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Morphological characterization of
Cryptosporidium parvum life-cycle stages in an
in vitro model system
Borowski, H.; Thompson, R.C.A.; Armstrong, T.; et.al.
https://researchportal.murdoch.edu.au/esploro/outputs/journalArticle/Morphological-characterization-of-Cryptosporidium-parvum-life-cycle/991005
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Borowski, H., Thompson, R. C. A., Armstrong, T., & Clode, P. L. (2010). Morphological characterization of
Cryptosporidium parvum life-cycle stages in an in vitro model system. Parasitology, 137(01), 13–26.
https://doi.org/10.1017/S0031182009990837
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 13

Morphological characterization of Cryptosporidium parvum

life-cycle stages in an in vitro model system

H. BOROWSKI 1, R. C. A. THOMPSON 1*, T. ARMSTRONG 1 and P. L. CLODE 2

1
WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Veterinary and Biomedical Sciences,
Murdoch University, South Street, Murdoch, WA 6150, Australia
2
Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, 35 Stirling Hwy, Crawley,
WA 6009, Australia

(Received 30 January 2009; revised 25 May, 15 June and 19 June 2009; accepted 22 June 2009; first published online 20 August 2009)

SUMMARY

Cryptosporidium parvum is a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the
potential to cause severe enteric disease. We describe the complete life cycle of C. parvum in an in vitro system. Infected
cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional
data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well
as determining their time of occurrence after inoculation. Numerous stages of C. parvum and their behaviour have been
visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host
interactions and the effect of C. parvum on host cells were also visualized. An improved understanding of the parasite’s
biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.

Key words: Cryptosporidium parvum, morphology, host cell interaction, phylogenetic affinity, gregarines, electron
microscopy.

INTRODUCTION phylogenomic analysis has since revealed that

Cryptosporidium is most closely related to gregarines
Cryptosporidium is a protozoan enteric parasite of
(Barta and Thompson, 2006). Cryptosporidium shares
humans and other vertebrates (Fayer et al. 1997).
many features in common with gregarines, including
Numerous Cryptosporidium species have been de-
an extracytoplasmic location and connection to the
scribed (Smith et al. 2005) most of which are specific
host cell via a myzocytosis-like feeding mechanism
to their vertebrate host. The species C. parvum is of
(Barta and Thompson, 2006). The primary differ-
medical and economic relevance as it affects both
ence between these two groups is that Crypto-
humans and cattle with its primary site of infection
sporidium induces the host cell to overlay it with the
being the gastrointestinal tract. It affects the epi-
host cell apical membrane (Barta and Thompson,
thelial lining of the ileum, resulting in self-limiting
2006 ; Butaeva et al. 2006). The parasite appears on
diarrhoea in immunocompetent individuals or in
the surface of cells, residing in a parasitophorous
life-threatening diarrhoeal diseases in immunocom-
vacuole (PV) between the cytoplasmic membrane
promised individuals.
and the apical membrane (Huang et al. 2004).
Belonging to the phylum of apicomplexan para-
Critically, the mechanisms of Cryptosporidium
sites, Cryptosporidium shares common life-cycle
pathogenesis are not fully understood, but both
features and morphological characteristics with
parasite stimuli as well as host immune responses are
other members of this phylum (Tetley et al. 1998).
thought to play critical roles (Barta and Thompson,
Initially, Cryptosporidium was categorized as a cocci-
2006). The life cycle and the mechanisms of infection
dian parasite (Levine, 1988). However, more recent
by Cryptosporidium have recently been reviewed
studies show that Cryptosporidium lacks key mor-
in detail by Smith et al. (2005) and Borowski et al.
phological characteristics of coccidians and is insen-
(2008). Importantly, previous studies by Hijjawi
sitive to anti-coccidial agents (O’Donoghue, 1995 ;
et al. (2001, 2004) described the life cycle of C. par-
Fayer et al. 1997 ; Carreno et al. 1999). Further
vum in vitro, using light microscopy while more
recent studies by Valigurova et al. (2008) described
the morphology of various life-cycle stages of 2 dif-
* Corresponding author : WHO Collaborating Centre ferent Cryptosporidium species from mice and toads
for the Molecular Epidemiology of Parasitic Infections, in vivo using electron microscopy.
Veterinary and Biomedical Sciences, Murdoch University,
South Street, Murdoch, WA 6150, Australia. Tel : +(08) The aim of this study was to expand on this earlier
9360 2466. Fax : +(08) 9360 6285. E-mail : a.thompson@ work and to gain a better understanding of the
murdoch.edu.au biology and relationship with host cells of the

