INTRODUCTION
The importance of Cryptosporidium has risen significantly largely due to its clinical impact and wide global distribution. It is recognized as a potentially severe opportunistic infection in people suffering from AIDS and malnourished children that can result in death (Putignani and Menichella, Reference Putignani and Menichella2010). The first in vivo ultra-structural study of the asexual stages of Cryptosporidium was performed by Hampton and Rosario (Reference Hampton and Rosario1966); the sexual development stages were later demonstrated to include gametogony and sporogony by Vetterling et al. (Reference Vetterling, Takeuchi and Madden1971) and Barker and Carbonell (Reference Barker and Carbonell1974). Since those reports, sequential extensive studies have investigated the life cycle and fine structural features of Cryptosporidium development. Furthermore, researchers have attempted to cultivate Cryptosporidium in vitro since 1983. The in vitro fine structural details of asexual development were first reported in the HT29·74 cell line by Aji et al. (Reference Aji, Flanigan, Marshall, Kaetzel and Aikawa1991) and observations of sexual gamonts in cell lines were demonstrated by Buraud et al. (Reference Buraud, Forget, Favennec, Bizet, Gobert and Deluol1991) and Rosales et al. (Reference Rosales, Mascaro and Osuna1993). Some studies reported the development of Cryptosporidium in different cell-free culture systems using the only commercially available Sporo-Glo™ immunofluorescence antibody and confocal microscopy (Boxell et al. Reference Boxell, Hijjawi, Monis and Ryan2008; Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010; Koh et al. Reference Koh, Clode, Monis and Thompson2013, Reference Koh, Thompson, Edwards, Monis and Clode2014). However, these methods failed to detect stages residing within meronts (Koh et al. Reference Koh, Thompson, Edwards, Monis and Clode2014) or to distinguish between the different developmental stages with its limited ability to show internal ultra-structures.
To date, there have been few if any investigations on the development of the sexual phase; the same is true of the fertilization process in cell-free culture. This manuscript reports for the first time the ultra-structural development of macrogamonts, microgametes and new oocysts in an effort to gain additional insight into the gametogony, sporogony and developmental processes of the extracellular stages of Cryptosporidium parvum in cell-free culture. Due to the lack of available data regarding the ultra-structure of the in vitro stages of the axenic system, the results described are compared with ultra-structure studies using cell lines and in vivo inoculation experiments.
MATERIALS AND METHODS
Cryptosporidium oocysts and genotyping
The purified C. parvum oocysts used during this study were kindly provided by the Leipzig Parasitology Institute, Germany. According to the supplier, the oocysts were purified using the ether extraction and saturated NaCl flotation method. The purified oocysts were stored in 50 mL of phosphate buffered saline supplemented with amphotericin B (0·0025 mg mL−1) and penicillin/streptomycin (100 U mL−1 and 0·1 mg mL−1, respectively) at 4 °C. The species was confirmed by sequence analysis of the SSU rRNA as previously described (Xiao et al. Reference Xiao, Singh, Limor, Graczyk, Gradus and Lal2001).
Decontamination (bleaching) of oocysts and excystation
Purified C. parvum oocysts (~106 oocysts) were bleached before use by placing them in a 10% sodium hypochlorite solution for 30 min on ice at 4 °C. Afterwards, the suspension was washed and centrifuged at 5000 g for 3 min at room temperature. The oocysts were re-suspended in freshly prepared and filtered excystation medium containing 0·25 g of trypsin and 0·75 g of sodium taurocholate in 100 mL of RPMI-1640 medium and incubated at 37 °C for 30 min with mixing every 5 min. Subsequently, the suspensions were pelleted by centrifugation and resuspended in cultivation medium. As a control, oocysts were fixed directly in 2·5% glutaraldehyde after decontamination and processed for transmission electron microscopy (TEM) as described below.
Cell-free cultivation of Cryptosporidium
A 12-well culture plate (Greiner Bio-One, CELLSTAR; Belgium) was prepared with 1·5 mL of cell-free maintenance media [Express Five® Serum Free Medium (SFM); 1×; Gibco by life technologies; Germany] per well with and without the following supplements per 100 mL, as previously described by Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzo, Ryan, Olson, Cox, Monis and Thompson2004): 0·02 g bovine bile; 0·03 g L-glutamine; 0·3 g sodium bicarbonate; 875 µg ascorbic acid; 50 µg calcium pantothenate; 25 µg folic acid; 0·1 g glucose; 100 µg 4-aminobenzoic acid; 1% fetal calf serum (Invitrogen); 0·36 g HEPES buffer; 10 000 units penicillin; 10 mg streptomycin; and 25 µg amphotericin B. The wells were inoculated with a suspension of C. parvum excysted oocysts or oocysts without prior exposure to excystation. The cultures were incubated at 37 °C, 5% CO2 for 1/2, 2, 4, 6, 8, 12 and 24 h and up to 216 h. The wells were inspected daily for any contamination, and the spent media was exchanged with fresh 100% Express Five SFM every 2–3 days.
