See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/273465084

Validation of cell-free culture using scanning

electron microscopy (SEM) and gene expression
studies
ARTICLE in EXPERIMENTAL PARASITOLOGY MARCH 2015
Impact Factor: 1.64 DOI: 10.1016/j.exppara.2015.03.002 Source: PubMed

CITATIONS

READS

82

4 AUTHORS, INCLUDING:
Rongchang Yang

Nawal Hijjawi

Murdoch University

Hashemite University

59 PUBLICATIONS 505 CITATIONS

28 PUBLICATIONS 986 CITATIONS

SEE PROFILE

SEE PROFILE

Una Ryan
Murdoch University
142 PUBLICATIONS 4,289 CITATIONS
SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate,

letting you access and read them immediately.

Available from: Rongchang Yang

Retrieved on: 08 April 2016

ARTICLE IN PRESS
Experimental Parasitology (2015)

Contents lists available at ScienceDirect

Experimental Parasitology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

Full length article

1
2
3
4
5
6

Validation of cell-free culture using scanning electron microscopy

(SEM) and gene expression studies
Q2 R. Yang a, Y. Elankumaran a, N. Hijjawi b, U. Ryan a,*
a School of Veterinary and Life Sciences, Vector- and Water-Borne Pathogen Research Group, Murdoch University, Murdoch, Western Australia 6150,
Australia
b Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, The Hashemite University, PO Box 150459, Zarqa 13115, Jordan

7
8
9
10
11
12
13

H I G H L I G H T S

14
15
16
17
18
19
20
21
22
23

Scanning electron microscopy

(SEM) characterisation of life cycle
stages in cell-free culture.
Comparison of gene expression in
cell culture and cell-free culture.
Gene expression cell-free culture
was delayed and was lower.
Data provide support for cell-free
culture of Cryptosporidium.

G R A P H I C A L

A B S T R A C T

Mer

Mer
2 m

S
T

24

Mer
Mer
Cryptosporidium life cycles stages in cell-free culture

25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55

A R T I C L E

I N F O

Article history:
Received 8 September 2014
Received in revised form 13 January 2015
Accepted 3 March 2015
Available online
Keywords:
Cryptosporidium
SEM
mRNA expression
RT-qPCR
18S rRNA
COWP
cp15
gp900

A B S T R A C T

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture
using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h,
122 h and 170 h, chemically xed and specimens were observed using a Zeiss 1555 scanning electron
microscope. The presence of sporozoites, trophozoites and type I merozoites were identied by SEM. Gene
expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR
(RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium
oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell
culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and
COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the rst to compare gene
expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum
in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture
and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP

56
57
58
59

Q1

* Corresponding author.
E-mail address: Una.Ryan@murdoch.edu.au (U. Ryan).
http://dx.doi.org/10.1016/j.exppara.2015.03.002
0014-4894/ 2015 Published by Elsevier Inc.

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

ARTICLE IN PRESS
R. Yang et al./Experimental Parasitology (2015)

in cell free culture compared with the cell culture system and was lower. Although three life cycle
stageswere conclusively identied, improvements in SEM methodology should lead to the detection of
more life cycle stages.
2015 Published by Elsevier Inc.