Parasitology (2010), 137, 13–26. f Cambridge University Press 2009

doi:10.1017/S0031182009990837 Printed in the United Kingdom
H. Borowski and others 14

economically and medically important species, investigate extracellular C. parvum stages present
C. parvum. In this study the human ileocecal epi- within the supernatant of infected cells, medium
thelial cell line HCT-8 was used as an in vitro model from cells was aspirated and added to an equal
to monitor the developmental process of C. parvum volume of 5 % glutaraldehyde in 2rPBS. Cellular
and to study the effects upon target cells. Crypto- material within this supernatant was subsequently
sporidium was observed to proliferate in our culture attached to poly-L-lysine coated glass cover-slips
system for 5 days. Hence, infected cells were moni- for SEM investigation by applying several drops
tored for this period, with data obtained using of concentrated supernatant and incubating for
scanning (SEM) and transmission (TEM) electron 20 min.
microscopy. From this, a more complete life cycle All samples were post-fixed in 1 % OsO4 in PBS,
of C. parvum has been visualized and the behaviour and then dehydrated in a graded series of ethanols
and morphological characteristics of numerous life- using a Pelco Biowave Microwave Processor.
cycle stages described for the first time with the aid Samples destined for SEM were then critical-point
of SEM. dried, mounted on stubs with carbon tabs, and
coated with 3 nm platinum for high-resolution
imaging. Samples destined for TEM were infil-
MATERIALS AND METHODS
trated and embedded in Spurr’s Resin. Thermonox
Cell culture cover-slips were removed under liquid nitrogen,
and samples re-embedded. Sections, approximately
The C. parvum cattle isolate used during this
100 nm thick, were cut on a diamond knife and
study was originally obtained from the Institute of
mounted on copper grids.
Parasitology, University of Zurich. Oocysts were
subsequently passaged through, and purified from,
infected ARC/Swiss mice as described by Meloni Imaging
and Thompson (1996). For routine passaging, SEM images were acquired at 3 kV using the in
HCT-8 cells were cultured in RPMI medium with lens secondary electron detector, on a Zeiss 1555VP
2 g Lx1 sodium bicarbonate, 0.3g Lx1 L-glutamine, field emission SEM. TEM sections were viewed
3.574 g Lx1 HEPES buffer (15 mmol Lx1) and 10 % unstained at 120 kV using a JEOL 2100 TEM.
fetal calf serum (FCS) at pH 7.4, 37 xC with 5 % CO2. Images were digitally acquired with a Gatan SC1000
ORIUS digital camera.
Pre-treatment of oocysts
C. parvum oocysts were bleached with 200 ml of RESULTS AND DISCUSSION

household bleach in 10 ml of water for 30 min at All recognized C. parvum life-cycle stages (Table 1)
room temperature (RT). Sterilized oocysts were were observed on the surface of epithelial cells or in
inoculated into excystation medium (0.5 % trypsin, the supernatant. Small trophozoites (<1 mm) were
pH of 2.5) for 30 min at 37 xC. Excysted oocysts were observed as early as 6 h post-inoculation with well-
resuspended in maintenance medium consisting of distinguishable meronts I and free merozoites type I
the RPMI medium described above plus 3 g Lx1 being observed after 24 h. This implies that oocyst
sodium bicarbonate, 0.2 g Lx1 bovine salt, 1 g Lx1 excystation and sporozoite invasion must have
glucose, 250 mg Lx1 folic acid, 1 mg Lx1 4 amino occurred immediately post-inoculation. Consistent
benzoic acid, 500 mg Lx1 calcium pentothenate, with this, previous studies by Forney et al. (1999),
8.75 mg Lx1 ascorbic acid and 1 % FCS. observed sporozoites invading host cells as early
as 5 min post-inoculation at the point of sporozoite
Cell line infection and cell-free culture emergence from the oocyst. Subsequent infection
increased over the next 2–3 days as a result of ongoing
Twenty-four h prior to an infection of cells with oocyst excystation, and the ability of C. parvum
C. parvum, HCT-8 cells were plated onto thermonox to replicate asexually. After 2 days post-infection,
cover slips in 24-well plates. Each cell was then only inoculated oocysts, sporozoites, trophozoites,
infected with C. parvum pre-treated oocysts meronts I and merozoites type I were observed in
(15 000 per cm2) in 1 ml of maintenance medium and culture. After 3 days, meronts II as well as mero-
maintained at 37 xC with 5 % CO2. Infected cultures zoites type II were seen, while gametocytes were
were sampled and processed for microscopy at 6 h, not observed until 4 days post-inoculation.
7 h, 24 h, 48 h, 72 h, 96 h and 120 h post-inoculation. This timeline of Cryptosporidium development
correlates with previous light microscopic data from
in vitro cultures of C. parvum (Hijjawi et al. 2001,
Sample preparation for electron microscopy
2002, 2004), as well as electron microscopy studies
Cover-slips with adherent cells were fixed in of Cryptosporidium sp. ‘ toad ’ and C. muris from
2.5 % glutaraldehyde in 1rPBS. Additionally, to experimentally infected toads and mice respectively
Morphological characterization of C. parvum in vitro 15