TEM specimen preparation and thin-sectioning
After cultivation, the parasites were pelleted and fixed with 2·5% glutaraldehyde for at least 24 h. C. parvum was concentrated by centrifugation and mixed with 2% low-melting-temperature agarose. After cooling, the agarose block was trimmed and fixed in 2·5% glutaraldehyde overnight at 4 °C. Then, the agarose pieces were stained with 1% osmium tetroxide for 3 h, dehydrated in an ethanol graded series and embedded in pure Epon using conventional procedures. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Zeiss EM-902 TEM at a voltage of 80 kV.
RESULTS
Serial TEM sections were exhaustively screened for the presence of several developmental stages of C. parvum in cell-free culture. Based on the TEM observations of the transformation of zoites into either male or female gamont stages, the elongated shape was usually maintained for a short period of time (Figs 1D, E and 3A, C). The characteristic organelles of these motile stages gradually disappeared and differentiated to sexual stages.
TEM of dividing microgamy and microgametes. (A) Microgamont (48 h) with 2 budding microgametes (arrows). (B, C) Section through microgamonts (120 h) and free in the medium (24 h); the nucleus has lobe projections (white arrow) and multiple budding compact nuclei (arrowheads). Note the extension of the outer pellicle (black arrow). (D) Extracellular microgamont (4 h) showing two bullet-shaped microgametes (arrowheads) with oblong electro-dense nuclei (Ni) and microtubules (Mt) and increased density of the nuclear chromatin (Nd). Note the ‘empty’ anterior large vacuole of rhoptry (asterisk) at the anterior end (AE). (E) Two gamonts in syzygy (1/2 h) joined together through the contact zone (long arrow). Note the cross-section of microgametes (arrowheads), the increased density of the nuclear chromatin, micronemes (Mn), and apical rings (three arrows). (F) Two associated extracellular microgamonts (48 h) with lobe projections of the nucleus (white arrow) and extension of the outer layer of the pellicle (asterisk). (G) Extracellular microgamont (12 h) with five compact nuclei near the periphery (arrowheads). (H) Microgamont at 2 h showing a number of compact nuclei (arrowheads). (I) Higher magnification of (E) shows the characteristic microtubules of microgametes near the nuclear envelope (Ne), microgamete nucleus, microgamete membrane (Mm) and inner and outer plasmalemma (Pl). (J) Detail of the inner bullet-microgamete near the nucleus presented in (F). (K) An enlargement of (G) showing the microtubules are in close proximity to the nuclear envelope and inner and outer plasmalemma. (L) Microgametes at high magnification in (H) consist of microtubules arranged in a circle close to the nuclear envelope. Bar = 1 µm (A, E, F, H), = 500 nm (G, B inset), and = 200 nm (B–D, I–L).
In the beginning of 1/2–2 h post-cultivation, the developmental stages of microgamonts within the cell-free medium (Fig. 1), within a residuum (Fig. 2) could be clearly differentiated. They measured 1·18 × 1·53 µm (range = 0·83–1. 7 × 1–2·6 µm; s.d. = ±0·19 × 0·35; n = 56 in the sectional plane) and were distinct from both the macrogamonts and meronts due to their peripheral high electron-dense compact nuclei. We identified two budding microgametes enveloped by a double membrane at the margin of the microgamont (Fig. 1A) and budding nuclei (3–10 out of 16) within the cytoplasm of microgamonts (Figs 1B, C and 2). Microgametes with characteristic compact nuclei and microtubules were also formed within the cytoplasm of the microgamonts (Fig. 1D–G, I–K), even though the cytoplasm was not relatively free of apical structures (i.e. apical rings, micronemes or ghost of rhoptry vacuole) (Fig. 1D and E).
Extracellular C. parvum microgamonts. (A, B) Sections through multinucleate organisms within the residuum (2 and 4 h) demonstrating nuclei (arrowheads) characteristic of early microgamonts. Note the condensation of chromatin at the nuclear membrane edge. (C) Three extracellular microgamonts (8 h) still attached to the residual body (RB) containing a number of budded condensed nuclei (arrowheads) within the mother cell. Note the lobe projection of the nuclei (arrow). Bar = 1 μm (B, C) and = 500 nm (A).
In the dividing microgamy, the nucleus exhibited peripherally condensed chromatin (Fig. 2B) with lobe projections (Figs 1B, C and 2C). These features were also noticed in Fig. 1D–F. Microgamete differentiation initiated with the formation of ‘beehive’ cytoplasmic projections close to the nuclei. Eventually, an additional outer plasmalemma layer of the outer and inner membranes and an internal microgamete membrane became apparent. Later, these membranes emigrated through the ruptured microgamont's pellicle. In cross-sections, we observed microtubules ~18 nm in diameter encircling the nuclear envelope of the electron-dense nucleus within the microgametes. The elongated/bullet-shaped microgametes were ~0·26 by 0·34 µm, possessed a compact nucleus 0·20 × 0·16 µm and were surrounded by a double-layered plasmalemma lying inside the microgamonts’ cytoplasm (Fig. 1D, F, J). Extracellular microgamonts paired up in caudo-caudal syzygy-like at the level of the nucleus (Fig. 1E, F, J), where occasionally a contact zone between the paired two gamonts was observed (Fig. 1E).
