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Complete development and long-term maintenance of Cryptosporidium
parvum human and cattle genotypes in cell culture
N.S. Hijjawi
a
, B.P. Meloni
b
, U.M. Morgan
a
, R.C.A. Thompson
a,
*
a
Division of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, W.A. 6150, Australia
b
Sir Charles Gairdner Hospital, QEII Medical Centre, Nedlands, W.A. 6009, Australia
Received 1 February 2001; received in revised form 15 March 2001; accepted 15 March 2001
Abstract
This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle
genotypes) in the HCT-8 cell line. Furthermore, for the ®rst time the complete life cycle was perpetuated in vitro for up to 25 days by
subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-
reinfection cycle of C.parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from
thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype
were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for
propagation of the complete life cycle of C.parvum in vitro. q2001 Australian Society for Parasitology Inc. Published by Elsevier Science
Ltd. All rights reserved.
Keywords:Cryptosporidium parvum; Complete life cycle; In vitro development; Human genotype; Cattle genotype; Auto-reinfection; Thin-walled oocyst;
HCT-8 cell line
1. Introduction
Cryptosporidium parvum is an enteric parasite that causes
diarrhoeal disease in humans and domesticated animals
world wide (O'Donoghue, 1995). The parasite infects intest-
inal epithelial cells resulting in self-limiting diarrhoea in
immunocompetent persons. However, it is more severe,
and potentially fatal, to the immunosuppressed population,
especially those with acquired immunode®ciency syndrome
(AIDS) (Peterson, 1992; O'Donoghue, 1995). Although a
large number of anti-parasitic drugs have been tested against
Cryptosporidium, no consistently effective chemotherapeu-
tic agent is available and a healthy, intact immune system
remains the only reliable defence (Theodos et al., 1998;
Tzipori, 1998).
There have been more than 25 reports describing cell
lines that support the growth of both sexual and asexual
stages of C.parvum in vitro. These studies showed that
different cells can be infected with C.parvum, in which
maximum growth was reached after 48±72 h, but then
gradually declined (Eggleston et al., 1994; Lawton et al.,
1997; Tzipori, 1998). Apart from the production of small
numbers of oocysts in some cell lines, it has not been possi-
ble to initiate re-infection of the cell line from these oocysts
(Lawton et al., 1997). The failure to perpetuate the infection
in vitro has been attributed to the lack of thin-walled oocyst
production, which is thought to be essential for the auto-
infection cycle in vivo (Current and Garcia, 1991).
Recent studies have shown that C.parvum is not a geneti-
cally uniform species but encompasses at least seven
distinct genotypes that appear to be host speci®c; human,
cattle, pig, marsupial, dog, ferret and mouse (Awad-el-
Kariem et al., 1995; Bonnin et al., 1996; Peng et al.,
1997; Morgan et al., 1995, 1997, 1998, 1999; Xiao et al.,
1999; Sulaiman et al., 2000). Humans are susceptible to
infection with the human and the cattle genotypes of C.
parvum, providing evidence of zoonotic transmission.
Regarding in vitro culturing of C.parvum, most success
has been obtained using cattle isolates (Villacorta et al.,
1996; Yang et al., 1996; Deng and Cliver, 1998), although
two attempts using oocysts from human patients with AIDS
have also been reported (Current and Haynes, 1984; Burand
et al., 1991). However, with the exception of the study by
Meloni and Thompson (1996) all C.parvum cultures have
been established without prior genotyping of the isolate
used.
International Journal for Parasitology 31 (2001) 1048±1055
0020-7519/01/$20.00 q2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
PII: S0020- 7519(01)00212-0
www.parasitology-online.com
* Corresponding author. Fax: 161-8-9360-2466.
E-mail address: andrew_t@numbat.murdoch.edu.au
(R.C.A. Thompson).
The establishment of the complete life cycle of C.parvum
in vitro will allow the evaluation of drug therapies against
different life-cycle stages, enhance research into the biology
of host cell±parasite interactions and enable ampli®cation of
parasite material for further immunological, biochemical
and molecular studies. It could also be used for the assess-
ment of viability of C.parvum oocysts isolated from envir-
onmental samples (Deng and Cliver, 1998; Di Giovanni et
al., 1999; Rochelle et al., 1999). Finally, the value of an in
vitro culture system for C.parvum will be further enhanced
if complete development of both human infective genotypes
(human and cattle) can be propagated.