Table 1. Distinguishing characteristics of Cryptosporidium life-cycle stages

Time
(h) Stage Size Morphological Feature Fig.

0 Oocyst 5r7 mm Ovular, smooth surface with cleft for 1A

sporozoite release
>24 Excysted oocyst 5r7 mm Perforated surface 1B
>3 Sporozoite 5r0.5 mm Rough surface, pointed apical region 1B–D
(elongated when in proximity to host cells),
rounded posterior region
>6 Early trophozoite <1 mm Smooth surface formed by the host cell 1E
apical membrane, hood like shape
>24 Trophozoite 2.5 mm Epicellular, smooth surface, electron dense 2A–G
band, feeder-organelle, PV, cytoplasmic
granulation, hood like shape
>24 Trophozoite — Merging of apical membranes 2D–G
clusters engulfing individual parasites
>24 Meronts I 1.5 mm Epicellular, smooth surface 5A,B
>24 Merozoites 0.4r1 mm Rod like shape, pointed 5E–H
Type I apical region, rough surface
>72 Meronts II 3.5 mm Epicellular, smooth thick membrane 6A
>72 Merozoites 0.5–1 mm Round, rough surface 6A–C
Type II
>96 Microgamont 2r2 mm Extracellular, densely packed with 8A–C
microgametes
>96 Microgamete 0.1 mm Spherical, rough surface 8A–C
>96 Macrogamont 4r5 mm Extracellular, ovular, rough surface 7A–C
48 Extracellular 2 mm Forms within parent stage, rough surface 9A
trophozoite
96 Extracellular <2 mm Round, extracellular 9B
meront accumulation of trophozoites

(Valigurova et al. 2008). This consistency with an begin to express the surface lectins needed for host
in vivo system confirms the validity of our in vitro cell adherence until after extended exposure to host
model system of C. parvum infection. cells or simply until a certain progression of time
In addition to the widely accepted life-cycle stages, after excystation stimuli are initiated. The outer
we observed stages that are not commonly reported membrane of excysted oocysts appeared rough
or known to date (Table 1). Such stages include (Fig. 1B). This is probably due to membrane per-
trophozoites that formed within parent stages with- foration during the excystation processes.
out host cell invasion, and extracellular accumu- Sporozoites observed in this study measured about
lations of trophozoites. 5 mmr0.5 mm and showed well-defined apical ends
(Fig. 1B–D). The hatching sporozoite shown in
Fig. 1B as well as the sporozoite on the surface of
Oocyst excystation and sporozoite host cell invasion
the host cell in Fig. 1D, both showed an enlarged
Following inoculation, intact C. parvum oocysts posterior region and a well-defined apical region,
were only ever found in the supernatant and these which appeared thin and elongated. Sporozoite
were ovular, 5 mmr7 mm in size and possessed a apical organelle discharge is known to mediate host
smooth surface (Fig. 1A). This correlates with pre- cell contact and according to previous findings
vious findings of Hijjawi et al. (2001). On the surface already occurs during excystation (Snelling et al.
of these oocysts a cleft was visible and presumably 2007). As the sporozoites shown in Fig. 1B and
it is along this cleft that the oocyst opens to release D appear to be making host cell contact, apical or-
sporozoites during the excystation process. Excysted ganelle discharge of molecules, which are discharged
oocysts were observed adhered to cells after 24 h but in the presence of host cells, might account for the
not before. It is known that oocysts possess surface occurrence of this thin and elongated apical region.
lectins that are thought to hinder their adherence to In contrast, sporozoites isolated from supernatant
host tissue in vivo until the target tissue is reached did not display this apical elongation and their shape
where they then mediate attachment to host cells remained largely regular along their entire length
(Kuznar and Elimelech, 2006). In the present study, (Fig. 1C). Sporozoites were only observed up to
oocysts were directly applied to target cells but did 48 h post-inoculation. As sporozoites are known to
not attach immediately. Perhaps without passage invade host cells within 5 min after excystation
through the gastrointestinal tract, oocysts do not (Forney et al. 1999) it can be assumed that after
H. Borowski and others 16