Beginning 12 h post-cultivation, we observed macrogamonts that were spherical to ovoid in shape, measured 1·09 (0·79–2·03) × 1·59 (1–2·44) μm (s.d. = ±0·27 × 0·4; n = 29 in the sectional plane) and were covered by two membranes (Fig. 3). In the early stages, the cytoplasm featured prominent amylopectin granules and lipid vacuoles (Fig. 3A and B). During the advanced stages, two types of wall forming bodies (WFB I and II) and amylopectin granules became visible at the periphery of macrogamonts’ cytoplasm. The ovoid WFB I consisted of uniform electron-dense contents with dark spots surrounded by a membrane (Fig. 3C), while WFB II was composed of slightly irregularly shaped, less dense material enclosed within a distinct membrane.
TEM of cell-free C. parvum macrogamonts. (A, B) Early extracellular macrogamonts (24 and 48 h) possess a large amylopectin granule (Am) and lipid body (Lb). (C) Macrogamont (12 h) showing high electron-dense WFB I with dark spots. (D) Mid-stage macrogamont (120 h) demonstrating WFB II at the periphery. Note the bundles of 3–4 longitudinal sub-pellicular microtubules (arrows). (E) Two early macrogamonts at 24 h with an eccentric nucleus and numerous accumulations of amylopectin granules. (F) Macrogamont at 120 h demonstrating WFB I and WFB II at the periphery. Bar = 500 nm (D, E), and = 200 nm (A–C, F).
Bullet-shaped microgametes penetrating immature macrogamonts were observed in the cell-free culture at 48 and 216 h; the microgametes were identified by their oblong nucleus and microtubules (Fig. 4A and B). Although the penetration was not total, the area beneath the contact site was associated with an electron-dense layer and was slightly dented. Microgametes were not observed completely inside the cytoplasm of these young macrogamonts; moreover, there was no exchange of nucleoplasm. The zygote or fertilized macrogamont was 1·5 × 2·9 µm and possessed a large number of amylopectin bodies (Fig. 4C). Oocyst wall formation commenced with the aggregation and fusion of each WFB I and WFB II into larger uniform irregularly shaped structures adjacent to the periphery. Breakup of the WFB I membranes with simultaneous reduction in the electron density of its contents (Fig. 5A) as well as branched intra-cytoplasmic tubules connecting the WFB II organelles to the periphery of the parasite (Fig. 8F) were noticed.
Fertilization and zygote in axenic culture. (A, B) Microgamete (Mic) attached to a young macrogamont (arrow) at 24 and 216 h. (C) Fertilized macrogamont or zygote containing numerous amylopectin granules, artefact (asterisk). Bar = 500 nm (C) and = 200 nm (A, B).
Early new oocyst formation after 120 h in cell-free culture (A) Electron micrograph showing early oocyst wall formation with the aggregates of WFBs. (B) TEM of non-sporulated TKW exhibiting a membrane-bounded residual body (RB) (arrowhead) with protein crystal inclusion (CI) and numerous amylopectin granules. (C) View of a thin-walled oocyst with developing sporozoites (S) and amylopectin granules with fine granular interior. Note the unit membrane surrounding the residual body (arrowhead). (D) Portion of the oocyst wall in (B) demonstrating L1–L4. Note the electron-pale-dense columnar projections on L1 and the electro-dense bulging structures (asterisk). (E) Section through the wall in (C) composed of 2 electron-dense layers with an electron-lucent layer in between. Note the lack of L4. Bar = 1 µm (B, C), = 500 nm (A), and = 200 nm (D, E).
Most of the parasitic stages observed 120 h post-cultivation were new oocysts measuring 3·5 (1·69–6·25) × 3·8 (1·77–7·35) μm in diameter (s.d. = ±0·7 × 0·8; n = 95 in the sectional plane). Three types of oocysts were present in the cell-free medium: typical thick-walled oocysts (TKW), thin-walled oocysts (Tw) and the developmental stages of thick immature oocysts (Figs 5–8). The fully sporulated TKW oocysts were easily identified by their distinctive wall and fine mesh of filamentous-like structures filling the newly formed oocysts, which later subsided along the surface of the inner L4 layer and disappeared. The oocysts were elliptical to nearly spherical in shape. Sporogony was noticed inside these oocysts resulting in the formation of 3–4 sporozoites within the amylopectin-rich residual body (Figs 6 and 7). The TKW oocysts varied in thickness from 36·41 to 161·2 (mean = 81·8; s.d. = ±17·4–28·8) nm and were composed of four layers: the thin outer electron-dense layer (L1) (mean 12·8 nm; range = 1·59–41·86; s.d. = ±7·7), the dark osmophilic electron-dense layer (L3) (mean 11·43 nm; range = 4·63–27·07; s.d. = ±10·9) that was interposed with a central electron-lucent layer (L2) (mean 8·79 nm; range = 2·87–29·30; s.d. = ±3·9) and a distinct inner thicker granular layer (L4) (mean 33·58 nm; range = 9·42–107·62; s.d. = ±13·45; n = 31 in the sectional plane) (Fig. 6). In contrast, non-sporulated TKW oocysts were distinguished as membrane-enclosed amylopectin-rich residual bodies that presented with irregular outer columnar projections on L1 and inner electro-dense bulging structures on L4 (Fig. 5B and D).