2. Materials and methods
2.1. Parasite isolates
The C.parvum cattle isolate (Swiss cattle C26) used
during this study was originally obtained from the Institute
of Parasitology, Zurich and has subsequently been passaged
through mice as previously described by Meloni and
Thompson (1996). The human isolate was obtained from a
local diagnostic laboratory from a patient in Perth, Western
Australia.
2.2. Genotyping of parasite material
The human and cattle genotypes of C.parvum were iden-
ti®ed according to the method described by Morgan et al.
(1997) based on direct PCR analysis and sequencing of the
18S rDNA. Genotyping of the human isolate was carried out
using DNA extracted from faecal oocysts and from parasite
material collected from HCT-8-infected cultures. The cattle
isolate (Swiss cattle C26) was previously genotyped by
Morgan et al. (1997) and furthermore, genotyping was
carried out for oocysts puri®ed from mice after being
infected from in vitro-derived oocysts of cattle origin.
2.2.1. Direct PCR analyses
Speci®c PCR primers that directly differentiate between
human and cattle genotypes on the basis of the size of the
PCR product were also used in the present study (Morgan et
al., 1997). The human-speci®c primer, CP-HR, which
ampli®es a 411-bp product from human isolates only, and
the cattle-speci®c primer, CP-CR, which ampli®es a 312-bp
product from animal isolates only, were used for the PCR
ampli®cation. The ampli®cation products were subjected to
electrophoretic separation in 1.5% agarose gels, stained
with ethidium bromide, visualised under UV light and
compared with positive controls of both cattle and human
genotypes.
2.2.2. Sequencing of the 18S rDNA
For further con®rmation of the human C.parvum geno-
type, DNA extracted from oocysts and infected HCT-8
cultures was used to amplify the 18S rDNA for sequencing
(Morgan et al., 1997).
2.3. Puri®cation of C. parvum oocysts
For in vitro culturing, C.parvum oocysts of the human
genotype were puri®ed from faeces using a routine puri®ca-
tion procedure (Morgan et al., 1995). Cryptosporidium
parvum oocysts of the cattle genotype were obtained by
infecting 7±8-day-old ARC/Swiss mice with 100 000±
120 000 oocysts. The oocysts were isolated and puri®ed
according to the procedure described by Meloni and
Thompson (1996), with the addition of a ®nal bleaching
step. On day 8 p.i., mice were euthanised (CO
2
inhalation)
and the jejunum, ileum, caecum, colon and rectum removed,
placed in sterile PBS/0.02% Tween-20 <4 ml/mouse) and
dissected into small segments. The segments were further
homogenised at 48C and sputasol (0.005 g/ml of the suspen-
sion) was added. The homogenate was then left at room
temperature (RT) for 90±120 min on a rotary mixer before
centrifugation at 2000 £gfor 8 min. The supernatant was
removed, 40 ml of PBS/0.02% Tween-20 and 10 ml of ether
added before mixing vigorously (20±30 s) and centrifuging
for 8 min at 2000 £g. The supernatant was removed and the
pellet resuspended in 4 ml PBS. For further puri®cation of
oocysts, the 4-ml suspension was layered on to a Ficoll
gradient (1%/0.5% Ficoll prepared in PBS containing 16%
sodium diatrizoate). Gradients were centrifuged at 2000 £g
for 30 min at RT. Oocysts were collected from the PBS/
0.5% Ficoll interface and washed twice with PBS and made
up to 10 ml with sterile PBS. They were then bleach treated
by adding sodium hypochlorite (200±300 ml/ml), and incu-
bated at RT for 20 min, washed twice with PBS and centri-
fuged at 2000 £gfor 8 min. Finally, puri®ed oocysts were
resuspended in cold sterile PBS and stored at 48C after
adding 15 ml/ml antibiotic solution containing ampicillin
(10 mg/ml) and lincomycin (4 mg/ml).
2.4. Pre-treatment of oocysts and culture media preparation
Oocysts were excysted to release sporozoites in a freshly
prepared, ®lter-sterilised (0.22 mm ®lter) excystation
medium composed of acidic H
2
O (pH 2.5±3) containing
0.5% trypsin and incubated in a water bath at 378C for 20
min with mixing every 5 min. Thereafter, the excystation
suspension was centrifuged at 2000 £gfor 4 min at RT.
