (Fig. 6). An exten- sion of the outer pellicle of the meront as a parasitophorous vacuole-like membrane was detected (Fig. 4g). Notably, developing meronts showing dividing nuclei were also observed inside the oocysts in association with cytoplasmic budding and infolding of the double membrane ( Fig. 3b-d), resulting in almost mature meronts (Figs 3d and 5). Meronts at all stages of differentiation were approxi- mately 2.34 × 2.7 m (range = 1.11-3.07 × ...
Citations
... These novel high-resolution images are the first to deliver such information regarding the asexual cell-free in vitro developmental stages of C. parvum, as well as sexual (Aldeyarbi and Karanis, 2014; Aldeyarbi and Karanis, submitted for publication) and gregarine-like stages Karanis, 2014, 2015), confirming the closeness of this species to gregarines. These data provide should prompt future studies to determine the physiological processes occurring within and the parasitic strategies of these extracellular parasites, which urgently need to be closely analyzed. ...
The stages of Cryptosporidium parvum asexual exogenous development were investigated at high ultra-structural resolution in cell-free culture using transmission electron microscopy (TEM). Early C. parvum trophozoites were ovoid in shape, 1.07×1.47μm(2) in size, and contained a large nucleus and adjacent Golgi complex. Dividing and mature meronts containing four to eight developing merozoites, 2.34×2.7μm(2) in size, were observed within the first 24h of cultivation. An obvious peculiarity was found within the merozoite pellicle, as it was composed of the outer plasma membrane with underlying middle and inner membrane complexes. Further novel findings were vacuolization of the meront's residuum and extension of its outer pellicle, as parasitophorous vacuole-like membranes were also evident. The asexual reproduction of C. parvum was consistent with the developmental pattern of both eimerian coccidia and Arthrogregarinida (formerly Neogregarinida). The unique cell-free development of C. parvum described here, along with the establishment of meronts and merozoite formation, is the first such evidence obtained from in vitro cell-free culture at the ultrastructural level.
... Cryptosporidium drug discovery has been hampered by lack of an in vitro culture system that can continuously culture the parasite and the lack of genetic tools to construct transgenic reporter parasites that would greatly facilitate screening efforts (Sharling et al., 2010). Recent developments in the in vitro cultivation have revealed that Cryptosporidium can complete its life cycle in media devoid of host cells (Aldeyarbi and Karanis, 2014;Boxell et al., 2008;Hijjawi et al., 2004Hijjawi et al., , 2010Kartashev et al., 2009) and both cell and cell-free cultures have demonstrated that Cryptosporidium may not be an obligate intracellular parasite and can in fact multiply extracellularly (Borowski et al., 2010;Hijjawi et al., 2002;Huang et al., 2014;Karanis et al., 2008;Koh et al., 2013;Rosales et al., 2005). These findings may reflect the fact that Cryptosporidium is closely related to gregarine protozoa (Barta and Thompson, 2006;Bull et al., 1998;Carreno et al., 1999;Hijjawi et al., 2002;Leander et al., 2003;Rosales et al., 2005). ...
... Immune labeling of life cycle stages in HCT-8 cell cultures and in cell-free cultures has been previously described and has demonstrated that cell free stages bind the same antibodies as cell culture stages (Boxell et al., 2008;Edwards et al., 2012). More recently, all life cycle stages from cell-free culture have been described using electron microscopy (Aldeyarbi and Karanis, 2014). The aim of the present study was to further validate cell-free culture using scanning electron microscopy (SEM) to characterise life cycle stages in more detail and to compare gene expression in cell-free versus conventional cell culture using genes used in a previous study of gene expression in Cryptosporidium cell culture (Jakobi and Petry, 2006). ...
The sexual stages and new oocysts development of
Cryptosporidium parvum
were investigated in a cell-free culture system using transmission electron microscopy (TEM). Sexual development was extremely rapid after inoculation of oocysts into the medium. The process began within 1/2–12 h and was completed with new oocyst formation 120 h post-inoculation. The macrogamonts were bounded by two membranes and had amylopectin granules and two distinct types of wall-forming bodies. The microgamonts had a large nucleus showing lobe projections and condensation of chromatin, giving rise to peripherally budding microgametes. The microgametes contained a large area of granular substance containing groups of microtubules surrounding the electron-dense nucleus. In some instances, the dividing microgamy was observed in cell-free cultures with no preceding merogonic process. Fertilization was observed with the bullet-shaped microgamete penetrating an immature macrogamont at 24 and 216 h. The new thin- and thick-walled oocysts had a large residuum with polysaccharide granules and sporogony noted inside these oocysts. Novel immature four-layer walled thick oocysts with irregular knob-like protrusions on the outer layer resembling the immature
Eimeria
oocysts were also observed. The present study confirms the gametogony and sporogony of
C. parvum
in cell-free culture and describes their ultra-structure for the first time.