Sporulating C. parvum thick-walled oocysts (TKW) after 120 h in axenic medium. (A, B) Mature new oocysts with three to four sporozoites (S) and centrally located residual body. Note the mesh of filamentous-like structures filling the new oocysts. (C) New TKW oocysts with double sutures at 120 h. (D) Sporulated oocysts with four sporozoites and an amylopectin-rich residual body with finely stained granule contents. Note the outer portion of the wall (L1) appears to be split along the axis of the translucent layer (L2) (as indicated by asterisk in D and A). (E) Cross-section of a wall with four layers (L1–L4). (F) Shows closed suture (Su) with an inner electron-lucent band (LB), an outer electron-dense band (DB) on each side, a central electron-lucent band (CLB) and inferior swelling (asterisk). Bar = 2 µm (A–C), = 1 µm (D) and = 200 nm (E, F).
In vitro C. parvum excystation from new thick-walled oocysts 120 h post-cultivation. (A, B) Ultra-thin sections of partially excysted oocysts exhibiting longitudinal and transverse sections of fully differentiated sporozoites (S). (C–E) Sporozoites excysting from new TKW oocysts. Note the conoid (C), single rhoptry (R), apical rings (arrowheads), micronemes and dense granules (Dg). (F) This field of view shows a sporozoite that has been distorted by the inward curling of a portion of the wall. Bar = 2 µm (D, E) and = 1 µm (A–C, F).
Developing C. parvum thick immature oocysts after 120 h. (A) Cross-sections of early thick immature oocysts with indentation of the inner double cytoplasmic membrane (Cm) of the zygote (arrows). Notice the knob-like protrusion (Kp) of the outer L1 and the under-lying layers L2, L3 and L4 representing the middle, inner-middle and inner layers of the oocyst wall, respectively. (B) Oocyst showing amylopectin granules, lipid body and wall-forming bodies. (C, D) Immature thick oocysts with a large nucleus and nucleolus (Nu). Note the separation of the cytoplasmic membrane from the inner wall (arrow). (E) A longitudinal section of an early conoid (arrow). (F) Intra-cytoplasmic tubules (arrow) of the zygote connecting the remnants of WFB II to the new forming oocyst layer. Bar = 1 µm (B, D), = 500 nm (A, C) and = 200 nm (E, F).
The sporulating thin-walled oocysts that are believed to play a role in autoinfection possessed a cytoplasmic membrane separating the zygote from the oocyst, which contained one curved longitudinal and cross-sectional sporozoit (Fig. 5C). The lipid body and amylopectin granules were recognized also within the membrane-bound residual body. In perpendicular sections, we observed that the thin wall was trilaminar and 27 (24–32·1; s.d. = ±0.6) nm thick with a thin outer layer (L1) 6·18 (5·41–6·51; s.d. = ±0·16) nm thick, followed by an L2 electron-lucent layer 10·21 (8·91–11·31; s.d. = ±0·16) nm thick and an electron-dense inner layer (L3) 12·18 (10·04–14·09; s.d. = ±0.6) nm thick (n = 3 in the sectional plane); these structures lacked the innermost layer (L4) of the TKW oocyst (Fig. 5E).
A fused suture was found directly below the outer 2 layers of the wall of the newly formed TKW oocysts. A central band (CLB) ran through the suture, beneath which an inferior amorphous swelling, inner-electron lucent band (LB) and outer dense band (DB) were observed on each side (Fig. 6F). During excystation, inward curling at the margins of the bands of the inner layer coincided with degeneration of an inferior substance. This persistent curling ultimately caused the outer layers to break and move apart, with internal spiralling of the suture's border up to 1/4 of the oocyst's circumference; this phenomenon allowed the sporozoites to escape. Single or double sutures were found at the poles (Fig. 6). The excysted sporozoites were banana-shaped and measured 1·6 (1·04–3·6) × 2·2 (1·32–6·29) μm (s.d. = ±0·7 × 1·04; n = 20 in the sectional plane). Each sporozoite had an apical complex, nucleus, single rhoptry, electron-dense bodies and was surrounded by a pellicle (Fig. 7).
The developing thick immature oocysts shown in Fig. 8 measured 3·56 (1·85–6·25) × 3·85 (2·4–6·79) μm (s.d. = ±0·7 × 1·04; n = 36 in the sectional plane). The zygote was surrounded by a festooned cytoplasmic membrane that separated it from the inner layer of the enveloped wall. This wall was composed of four layers (L1–L4) progressing from the inside to the outside: a relatively homogeneous thin electron-dense L4 inner layer (16·75–31·17 nm thick), electron-pale L3 middle layer (94·03–129·03 nm thick), a more electron-translucent L2 layer (43·03–106·59 nm thick) and a relatively electron-dense L1 outer layer with knob-like projections that varied in thickness from 49·71 to 151·60 nm. The thickness of the L2, L3 and L4 layers was constant throughout most of the oocyst wall. The electron density of L3 and L4 increased during maturation, whereas the outermost layer L1 disintegrated during the late stages, leaving a very thin osmophilic layer. Immature thick oocysts displayed a large oval-rounded shaped nucleus (~1·5 µm in diameter) that was located centrally (Fig. 8A) or eccentrically (Fig. 8C, and D) and enclosed an electron-dense nucleoplasm. The cytoplasm contained densely packed amylopectin granules, lipid granules, a number of clearly electron translucent vacuoles and remnants of WFB I, WFB II.