Fig. 1. Oocyst excystation and sporozoite host cell invasion. (A) Intact oocyst from supernatant, 3 h post-inoculation.
(B) Oocyst excystation in vitro, 48 h post inoculation. (C) Free sporozoite isolated from supernatant 3 h
post-inoculation. (D) Free sporozoite on host cell, 7 h post-inoculation. Arrows indicate the apical regions of
sporozoites. (E) Encapsulated by the host cell apical membrane, invading sporozoites transform into the trophozoite
stage epicellularly, at 6 h and 24 h respectively. Scale bars : (A) 4 mm; (B) 2 mm; (C,D,E) 1 mm.

48 h all oocysts have excysted and that no new followed by parasite development occurs rapidly
sporozoites are produced. Invasion of host cells by after parasite-host tissue contact.
sporozoites is shown in Fig. 1E. Invading sporozoites
eventually become completely encapsulated by the
Trophozoites on the host cell surface
host cell apical membrane, which initially appeared
48 h post-inoculation
as a hood-like structure at this early invasion stage
(Fig. 1E). A similar process has also been described During the invasion process, C. parvum sporozoites
in the in vivo toad system by Valigurova et al. (2008). always remain epicellular and appear to transform
As part of the infection process, sporozoites trans- into the trophozoite stage extracytosolic. Thus, stages
form into the trophozoite stage and undergo mer- that result from host cell invasion show a similar
ogony, which leads to trophozoite growth. This cellular location as gregarines, with the only differ-
initial trophozoite stage measured <1 mm in diam- ence being that Cryptosporidium becomes encapsu-
eter (Fig. 1E) and was observed as early as 6 h lated by a host cell apical membrane (Fig. 2A–G ;
post-inoculation, showing that host cell infection Barta and Thompson, 2006). Trophozoites shown in
Morphological characterization of C. parvum in vitro 17

Fig. 2. Trophozoites on the host cell surface 48 h post-inoculation. (A) Cross-section through an early trophozoite
showing the feeder-organelle (arrow) attachment to the host cell cytosol. (B) Mature trophozoite. (C) Cross-section
through a mature trophozoite revealing the electron-dense band (arrow) that separates the parasite from the
host cell. (D,E,F,G) Accumulations of trophozoites on the host cell surface. Scale bars: (A) 0.5 mm ; (B,C,G) 1 mm ;
(D,E,F) 2 mm.

Fig. 2A–G may have developed from either spor- Trophozoites were always found to reside within
ozoites or from merozoites type I after host cell in- a PV and to be engulfed by the host cell apical
vasion. Trophozoites varied in size depending upon membrane, which displayed a smooth surface.
their developmental stage. Early trophozoites ob- In later stages of trophozoite development, the basal
served from 6 h post-inoculation onwards, were membrane developed a hood-like structure and cyto-
f1 mm in size (Fig. 1E), whereas well-developed plasmic granulation occurred leading to merozoite
trophozoites observed after 24 h post-inoculation, development within the trophozoite (Fig. 2A and B).
were up to 2.5 mm (Fig. 2B). These mature tropho- Single trophozoites were regularly seen attached
zoites appeared attached to the surface of cells, but to infected cells (Fig. 2B), but accumulations of 2
were separated from the host cell via an electron- or more trophozoites were more frequently observed
dense band (Fig. 2C). The formation of a feeder or- (Fig. 2D–G). The 4 trophozoites shown in Fig. 2D
ganelle structure is hypothesized for all C. parvum are likely to have developed from sporozoites that
stages and is shown here in Fig. 2A. Previous mor- simultaneously excysted from a single oocyst. How-
phological studies by Huang et al. (2004) have already ever, the 6 trophozoites observed in Fig. 2E are
described this extracytosolic location of the parasite more likely to have resulted from merozoite type I
and its feeder organelle attachment. Our data show host cell invasion, as one meront I is believed to
sporozoites becoming engulfed by the host cell apical contain 6 or 8 merozoites (Hijjawi et al. 2001).
membrane. This phenomenon was proposed (Perkins A merging of membranes engulfing 2 or more para-
et al. 1999 ; Elliott and Clark, 2000 ; Pollok et al. 2003 ; sites was also often observed (Fig. 2D–G). This leads
Chen et al. 2004 a, b, 2005 ; Hashim et al. 2006) and to the assumption that clusters of 2 or more tropho-
recently confirmed by Valigurova et al. (2008). It has zoites result from invasion of infective zoites (sporo-
been suggested that the parasite induces the host cell zoites or merozoites) in closest proximity. Hijjawi
to encapsulate itself with the host cell apical mem- et al. (2004) made the observation that zoite stages
brane (Borowski et al. 2008) to escape the hosts de- commonly accumulate in cell-free cultures. Possibly,
fence mechanisms. there is a weak unspecific binding between infective
H. Borowski and others 18