Oocysts were resuspended in maintenance medium (100
ml RPMI-1640) containing 0.03 g l-glutamine, 0.3 g
sodium bicarbonate, 0.02 g bovine bile, 0.1 g glucose, 25
mg folic acid, 100 mg 4-aminobenzoic acid, 50 mg calcium
pantothenate, 875 mg ascorbic acid, 1% FCS, 15 mM
HEPES buffer, 10 000 units penicillin G and 0.01 g strep-
tomycin, adjusted to pH 7.4. The percent excystation, for the
human and cattle genotypes, was calculated by scoring at
least 300 oocysts as empty or intact after 3 h incubation in
maintenance medium. The percent excystation was calcu-
N.S. Hijjawi et al. / International Journal for Parasitology 31 (2001) 1048±1055 1049
lated as the number of empty oocysts/number of empty 1
intact oocysts.
2.5. Preparation and infection of host cells
HCT-8 cells (ATCC; CCL244) were grown in RPMI-
1640, 10% FCS in 25-cm
2
¯asks and seeded 24 h prior to
infection to allow them to reach monolayer. HCT-8 cells
were infected by removing the existing media and adding
maintenance medium containing 50 000 pre-treated oocysts
(2000 oocysts/cm
2
). Flasks were kept at 378C in a candle jar
(18.8% O
2
, 1.97% CO
2
). During in vitro cultivation, the
medium was changed every 2±3 days to maintain the pH
within the range 7.2±7.6.
2.6. Subculturing to new cell monolayers
Subculturing was carried out by collecting supernatant or
scraped cells from infected cultures which were used to
inoculate a new cell monolayer as described above. After
2 h, the supernatant and the cell debris were removed by
washing the cell monolayer with pre-warmed PBS at pH 7.2,
resupplied with maintenance medium and the ¯asks incu-
bated at 378C in a candle jar where the media was changed
every 2±3 days. Supernatant only was used later in the study
since the cells and cell debris appeared to adhere ®rmly to
the cells in the new monolayer and interfere with parasite
development.
2.7. Puri®cation of thin-walled oocysts from in vitro
cultures
An attempt to purify the thin-walled oocysts from in vitro
cultures was carried out during the present study. Super-
natant was collected from culture ¯asks after passaging
the parasite for 12 days, centrifuged, pellets resuspended
in PBS (pH 7.2) and puri®ed using Ficoll gradient centrifu-
gation as described above.
2.8. Infectivity of culture-derived oocysts to mice
Culture medium from four 75-cm
2
culture ¯asks
(approximately 200 ml) was collected from 5-day-old
cultures infected with oocysts (2000 oocysts/cm
2
)ofC.
parvum (cattle and human genotypes). As a control,
RPMI-1640 maintenance medium containing 100 000 pre-
treated oocysts, were also incubated for 5 days under the
same conditions in cell-free ¯asks without media changes.
This was done to be sure that the infectivity in mice resulted
from the thin-walled oocysts produced in vitro and not from
the non-excysted oocyst which remained in culture from the
initial inoculum. The culture media from the infected mono-
layers and control ¯asks were centrifuged at 2000 £gfor 5
min and the pellet reconstituted in 2 ml PBS before being
inoculated intragastrically into 7±8-day-old ARC/Swiss
mice (100 ml/mouse). Eight days p.i. mice were processed
for oocyst puri®cation as described above.
2.9. Examination of HCT-8 cells infected with C. parvum
oocysts (human and cattle genotypes)
Cultures were examined daily at magni®cations ranging
from £150 to £600 using an inverted light microscope
(Olympus IMT-2) ®tted with a heating chamber. Nomarski
phase-contrast microscopy (Olympus BX50) and Optimas
image analysis (MS-DOS operating system) were used for
capturing images of C.parvum stages. Because of the dif®-
culty in capturing the images of the different stages, mono-
layers infected with C.parvum (cultured on 25-mm
2
coverslips or scraped intact from ¯asks) were compressed
onto a glass slide, examined and photographed under oil
immersion £1000 magni®cation).