DISCUSSION
After ~105 years, Tyzzer's statement on Cryptosporidium that ‘This parasite undergoes the greater part of its development either attached to the surface of the epithelium or free in the lumen of the gastric glands so that its habit of life is similar in many respects to that of parasites of the order Gregarinida, of which no representative has been described hitherto in vertebrates’ (Tyzzer, Reference Tyzzer1910) was confirmed by the subsequent ultra-structural, immunofluorescent and phylogenomic studies that demonstrated the peculiarities of Cryptosporidium, thereby distinguishing it from other coccidians which were reviewed in detail by Karanis and Aldeyarbi (Reference Karanis and Aldeyarbi2011).
In the present study, the development of microgametes as nuclear protrusions at the gamont surface was similar to the reports of in vivo ultrastructural studies (Goebel and Braendler, Reference Goebel and Braendler1982; Ostrovska and Paperna, Reference Ostrovska and Paperna1990; Cheadle et al. Reference Cheadle, Toivio-Kinnucan and Blagburn1999; Dubey et al. Reference Dubey, Markovits and Killary2002; Melicherová et al. Reference Melicherová, Ilgová, Kváč, Sak, Koudela and Valigurová2014). This classical process was identical across the coccidia with the exception of the sarcosporidia and haemosporidia (Vetterling et al. Reference Vetterling, Pacheco and Fayer1973) in which the nuclei most likely emerge during the initial proliferative phase with repeated nuclear divisions, followed by marked peripheral condensation of the chromatin (Ferguson, Reference Ferguson2009). Then, the microgametes reach the periphery of the gamont and form by budding from a portion of the cytoplasm (Goebel and Braendler, Reference Goebel and Braendler1982; Ferguson, Reference Ferguson2009). However, the description of the new type of microgamete formation presented here in this study can be applied to other coccidians. First, the nuclear chromatin of the microgamonts increased and the nucleus became lobulated with an intact nuclear envelope. This step was previously described for Sarcocystis by Vetterling et al. (Reference Vetterling, Pacheco and Fayer1973) and Mehlhorn and Heydorn (Reference Mehlhorn and Heydorn1979) and for the haemosporidia Parahaemoproteus by Desser (Reference Desser1972), where microgametes are formed by means of splitting of the nuclear substance. In contrast, in Eimeria many nuclei exist before the microgamete's formation (Scholtyseck et al. Reference Scholtyseck, Mehlhorn and Hammond1972). The microgametes were similar to those reported by others (Bird and Smith, Reference Bird and Smith1980; Goebel and Braendler, Reference Goebel and Braendler1982; Landsberg and Paperna, Reference Landsberg and Paperna1986; Uni et al. Reference Uni, Iseki, Maekawa, Moriya and Takada1987; Ostrovska and Paperna, Reference Ostrovska and Paperna1990; Melicherová et al. Reference Melicherová, Ilgová, Kváč, Sak, Koudela and Valigurová2014), except that they were found within the cytoplasm of microgamonts and the apical cap and adhesion zone were not identified.
In the present study, the zoites developed directly to sexual stages during the early hours post-inoculation, and a merogonic process was not recognized in some instances. This is the first time for such a process to be described for Cryptosporidium; however, a similar process was previously described in Sarcocystis, where development of the sexual stages in human tissue culture was not preceded by merogony (Mehlhorn and Heydorn, Reference Mehlhorn and Heydorn1979). This finding also supports the earlier observations in Protococcidia, where the sexual stages arise directly from the transformation of sporozoites due to the lack of a merogonic process (Scholtyseck et al. Reference Scholtyseck, Mehlhorn and Hammond1972). Indeed, the rate of development of the sexual stages was quite rapid, in agreement with the results of Vetterling et al. (Reference Vetterling, Pacheco and Fayer1973) and Mehlhorn and Heydorn (Reference Mehlhorn and Heydorn1979) for Sarcocystis sp. and Current and Haynes (Reference Current and Haynes1984) and Yang et al. (Reference Yang, Healey, Du and Zhang1996) for Cryptosporidium sp. as the gametogony was distinguishable by 12–24 and 48 h in cell culture via Nomarski microscopy and TEM, respectively. Moreover, Buraud et al. (Reference Buraud, Forget, Favennec, Bizet, Gobert and Deluol1991) recognized the fine structure of macrogametes at 120 h in Caco-2 cells.