Fig. 3. The parasite’s effect on host cell microvilli. (A) Gliding trail composed of elongated microvilli between
an excysted oocyst and trophozoites 3 days post-inoculation. (B,C) Abnormal microvilli clusters surround trophozoites
48 h post-inoculation. Scale bars : 2 mm.

zoites (Fig. 6A–C). Chen et al. (2000) reported that to establish itself in its niche (Bonnin et al. 1995),
zoite stages express surface lectins that have adhesive it can be suggested that gliding sporozoites use
properties. It is possible therefore, that these lectins microvilli material for their gliding motion and thus
also facilitate the attachment of infective zoites to cause this abnormality seen in gliding trails. As
each other. Taken together, these data suggest that gliding trails were not observed in all cases of oocyst
excysted sporozoites and merozoites can be adherent excystation, it appears that not all sporozoites glide
to each other during host cell attachment and in- along the host membrane surface until they are ready
vasion of cells (Fig. 6C). This close proximity of to invade a cell at a particular area. Some sporozoites
invading stages results in the merging of membranes are thought to invade directly at their origin of ex-
between individuals, forming clusters of rapidly cystation (Fig. 2F), exhibiting gliding movements
growing trophozoites. in a small area only, whereas other sporozoites might
even travel through the culture medium (Fig. 1C)
before establishing host cell contact and invading
The effect on host cell microvilli
cells some distance from excystation.
Protozoan parasites possess gliding motility. Gliding Microvillus abnormality has not only been ob-
motility of sporozoites has been observed in previous served in gliding trails, but also around developing
studies (Barnes et al. 1998 ; Riggs et al. 1999 ; Wetzel trophozoites (Fig. 3B and C). Abnormally abun-
et al. 2005) leading to the suggestion that gliding dant microvilli were frequently found to surround
along the host cell surface is a prerequisite to host trophozoites (Fig. 3B) and in some cases were
cell invasion. C. parvum gliding trails along host cell significantly elongated (Fig. 3C). As the expression
surfaces were observed in this study (Fig. 3A). of microvilli around trophozoite clusters was found
These gliding trails were evident as trails of elon- to be higher than in non-infected areas, C. parvum
gated microvilli between an excysted oocyst and might even induce the production of microvilli to
newly formed trophozoites ; extending up to 15 mm. satisfy its need for microvillar components. Micro-
As C. parvum is known to utilize microvilli material villi of infected cells were also seen to appear to
Morphological characterization of C. parvum in vitro 19

Fig. 4. Cryptosporidium parvum binary fission and syzygy. (A,B) Binary fission of C. parvum stages 4 days
post-inoculation. (C,D) C. parvum syzygy 5 days post-inoculation. Basal discs are indicated by arrows.
Scale bars : (A,B,C) 2 mm ; (D) 1 mm.

become incorporated into the membrane engulfing become more extracellular. This is evident in stages
the parasites and to aid in securing the parasite to that appear to have little or no attachment to host
the host cell surface (Fig. 2B and G). This phenom- cells (Fig. 4A–C). All of these stages are likely to
enon has not been described before, but microvillar be microgamonts, one of which is clearly visible in
material has been identified in the membrane com- Fig. 4A. Microgamonts occur later in the life cycle
ponents engulfing the parasite using molecular tech- once sexual reproduction has been initiated. Thus,
niques (Bonnin et al. 1995). these stages show a more extracellular location. Our
observations raise the question whether C. parvum
stages with more epicellular location are still attached
Binary fission and syzygy in the C. parvum life cycle
to and encapsulated by the host cell apical mem-
Additional C. parvum stages were also found that brane, or have broken contact with host cells, but still
were characteristically different from those described retain the surrounding outer host cell membrane.
above. Figure 4A and B raise the possibility that Our data also indicate that C. parvum might
C. parvum may undergo binary fission, i.e. the employ syzygy (Fig. 4C). Syzygy is defined as the
splitting of a parent cell into 2 daughter cells, other association of gamonts (pre-gametes) end-to-end or
than during the course of merogony and shizogony. in lateral pairing prior to the formation of gametes
In Fig. 4A, two microgamonts that seem to be in that may be employed to ensure genetic diversity and
the process of binary fission, are engulfed by a single has been described in gregarines (Landers, 2001 ;
membrane that may have initially encapsulated Lacombe et al. 2002 ; Barta and Thompson, 2006 ;
the parent trophozoite on the host cell surface. The Toso and Omoto, 2007). Thus, it is reasonable that
finding of 2 parasites that appear to be dividing from syzygy may occur in closely related Cryptosporidium
1 parent and seem to be the same life-cycle stage species. Connecting discs between adjacent parasites
(Fig. 4A), demonstrates that C. parvum possibly are visible (Fig. 4C and D) and these appear to be
undergoes asexual reproduction other than in the the same as the basal discs already described in
course of merogony. As explained below, as C. par- Cryptosporidium by Valigurova et al. (2008). The
vum progresses through its life cycle, it appears to C. parvum stages involved in syzygy shown in
H. Borowski and others 20