3. Results
3.1. Genotyping
Analysis of the human isolate with the diagnostic primer
(CP-HR) ampli®ed a 411-bp product from faecal oocysts
and infected HCT-8 cultures after 72 h p.i. Furthermore,
sequencing of the 18S rDNA (DNA extracted from oocysts
and HCT-8 cultures after 72 h of infection) con®rmed that
this isolate exhibited the human genotype recognition
sequence according to Morgan et al. (1997). The genotype
of the cattle isolate, used during this study, which has been
continuously passaged in mice in our laboratory, was initi-
ally determined by Morgan et al. (1997) using the above two
methods and con®rmed regularly ever since.
3.2. Excystation of C. parvum oocysts prior to culturing
The excystation rate of the cattle and human genotypes
was 88 and 86%, respectively, after 3 h incubation in excys-
tation medium. Fig. 1a±d shows the sequential stages of
excystation of sporozoites from pre-treated oocysts of the
cattle genotype, which did not differ from the oocysts of the
human genotype.
3.3. Observation of HCT-8 cells infected with C. parvum
Human and cattle genotypes of C.parvum were observed
to complete their life cycle in HCT-8 cells (from sporozoites
N.S. Hijjawi et al. / International Journal for Parasitology 31 (2001) 1048±10551050
Fig. 1. Nomarski interference-contrast photomicrographs of the steps of
excystation of Cryptosporidium parvum oocysts (cattle genotype) after
treatment with trypsin and bile salts. (a) Intact thick-walled oocyst before
the treatment. (b) Partially excysted oocyst with the suture(s) partially
opened. (c) Free sporozoite released from the suture. (d) Shell of an
empty oocyst with residuum (r) and an open suture(s). Bar: 5 mm.
to sporulated oocysts) under the culturing conditions
described above (Figs. 1±5). The various developmental
stages were maintained in culture for up to 25 days by the
passage of parasite life-cycle stages from the supernatant of
5-day-old infected monolayers to fresh HCT-8 cell mono-
layers.
3.4. Description of the various developmental stages of C.
parvum (human and cattle genotypes) in culture
During the ®rst 24 h p.i., intracellular circular forms
likely to be trophozoites/uninucleate meronts (Figs. 2 and
3) formed as an early stage in the life cycle of C.parvum.At
this time the infection appeared to be restricted to the site of
initial sporozoite infections (Fig. 2). From 48±72 h p.i.
approximately 70±80% of cells became infected and all
the developmental stages could be clearly identi®ed.
These included merozoites, meront I, meront II, macroga-
metes, microgametocytes and oocysts. No major differences
could be seen in the in vitro development of the life-cycle
stages of C.parvum between the human and cattle geno-
types. However, the time needed to complete the life cycle
was much shorter in the human genotype. Unsporulated and
sporulated oocysts were seen in situ after 72 h p.i. for the
human genotype and after 5 days p.i. for the cattle genotype.
The stages observed in culture were characterised as
follows.
3.4.1. Merozoites
The merozoites were usually identi®ed during their
attempts to penetrate cells after 48-h p.i. Merozoites are
thread-shaped with rounded anterior and posterior ends.
We consistently observed merozoites inside HCT-8 cells
(Fig. 4a,b) and while they were released from the cells.
Once merozoites were released they penetrated other cells
rapidly. The merozoites displayed vigorous gliding and ¯ex-
ing movements and continuously attempted to invade cells.
3.4.2. Trophozoites
Round or oval intracellular forms, 2:7£2:7mmin
diameter, and considered as a transitional stage from spor-
ozoites and merozoites to meronts (Figs. 2 and 3).
3.4.3. Meront I
Developing meronts, 3:75 £4mm in diameter, with six or
eight merozoites (Figs. 3 and 4a,b) were observed in culture
and on one occasion six merozoites were counted while
N.S. Hijjawi et al. / International Journal for Parasitology 31 (2001) 1048±1055 1051
Fig. 2. Light photomicrograph of HCT-8 cell monolayer after 24 h of
infection with Cryptosporidium parvum (cattle genotype). Circular struc-
tures are trophozoite stage (t). Bar: 200 mm.