The pairing of the extracellular microgametes by their posterior ends resembles the caudo-caudal syzygy of the archigregarines Selenidiidae Selenidium pendula, Selenidium hollandei (Desportes and Schrével, Reference Desportes, Schrével, Desportes and Schrével2013) and Selenidium pennatum (Kuvardina and Simdyanov, Reference Kuvardina and Simdyanov2002). In contrast, the gamonts of the Mattesia grandis of Arthrogregarinida (formerly Neogregarinida) and some species of eugregarines come to apposition by their apical region (fronto-frontal syzygy) as Urosporoidea (formerly eugregarines) and Gonospora sp. (Vavra and McLaughlin, Reference Vavra and McLaughlin1970; Desportes and Schrével, Reference Desportes, Schrével, Desportes and Schrével2013). The exact identity of such pairing in Cryptosporidium remains unknown, but we suggest that this could be due to affinity between Cryptosporidium stages/gamonts rather than biological purposes as in gregarines (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2014, Reference Aldeyarbi and Karanis2016a ).
In this study, the remnants of apical complexes including the ghosts of rhoptries were identified in early microgamonts. Such empty vacuoles were visualized in the intracellular stages of Toxoplasma (Porchet-Hennere and Nicolas, Reference Porchet-Hennere and Nicolas1983) and Plasmodium berghei yoeli in cell culture (Ladda et al. Reference Ladda, Aikawa and Sprinz2001) and in extracellular Gregarina steini trophozoites (Valigurová et al. Reference Valigurová, Hofmannová, Koudela and Vávra2007), but have never been reported in early Cryptosporidium microgamonts. Interestingly, the apical complex was also preserved in the syzygy associates for a long time after cleavage of the nucleus in the archigregarines S. pennatum gamonts (Kuvardina and Simdyanov, Reference Kuvardina and Simdyanov2002). The extension of the microgamont's pellicle appeared to play a role in the dispersion of microgametes after its rupture and/or the parasite's adjustment for nutrient acquisition through increasing its surface area, as reported in other axenic Cryptosporidium asexual and gregarine-like stages (Aldeyarbi and Karanis Reference Aldeyarbi and Karanis2014, Reference Aldeyarbi and Karanis2016a , Reference Aldeyarbi and Karanis b ).
There is no nuclear division during macrogamont development; however, as the parasite grows, large numbers of lipid droplets and polysaccharide granules are synthesized and stored in the cytoplasm (Ferguson, Reference Ferguson2009). Similarly, this feature was also detected in the early macrogamonts. All of the other fine features described here including the presence of amylopectin granules and WFBs were documented in Cryptosporidium and related coccidians in earlier in vivo and in vitro reports (Vetterling et al. Reference Vetterling, Takeuchi and Madden1971; Barker and Carbonell, Reference Barker and Carbonell1974; Bird and Smith, Reference Bird and Smith1980; Goebel and Braendler, Reference Goebel and Braendler1982; Lefkowitch et al. Reference Lefkowitch, Krumholz, Feng-Chen, Griffin, Despommier and Brasitus1984; Landsberg and Paperna, Reference Landsberg and Paperna1986; Ostrovska and Paperna, Reference Ostrovska and Paperna1990; Buraud et al. Reference Buraud, Forget, Favennec, Bizet, Gobert and Deluol1991; Paperna and Vilenkin, Reference Paperna and Vilenkin1996; Yang et al. Reference Yang, Healey, Du and Zhang1996; Spano et al. Reference Spano, Puri, Ranucci, Putignani and Crisanti1997; Aydin, Reference Aydin1999). Moreover, the presence of amylopectin granules and WFBs has been also described in related coccidians (Scholtyseck et al. Reference Scholtyseck, Mehlhorn and Hammond1971; Speer and Duszynski, Reference Speer and Duszynski1975; Varghese, Reference Varghese1975; Sibert and Speer, Reference Sibert and Speer1980; Dubey et al. Reference Dubey, Lindsay and Speer1998).
The unexpected sexual development of the zoites within the cell-free culture medium could represent a type of adaption or a protective mechanism by which Cryptosporidium can enhance its proliferative potential while still in situ through merogony or gamogony. Comparable behaviour was reported via TEM by Speer and Dubey (Reference Speer and Dubey2001) for Sarcocystis neurona, where most of the merozoites remained within the host cell until they had primarily developed and initiated another generation of merogony. To the best of our knowledge, such observations have not been previously reported elsewhere for Cryptosporidium, and further studies are required to gain more insight into this phenomenon.