Fig. 5. Meronts I and merozoites type I. (A,B) Developing meronts I with internal merozoites type I. (C,D)
Mature meronts I at the stage of merozoite I excystation. (E,F,G) Free merozoites type I showing well-defined apical
regions (arrow) at 6 h of culture. (H) Merozoite type I host cell invasion 6 h post-inoculation. Scale bars: (A,B,C,E,F)
1 mm; (D) 2 mm ; (G,H) 0.5 mm.

Fig. 4C and D are probably microgamonts. As sy- apical membrane (Fig. 5A and B). Developing mer-
zygy was first observed when sexual life-cycle stages onts measured approximately 1.5 mm in diameter
occurred in culture, it can be suggested that pre- (Fig. 5A and B), whereas mature meronts at the stage
dominantly gamont stages (here microgamonts) of merozoite release were larger, measuring 2.5 mm
employ syzygy. This correlates with findings on (Fig. 5C and D). From this, it appears that tropho-
gregarines and further supports the affinity of zoites developing into meronts I undergo merogony
Cryptosporidium with these apicomplexans (Landers, and begin to form internal merozoites, before they
2001 ; Toso and Omoto, 2007). have reached their full size. Merozoites seemed to
be aligned in a parallel orientation within intact
meronts I (Fig. 5A and B) which is consistent with
Meronts and merozoites
observations by Hijjawi et al. (2001). Once excysted,
Trophozoites that result from sporozoite host cell the merozoites type I in a meront numbered 6 or 8,
invasion develop into meronts I. Meronts I with which also correlates with previous findings by
visible internal merozoites type I, were observed Hijjawi et al. (2001). Excysting merozoites type I
as early as 24 h post-inoculation (Fig. 5A and B). showed a rod-like shape with a pointed apical region.
Like trophozoites, meronts I were found to be In contrast to excysting oocysts, the membranes
attached to host cells and engulfed by the host cell of meronts I appear not to become perforated to
Morphological characterization of C. parvum in vitro 21

Fig. 6. Meront II and merozoites type II. (A) Excysting meront II with internal merozoites type II (arrow) 3 days
post-inoculation. (B) Merozoite type II host cell invasion. (C) Pairing of a merozoite type II with a merozoite type I 9 h
post-inoculation. Scale bars: (A) 1 mm ; (B,C) 0.5 mm.

facilitate infective zoite release. During merozoite (Chen et al. 2000) might mediate this attachment to
release, the membranes engulfing the parasite still host tissue. Merozoites appear initially to adhere to
appeared smooth (Fig. 5C and D) and seemed to host cells along their full body length, but become
either open up (Fig. 5 D) or rupture (Fig. 5C). When stouter as cellular invasion occurs (Fig. 5H). Thus
merozoites type I are released from the meront a it can be hypothesized that after initial host cell
residual body is left behind (Fig. 5C). Similar ob- attachment involving surface lectins, a re-orientation
servations have been made on the Cryptosporidium of merozoite organelles occurs, bringing the apical
sp. ‘ toad ’ model (Valigurova et al. 2008). complex into host cell contact to initiate cellular
Free merozoites type I were seen in cell culture invasion.
from 24 h post-inoculation onwards (Fig. 5E–H). Once merozoites type I are present, the parasite
Merozoites, which previously were probably in- employs 2 ways to replicate in its host which both
correctly referred to as microgametes (Thompson occur concurrently : (i) it progresses via asexual re-
et al. 2005) type I were 0.4 mmr1 mm, with both a production by the formation of meronts I and (ii) it
rounded and a pointed end (Fig. 5E–H). The pointed progresses via sexual reproduction by formation of
end appeared similar to that of sporozoites, which meronts II which then further develop into micro-
houses the apical organelles for host cell invasion and macrogamonts (Borowski et al. 2008).
(Tetley et al. 1998). These free merozoites must Meronts II as well as merozoites type II were
be adhered to the host cells in some manner observed in culture from 72 h post-inoculation.
(Fig. 5E–5H), otherwise they would have been Meronts II were found to be bigger than meronts I
removed during sample preparation. Surface lectins measuring 3.5 mm in diameter (Fig. 6). Meronts II
which have been detected on infective zoite stages appeared to possess a thicker outer membrane than
H. Borowski and others 22