Fig. 3. Light photomicrograph of HCT-8 cells after 48 h infection with
Cryptosporidium parvum (cattle genotype). Note the presence of uninucle-
ate meront/trophozoite stage (t) and immature type I meront (m). Bar: 200
mm.
Fig. 4. Nomarski interference-contrast photomicrographs of stages of endo-
genous development of Cryptosporidium parvum in HCT-8 cell (both from
human and cattle genotypes). (a) Immature type I meront with eight mero-
zoites (m) in focus. (b) Mature type I meront with six merozoites (m). (c)
Immature type II meront with four of the nuclei in focus. (d) Microgamonts
with microgametocytes and a large residuum (r). (e) Macrogamont contains
a large eccentric nucleus (n). Bar: 5 mm.
excysting. The merozoites in type I meronts are arranged
parallel to each other like segments of an orange.
3.4.4. Meront II
Meront II, 3:1£2:8mm in diameter, can be differentiated
from meront I by having four merozoites but we never
observed the release of four merozoites from type II meronts
(Fig. 4c).
3.4.5. Microgamonts
Microgamonts, 5:6£3:4mm in diameter, were observed
containing 14±16 non-¯agellated microgametes (Fig. 4d)
occupying most of the cell around a residuum. Free micro-
gametes as described by Current and Reese (1986) were
bullet-shaped, and displayed a jerky gliding movement in
the culture supernatants presumably after disruption of the
microgamont. Budding on the surface of microgamonts was
observed, and this might be an attempt by microgametes to
escape from the host cell.
3.4.6. Macrogamonts
This stage was distinguished by being large in size 4£4
mm in diameter) and having a large peripheral nucleus (Fig.
4e).
3.4.7. Oocysts
Culture-derived oocysts lacked an outer thick wall, which
is usually present in oocysts puri®ed from faeces (Fig. 5g),
and were identi®ed as thin-walled oocysts with a 5 £5mm
diameter (Fig. 5d±f). Unsporulated, as well as sporulated
oocysts were observed in situ (Fig. 5a±c) and in culture
supernatant. Some oocysts were seen empty and some had
only two sporozoites and a residuum; however, some spor-
ozoites were observed while excysting from the thin-walled
oocysts (Fig. 5f). Thin-walled oocysts were puri®ed from
the culture supernatant using Ficoll gradient.
3.4.8. Sporozoites
Sporozoites were ®rst observed within thin-walled
oocysts 72 h p.i. and thereafter (Fig. 5b±f). They are 5:2£
1:2mm in diameter and characterised by having a comma
shape with a rounded posterior end and a pointed, tapered
anterior end. Sporozoites exhibited a gliding movement and
many were seen actively moving inside the oocyst and
excysting, and ¯oating in the culture media.
Measurements of stages observed in culture were made
on captured images and were within the ranges of these
stages described by Current and Reese (1986) in vivo.
3.5. Infectivity of thin-walled oocysts to mice
Culture-derived oocysts of the cattle genotype were
infective to 7±8-day-old ARC/Swiss mice. An oocyst
yield of 8:3£105(collected from six mice) was obtained
after puri®cation as described in Section 2. However, under
the same conditions, culture-derived oocysts of the human
genotype and from control ¯asks (cattle genotype oocysts
incubated without the HCT-8 cells) failed to infect mice.
Oocysts puri®ed from human faeces (human genotype) also
failed to infect mice.
4. Discussion
The present study describes, for the ®rst time, the complete
development and long-term maintenance of C.parvum of
both cattle and human genotypes in vitro. The human geno-
type, which is restricted to humans, has never been propa-
N.S. Hijjawi et al. / International Journal for Parasitology 31 (2001) 1048±10551052
Fig. 5. Nomarski interference-contrast photomicrographs showing the stages of oocysts of Cryptosporidium parvum (cattle and human genotypes) develop-
ment in HCT-8 cell line. The earliest of these images was taken after 72 h p.i. (a) Unsporulated oocyst in intracellular location (in situ) with residuum (r). (b,c)
Sporulated oocyst in situ detected in HCT-8 cells after 72 h of infection with C.parvum (human genotype); note the presence of three sporozoites (sp) in focus
in (c). (d) Thin-walled oocyst, puri®ed using Ficoll gradient, from a 12-day-old culture (cattle genotype). (e) Fully sporulated thin-walled oocyst with four
sporozoites (sp) and residuum. (f) Sporozoite in an attempt to excyst from a thin-walled oocyst. (g) Thick-walled oocyst after puri®cation from faeces of a
human patient infected with C.parvum; note the presence of the outer thick-wall (ow) which is absent from the thin-walled oocyst in (d). Bar: 5 mm.