The fertilization process identified here was similar to that described by Goebel and Braendler (Reference Goebel and Braendler1982) for Cryptosporidium sp. in the mouse small intestine, where the penetration of the microgametes into macrogamonts and the deposition of electro-dense material beneath the contact site were documented. In other Cryptosporidium in vivo studies, microgametes were reported to be connected to the parasitophorous vacuole of the macrogamete through the association of a channel-like structure (Aydin, Reference Aydin1999) or infiltrated osmophilic material below the contact site (Goebel and Braendler, Reference Goebel and Braendler1982). The zygote or fertilized macrogamonts presented here were similar to those described in previous Cryptosporidium in vivo studies (Bird and Smith, Reference Bird and Smith1980; Aydin, Reference Aydin1999). A multinucleated zygote of C. parvum in HCT-8 cells at 90 h was documented by Rosales et al. (Reference Rosales, Cordón, Moreno, Sánchez and Mascaró2005) using TEM. Our data demonstrate for the first time the fertilization process and zygote formation in cell-free culture. Although little is known about fertilization, it has been established that after entry of the microgamete into the macrogamont cytoplasm it either passes its nuclear contents as demonstrated for Plasmodium yoelii and Sarcocystis (Sinden et al. Reference Sinden, Canning and Spain1976; Entzeroth, Reference Entzeroth1982) or enters the macrogamont's nucleus; in the latter case, only the 2N nucleus of the fertilized macrogamont undergoes meiosis to produce four 1N sporozoites (Fayer, Reference Fayer, Fayer and Xiao2008). Our data for sporogony are in agreement with previous observations that suggested that C. parvum was most numerous from 72 to 120 h in different cell cultures via TEM (Rosales et al. Reference Rosales, Mascaro and Osuna1993; Yang et al. Reference Yang, Healey, Du and Zhang1996) or Nomarski interference contrast images (Current and Haynes, Reference Current and Haynes1984; Buraud et al. Reference Buraud, Forget, Favennec, Bizet, Gobert and Deluol1991). In contrast, the fine structure of the C. muris immature and sporulated oocysts was recognized at 24 and 48 h in AGS cells, respectively (Choi et al. Reference Choi, Hong, Chai, Park and Yu2004). In the present study, both WFB I and WFB II assumed a more peripheral position and aggregated into uniform irregular structures with intra-connection of the WFB II organelles to the periphery. However, the exact sequential process by which these two granules form the wall has not been identified. Remarkably, a direct correlation between the inner layer of the Cryptosporidium oocyst wall, WFB I and the Cryptosporidium oocyst wall protein 1 was suggested (Spano et al. Reference Spano, Puri, Ranucci, Putignani and Crisanti1997), in contrast to ultra-structural observations relating WFB I to the formation of the outer oocyst wall layer in other apicomplexans (Belli et al. Reference Belli, Smith and Ferguson2006). Homologs of this cysteine-rich oocyst wall protein were also found in other apicomplexan parasites, such as Toxoplasma gondii (Templeton et al. Reference Templeton, Lancto, Vigdorovich, Liu, London, Hadsall and Abrahamsen2004) and Gregarina niphandrodes (Omoto et al. Reference Omoto, Toso, Tang and Sibley2004). The presence of amylopectin granules with finely stained natural content within the developing oocysts shown by our data is supported by other studies on C. parvum (Harris et al. Reference Harris, Adrian and Petry2004) and other coccidians (Ferguson et al. Reference Ferguson, Birch-Andersen, Hutchison and Siim1977). The three electron-dense layers with the intermediate electron-translucent layer of the TKW oocysts identified here are identical to micrographs observed in previous studies on purified oocyst walls viewed by negative staining or thin sectioning EM (Reduker et al. Reference Reduker, Speer and Blixt1985a ; Harris and Petry, Reference Harris and Petry1999; Jenkins et al. Reference Jenkins, Eaglesham, Anthony, Kachlany, Bowman and Ghiorse2010). The minimum measured wall thickness was almost similar to the thickness measured by Reduker et al. (Reference Reduker, Speer and Blixt1985a ) (31·6 nm), but was nearly 3/4 of the thickness measured by Jenkins et al. (Reference Jenkins, Eaglesham, Anthony, Kachlany, Bowman and Ghiorse2010) (50 nm) and was two- to three-times the maximum measured wall thickness reported in both studies (72·9 and 58·2 nm, respectively). Based on the descriptions in other studies, the thin wall could be interpreted as a single unit membrane (Current and Reese, Reference Current and Reese1986; Aydin, Reference Aydin1999) as a result of an anomaly in oocyst formation due the lack of the characteristic wall-forming bodies (Fayer, Reference Fayer, Fayer and Xiao2008) or could represent a misinterpretation of a detached parasitophorous sac containing stages (Melicherová et al. Reference Melicherová, Ilgová, Kváč, Sak, Koudela and Valigurová2014). The trilaminar layers of the thin-walled oocysts described in the present study correspond to the observations in other related species, where the most inner thick layer was missing in the C. baileyi thin-walled oocyst type (Cheadle et al. Reference Cheadle, Toivio-Kinnucan and Blagburn1999). This is the first time such an ultra-structural difference between TKW and Tw oocysts has been reported for C. parvum. The reported filamentous array on the inner surface of the oocysts via negative staining EM by Harris and Petry (Reference Harris and Petry1999) could explain the mesh of filamentous-like structures observed in the newly formed oocysts. Indeed, this filamentous array can be greatly depleted by digestion with proteinase K and trypsin (Harris and Petry, Reference Harris and Petry1999). Hence, this array could be unnoticeable in TEM micrographs after the use of trypsin in the excystation medium. The varying electro-dense bulging structures noticed on the inner layer were similar to those structures identified by Reduker et al. (Reference Reduker, Speer and Blixt1985b ) using scanning electron microscope.