Fig. 7. Macrogamonts and their attachment zones. (A) Macrogamont with feeder organelle (arrow) 4 days
post-inoculation. (B) Developing macrogamont with feeder organelle (arrow). (C) Cryptosporidium parvum attachment
zones 5 days post-inoculation. Scale bars: (A,C) 2 mm ; (B) 1 mm.

meronts I (Fig. 6A). Merozoites type II were round already broken host cell contact leaving the host
and measured between 0.5 mm and 1 mm in diameter cell-derived membrane that once surrounded it
(Fig. 6A–C). Similar to meronts I, the membrane behind. Such empty ‘ attachment-zones ’ often show
engulfing the merozoites appeared smooth and not a ring-like structure (Fig. 7C), presumably where
perforated for their release (Fig. 6A) suggesting the feeding organelles were attached. These findings
host cell origin of this membrane. correlate with observations by Valigurova et al.
Similar to the adhesion of sporozoites to each (2008) who described a similar extracellular location
other via surface lectins, merozoites type I and type of macrogamonts, their detachment from host cells,
II can also adhere to each other and co-invade cells the granular structure of their feeder organelles, as
(Fig. 6B and C). well as detachment zones in vivo, in their Crypto-
sporidium sp. ‘ toad ’ model.
Microgamonts were rounder and smaller than
Microgamonts and macrogamonts
macrogamonts, measuring about 2 mmr2 mm
Merozoites type II that are released in culture, (Fig. 8A–C). They contained a large number of
invade cells to transform into either micro- or microgametes (Fig. 8A and C), which are released to
macrogamonts. Four days post-inoculation micro- fertilize macrogamonts. This observation is not
and macrogamonts were observed for the first time consistent with findings by Hijjawi et al. (2001), who
(Figs 7 and 8). Both gamont stages appeared to have identified only 16 microgametes in 1 microgamont
less contact with host cells than trophozoites or with light microscopy. This difference is likely to be
meronts. This supports our suggestion that the fur- due to the difficulty resolving such structures with
ther the parasite progresses in its life cycle, the more light microscopy. Microgametes measure about
extracellular it appears to become. Macrogamonts 0.1 mm in diameter and are spherical. Hijjawi et al.
were ovular and measured about 4 mmr5 mm (2001) believed that they were non-flagellated which
(Fig. 7A). Feeder organelle attachment of a macro- is confirmed by our SEM study. The microgamonts
gamont is visible in Fig. 7A. The surface of this shown in Fig. 8A seem to be surrounded by pre-
macrogamont appeared rough, suggesting that it has sumably host cell-derived membrane. Fig. 8C shows
Morphological characterization of C. parvum in vitro 23

Fig. 8. Microgamonts. (A,B) Microgamonts without stalk. The arrows in A indicate a cleft along which the
macrogamonts open. (C,D) Microgamonts with stalk (arrow). Scale bars: (A,B) 1 mm ; (C,D) 2 mm.