gated or maintained in culture for such a long period (25
days). Furthermore, apart from the study by Meloni and
Thompson (1996), no previous workers genotyped Cryptos-
poridium isolates prior to in vitro culturing. Although there
have been two studies describing the complete development
of C.parvum originating from human patients with AIDS
(Current and Haynes, 1984; Burand et al., 1991), neither of
these isolates were genotyped. A number of human cases
have been reported to be due to infection with the cattle
genotype (Morgan et al., 1997; Peng et al., 1997; Widmer
et al., 1998). Research in our laboratory, screening a large
number of faecal samples, has shown that approximately
17% of isolates of Cryptosporidium infecting humans
display the cattle genotype (Morgan et al., 1998). Further-
more, recent genetic evidence showed that patients with
AIDS might be susceptible to a wide range of Cryptospor-
idium species and genotypes to which an immunocompetent
individual is not susceptible to (Widmer et al., 1998; Morgan
et al., 2000). Consequently, genotyping should be an essen-
tial step before culturing different isolates of Cryptospori-
dium to allow interpretation of differences in their
development, pathogenesis, host cell interactions and
susceptibility to chemotherapeutic agents. Several attempts
to propagate the human genotype of C.parvum in neonatal
mice or cattle proved to be unsuccessful (Meloni and Thomp-
son, 1996; Peng et al., 1997; Widmer et al., 1998; present
study), although a recent study described the successful
propagation of the human genotype in gnotobiotic piglets
(Widmer et al., 2000). However, the maintenance of the
parasite in animal hosts is labour-intensive, expensive, and
it is not easy to purify the oocysts from large animals such as
pigs and calves. In addition, they will not support the growth
of the parasite for prolonged periods of time. Thus the present
in vitro culturing system should provide a model for the
maintenance and propagation of both the C.parvum human
and cattle genotypes.
In the present study, the in vitro cultivation of C.parvum
was based on the method of Meloni and Thompson (1996),
but with some modi®cations to pH. The pH appeared to play
an important role in successfully maintaining the growth of
C.parvum in vitro. The pH was monitored and maintained
within an optimum range (7.2±7.6). This was achieved by
changing the media regularly, every 2±3 days, and adding
HEPES buffer at 15 mM. An optimum pH is likely to be
important for both cell invasion and release of parasite stages
(Upton et al., 1995; Meloni and Thompson, 1996) as well as
for the stability of the host cell monolayer. Sloughing of the
host cell monolayer was observed on several occasions
following sudden drops or rises in pH. The low number of
progeny oocysts in vitro in previous studies might be due, at
least in part, to pH ¯uctuations. It has been suggested that
microgamete inactivity at suboptimal pH might be the reason
for the low yield of oocysts of C.parvum in vitro (Upton,
1997). With Plasmodium spp., it has been shown that micro-
gamete emergence and ex¯agellation is a pH dependent
process (Kamamoto et al., 1991). Furthermore, the yield of
Eimeria tenella oocysts in cell culture was increased three-
fold with better pH regulation (Doran, 1971).
A healthy, 1±4-day-old intact host cell monolayer also
appeared to be an important factor for C.parvum growth
in vitro. During the ®rst 4 days p.i., the infection reached
maximum levels, after which time large quantities of cell
debris were evident in the culture medium. The presence of
cell debris and the reduced ability of the merozoites/spor-
ozoites to infect old cells are likely factors contributing to
reduced parasite development. Older cells are known not to
be very susceptible to infection with species of coccidia
(Dvorak and Crane, 1981; Dvorak and Howe, 1977). During
the present study, the problem of overgrowth and degenera-
tion of host cells was avoided by subculturing. Multiple
subculturing proved to be successful and resulted in the
maintenance of the parasite in vitro for up to 25 days.