The fine structural analysis of the suture described here in the newly formed oocysts confirms previous reports by others for C. parvum (Reduker et al. Reference Reduker, Speer and Blixt1985a ; Jenkins et al. Reference Jenkins, Eaglesham, Anthony, Kachlany, Bowman and Ghiorse2010), C. muris (Uni et al. Reference Uni, Iseki, Maekawa, Moriya and Takada1987; Dubey et al. Reference Dubey, Markovits and Killary2002) and Toxoplasma (Ferguson et al. Reference Ferguson, Birch-Andersen, Siim and Hutchison1979) in terms of its location and the arrangement of the electron-lucent and electron-dense bands. The oocyst wall spanned up to 1/4 of the circumference, which was comparable with findings reported by Reduker et al. (Reference Reduker, Speer and Blixt1985b ) (1/3–1/2). Interestingly, the double suture identified here was formerly reported several years ago by Ostrovska and Paperna (Reference Ostrovska and Paperna1990) in cryptosporidiosis of a lizard (Fig. 32 in their publication). The frequent separations in the electron-translucent layer of the oocysts identified in the present micrographs were similarly noted by Jenkins et al. (Reference Jenkins, Eaglesham, Anthony, Kachlany, Bowman and Ghiorse2010) and reflect a weak point in this layer.
The early developmental stages of the thick immature oocysts have not previously been evident in the very few published electron micrographs of Cryptosporidium. However, they resemble the immature oocysts of Eimeria magna reported by Bouchet and Boulard (Reference Bouchet and Boulard1991) (Figs 1–3 in their publication), the sporulated oocysts of Eimeria punctata reported by Shah (Reference Shah1963) (Fig. 3 in their publication) and the sporocysts of Eimeria callospermophili reported by Roberts et al. (Reference Roberts, Speer and Hammond1970) (Figs 12–15 in their publication) in that they have conspicuous pits on the outer layer and a residual body containing vacuoles and lipoid granules but lack a stieda body and substiedal body. The presence of these types of oocysts in Cryptosporidium indicates that while they are unique, they still share some features with the Apicomplexans.
Kar et al. (Reference Kar, Daugschies, Cakmak, Yilmazer, Dittmar and Bangoura2011) reported that pre-treatment with sodium hypochlorite markedly enhanced the excystation of C. parvum oocysts. This result is in agreement with our findings that empty or partially empty oocysts with only a residual body could be identified in trials where the excystation phase was omitted (data not shown) after decontamination with hypochlorite solution. This finding supports the view that the addition of trypsin does not increase the amount of sporozoites that excyst (Rosales et al. Reference Rosales, Mascaro and Osuna1993). Moreover, it reflects the ability of sodium hypochlorite (Smith et al. Reference Smith, Rosely and Grimason2005; Kar et al. Reference Kar, Daugschies, Cakmak, Yilmazer, Dittmar and Bangoura2011) and parasite-derived triggers such as cysteine and serine proteases (Borowski et al. Reference Borowski, Clode and Thompson2008; Clode et al. Reference Clode, Koh and Thompson2015) to enhance excystation. This finding was clearly justified based on observations of sporozoites released from the newly formed oocysts and supports the view of Arrowood (Reference Arrowood2002), who stated that ‘excystation will often proceed in the cell culture medium soon after inoculation onto host cells, even without specific treatment to trigger it’. Different levels of excystation were exhibited because not all of the zoites were released at the same rate or time, and some lazy sporozoites could remain unexcysted for several days. These differences in excystation were also described by several authors for Cryptosporidium in in vitro (Campbell et al. Reference Campbell, Robertson and Smith1992; Robertson et al. Reference Robertson, Campbell and Smith1993; Smith et al. Reference Smith, Rosely and Grimason2005) and cell-free culture studies (Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010). There does not appear to be a fixed hierarchy of triggers or synergism, even though temperature elevation to 37 °C seems to be essential for excystation as previously reported (Smith et al. Reference Smith, Rosely and Grimason2005). Culture medium supplements did not greatly enhance parasite development in vitro because the same life stages were observed at similar intervals compared with trials without supplements.
In light of the much recent morphological and sequence tree evidence; Cryptosporidium was transferred from Coccidia to class Gregarinomorphea as new subclass Cryptogregaria (Cavalier-Smith, Reference Cavalier-Smith2014). By combining the present evidence and future evolutionary and phylogenetic studies, we can accomplish a re-evaluation of the life cycle and gain clearer insight into the position of Cryptosporidium within the Apicomplexa phylum. The present findings will not only provide a means for the rapid evaluation of potentially useful prophylactic and therapeutic compounds, but will also provide the opportunity to study its biological development, metabolism and behaviour.
ACKNOWLEDGEMENTS
This paper is a part of the doctoral thesis of Hebatalla Aldeyarbi through a PhD scholarship funded by the Deutscher Akademischer Austauschdienst (DAAD) and the Egyptian Ministry of Higher Education and Research. We acknowledge the skilled technical assistance of Mrs. Eveline Janssen and Mr. Christian Hoffmann. Electron microscope facilities were made available by Prof. Wolfram F. Neiss, Center of Anatomy, Cologne University, Germany.
FINANCIAL SUPPORT
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