a feeder organelle attachment of the same origin as Development of C. parvum in host cell culture
that of the macrogamont in Fig. 7A. Approximately without invasion of host cells
half of all microgamonts seen in culture possessed
a stalk-like structure that either appeared as if the The present study is consistent with the intra-
stalk had broken (Fig. 8D) or seemed to attach the cellular life cycle of C. parvum described by Hijjawi
parasite to the host cell (Fig. 8C). Again, a similar et al. (2001). The stages described above all resulted
finding has also been reported by Valigurova et al. from host cell invasion, yet, complete cell-free de-
(2008). This stalk on microgamonts might result velopment of C. parvum has also been documented
from the parasite being released from the attachment at the light microscope level (Hijjawi et al. 2004)
zones described above in a similar manner to macro- in vitro. From our own observations, we hypothesize
gamonts. The microgamonts that possess a stalk that extracellular life-cycle stages might be part of
(Fig. 8C and D) appeared to have less host cell con- the C. parvum life cycle, or co-exist resembling
tact compared to those which did not show such a rudimentary stages of an ancestral life cycle. Our data
structure (Fig. 8A and B). Additionally, their mem- show that C. parvum can develop extracellularly in
brane appeared perforated (Fig. 8C) in contrast to the presence of host cells in an in vitro model, without
microgamonts without this stalk. invasion. We have observed stages of C. parvum that
Like trophozoites, microgamonts were also ob- have been described before at the light microscopic
served to occur in clusters of 2 or more. The ac- level but are not as yet considered as part of the life
cumulation of microgamonts (Fig. 8D) probably cycle.
resulted from microgamonts that had released from Life-cycle stages of C. parvum that developed
their attachment zone and adhered to each other, extracellularly showed a rough surface like that of
whereas the 2 adjacent microgamonts in Fig. 8B are previously described free parasite stages (Fig. 5E–H
more likely to be a result of merozoite type II host and Fig. 6A–C), and they did not appear to be
cell invasion in close proximity. engulfed by the host cell apical membrane.
H. Borowski and others 24

undergoing merogony and transforming into a

trophozoite stage, without invasion of host cells
(Fig. 9A). This suggestion is supported by our
findings of a proposed extracellular meront 4 days
post-inoculation (Fig. 9B). The meront measured
approximately 8 mm in diameter and was round in
shape. The zoite stages observed within the meront,
measured up to 2 mm and appeared to be densely
packed. It is likely that this extracellular meront
has formed through the clumping of infective zoite
stages in culture, of which each single one has under-
gone merogony to form a trophozoite. In support of
this, accumulations of merozoites and/or tropho-
zoites, as well as the formations of meronts in cell-
free culture, have already been described (Hijjawi
et al. 2004).
Critically, how extracellular trophozoites and
meronts progress in their life cycle without cellular
invasion is still not understood. Each extracellular
trophozoite might progress in a manner typical of
an intracellular trophozoite, or each single tropho-
zoite might transform into the next life-cycle stage.
The observation that C. parvum also develops ex-
tracellularly, despite the presence of host cells, fur-
ther supports the proposal that Cryptosporidium
has a close affinity with gregarines (Carreno et al.
1999 ; Barta and Thompson, 2006 ; Valigurova et al.
2007).

CONCLUSIONS

For the first time, our study reveals the morphology

of each stage in the currently accepted life cycle of
C. parvum. The development of life-cycle stages
described here extends previous reports that were
based on light microscopic findings (Hijjawi et al.
2001, 2002, 2004). Data from our in vitro model
Fig. 9. Development of Cryptosporidium parvum in system are further supported by similar morpho-
host cell culture without the invasion of host cells. logical observations from in vivo studies on C. muris
(A) Trophozoite development within an inoculated and Cryptosporidium sp. ‘ toad ’ (Valigurova et al.
oocyst after 2 days. (B) Possible extracellular meront 2008). Surprisingly, the species C. parvum observed
after 4 days of culture. Scale bars: (A) 2 mm ; (B) 1 mm. in our study shows more similarities to the species
Cryptosporidium sp. ‘ toad ’ than to C. muris
(Valigurova et al. 2008).
After 48 h inoculation, we observed what we be- Apart from revealing the morphology of accepted
lieve to be trophozoite development directly within life-cycle stages in C. parvum our study describes
an oocyst (Fig. 9A). The membrane of the oocyst previously unreported features of C. parvum life-
releasing the trophozoites appeared rough and per- cycle stages, which include their morphological
forated, a characteristic already observed during structure and interaction with host cells. Further,
the release of sporozoites (Fig. 1B). Similar to these extracellular stages of C. parvum are reported and
so-called intracellular trophozoites, trophozoites characterized for the first time in an in vitro model
that formed directly within an oocyst measured ap- of cultured host cells at an electron microscopic
proximately 2 mm. As these stages were observed as level.
early as 48 h post-inoculation (Fig. 9A) it can be It is not clear whether extracellular stages of
hypothesized that the trophozoites developed within C. parvum occur in vivo, and whether they are part
an inoculated oocyst, and were eventually released of the parasite’s life cycle or represent rudimentary
into culture. stages of an ancestral life cycle. Future studies are
From our observations it can be suggested that needed to identify the existence of these stages in vivo
each sporozoite and each merozoite is capable of and clarify their nature.
Morphological characterization of C. parvum in vitro 25

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