Previous failures to maintain the intensity of infection
with C.parvum in vitro have been attributed to the poor/
limited production of thin-walled oocysts which are consid-
ered to be an essential factor for auto-reinfection (Current
and Garcia, 1991). Earlier attempts at subculturing in our
laboratory using the cattle genotype were unsuccessful
(unpublished observations). This may have been attributed
to the time subculturing was carried out. Earlier attempts at
subculturing were performed at a time when oocysts were
unlikely to be present (2±3 days p.i.). These ®ndings suggest
that thin-walled oocysts, from which new invasive sporo-
zoites were released, and not merozoites are essential for
perpetuating the life cycle of C.parvum in vitro. Auto-rein-
fection through the production of thin-walled oocysts may
explain why Cryptosporidium infections in immunode®-
cient individuals develop into persistent, life-threatening
diarrhoea with infection of extra-intestinal sites such as
respiratory and biliary ducts (Current and Haynes, 1984).
Apart from Current and Haynes (1984), who described the
presence of some thick-walled oocysts in an intracellular
location in a human foetal lung cell line, in all other studies
including the present, C.parvum oocysts produced in vitro
were thin-walled (Fig. 5a±f). From the present study it
appears likely that such thin-walled oocysts are the stage
responsible for perpetuating the cycle in vivo. Failure in
the production of thick-walled oocysts in culture might be
due to the absence of an essential ingredient from the culture
medium (such as a speci®c enzyme, hormone or growth
factor, which is normally present in vivo) or the lack of an
effective immune response. It is possible that in vivo, the
parasite responds to the development of an immune response
by producing thick-walled oocysts, thus curtailing the auto-
infective cycle, which continues in individuals with impaired
immunity. This hypothesis could be tested by adding
immune serum or other immunological factors against C.
parvum to the culture medium. Many molecules on the
surface of sporozoites and merozoites are strongly immuno-
genic; however, the parasite appears to respond to this chal-
lenge (immune response) by developing rapidly into thick-
walled oocysts, which can infect new susceptible hosts.
N.S. Hijjawi et al. / International Journal for Parasitology 31 (2001) 1048±1055 1053
The thin-walled oocysts (cattle genotype) derived from
culture were infective to mice and produced a yield of 8:3£
105oocysts (from six mice at 8 days p.i.), which is higher than
the initial inoculum (6 £105oocysts) used to infect the cell
monolayers. Culture-derived oocysts of the human genotype
failed to infect mice, a result consistent with earlier in vivo
studies which showed that the human genotype does not read-
ily infect mice (Current and Reese, 1986; Pozio et al., 1992;
Meloni and Thompson, 1996; Peng et al., 1997; Widmer et
al., 1998). Although in vivo infections with thick-walled
oocysts usually give a much higher yield (3±4 million
oocysts/mouse) (Meloni and Thompson, 1996), the future
standardisation of the in vitro technique described here
may lead to large-scale production of oocysts and obviate
the need to maintain the parasite in animal models.
During the present study, although no attempts were
made for the quantitation of the different developmental
stages encountered in vitro, the human genotype appeared
to be much more aggressive in its growth in HCT-8 cells
than the cattle genotype. After 72 h p.i. with oocysts of the
human genotype, HCT-8 cells appeared to be perforated
with large numbers of parasitic stages, especially mero-
zoites that could be seen continuously attempting to pene-
trate the cells. Furthermore, the human genotype completed
the life cycle with the production of oocysts within 72 h,
whereas oocysts of the cattle genotype could not be detected
in culture before 5 days p.i. The reason for this difference
might be that the human genotype has a faster growth rate
than the cattle genotype and/or is better adapted to grow in
the HCT-8 cell line, which is of human origin.
The culture system described in the present study for the
in vitro maintenance of C.parvum will provide a model to
investigate host cell±parasite interactions and drug ef®cacy
with different genotypes and life-cycle stages. It may also
prove valuable for investigating the pathogenesis of the
parasite in the absence of an effective immune response.
The system can also be used for the large-scale production
of oocysts and to assess the viability of C.parvum oocysts
from environmental samples.
Acknowledgements
We should like to thank Russ Hobbs for his excellent
assistance with the illustrations and Aileen Eliott for her
diagnostic expertise. We are also grateful to George Di
Giovanni for helpful discussions at the commencement of
these studies. The ®nancial assistance of Murdoch Univer-
sity and the Pig Research and Development Corporation is
acknowledged.
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