1. Introduction
Protozoan parasites of the genus Cryptosporidium infect the epithelial cells of the intestinal tract of many mammals including
humans and cause the gastrointestinal disease cryptosporidiosis
(OHara and Chen, 2011). In immunocompetent individuals
cryptosporidiosis is characterised by self-limiting diarrhoea, nausea,
vomiting, fever and abdominal pain (Leitch and He, 2011). In immunosuppressed individuals, the infection can spread to
extraintestinal, hepatobiliary, pancreatic and pulmonary regions of
the body leading to chronic disease and wasting (Leitch and He,
2011).
Cryptosporidium is transmitted mainly via the faecal oral route
and contaminated water and has an infectious dose of as low as 10
100 oocysts (Baldursson and Karanis, 2011; Chappell et al., 2006).
Currently, nitazoxanide (NTZ) is approved for treatment of
cryptosporidiosis in children and immunocompetent adults in the
U.S.A., however treatment failures are common and NTZ is not effective without an appropriate immune response and is therefore
ineffective for the treatment of immunocompromised individuals
and young children (Gargala, 2008; Rossignol, 2010).
Once ingested, oocysts release sporozoites in the intestine, where
infections are predominately localised to the jejunum and ileum.
Cell invasion by the sporozoite is followed by intracellular development to a trophozoite stage, which undergoes asexual proliferation
to produce two different types of meronts. Merozoites released from
type I meronts enter other intestinal epithelial cells and either
develop into type II meronts or complete another cycle of type I
meronts. Merozoites from type II meronts then multiply sexually
to produce microgamonts and macrogamonts. The microgamonts
fertilise the macrogamonts producing zygotes, which mature into
oocysts (Hijjawi, 2010).
Cryptosporidium drug discovery has been hampered by lack of
an in vitro culture system that can continuously culture the parasite and the lack of genetic tools to construct transgenic reporter
parasites that would greatly facilitate screening efforts (Sharling et al.,
2010). Recent developments in the in vitro cultivation have revealed that Cryptosporidium can complete its life cycle in media
devoid of host cells (Aldeyarbi and Karanis, 2014; Boxell et al., 2008;
Hijjawi et al., 2004, 2010; Kartashev et al., 2009) and both cell and
cell-free cultures have demonstrated that Cryptosporidium may not
be an obligate intracellular parasite and can in fact multiply extracellularly (Borowski et al., 2010; Hijjawi et al., 2002; Huang et al.,
2014; Karanis et al., 2008; Koh et al., 2013; Rosales et al., 2005). These
ndings may reect the fact that Cryptosporidium is closely related
to gregarine protozoa (Barta and Thompson, 2006; Bull et al., 1998;
Carreno et al., 1999; Hijjawi et al., 2002; Leander et al., 2003; Rosales
et al., 2005).
Cell-free culture of Cryptosporidium is controversial, as some
authors have attempted to propagate Cryptosporidium spp. in cell
free culture, without success (Girouard et al., 2006). However, multiplication of Cryptosporidium DNA from cell-free cultures has been
reported (Hijjawi et al., 2010; Zhang et al., 2009) and various
Cryptosporidium developmental stages (sporozoites, trophozoites,
type I and II meronts) have been identied from biolms (Koh et al.,
2013). Immune labeling of life cycle stages in HCT-8 cell cultures
and in cell-free cultures has been previously described and has demonstrated that cell free stages bind the same antibodies as cell culture
stages (Boxell et al., 2008; Edwards et al., 2012). More recently, all
life cycle stages from cell-free culture have been described using

electron microscopy (Aldeyarbi and Karanis, 2014). The aim of the

present study was to further validate cell-free culture using scanning electron microscopy (SEM) to characterise life cycle stages in
more detail and to compare gene expression in cell-free versus conventional cell culture using genes used in a previous study of gene
expression in Cryptosporidium cell culture (Jakobi and Petry, 2006).

2. Materials and methods

2.1. Cryptosporidium isolate used in the present study
The C. parvum cattle isolate SC26 was used for SEM and gene expression studies. The isolate was originally obtained from the
Institute of Parasitology, University of Zurich and has been passaged and puried at Murdoch University following the protocol
described by Meloni and Thompson (1996). Puried oocysts of
C. parvum were stored in sterile 1 PBS at 4 C before use.
2.2. Bleaching and excystation of C. parvum oocysts
Puried oocysts were bleached in 10 mL of sterile water containing 0.25% sodium hypochlorite for 20 min at room temperature
and excysted as previously described (Hijjawi et al., 2010). Briey,
oocysts were resuspended in acidic water (pH 2.53) containing 0.5%
trypsin/EDTA to stimulate excystation and incubated at 37 C for
30 min with mixing every 5 min. Thereafter, the excystation suspension was centrifuged at 2000 g for 8 min and resuspended in
sterile water. As a control, intact but heat inactivated oocysts (incubated at 90 C for 30 min) were also processed for both cell-free
and HCT-8 cultivation.

2.3. Filtration of excysted oocysts to purify sporozoites

Following excystation of oocysts, the suspension was re-eluted
in 5 mL of maintenance medium and incubated at 37 C in a water
bath for at least 2 h to allow the full release of sporozoites from the
oocysts. This suspension was then ltered through a syringe lter
unit (Pall Scientic, New York, U.S.A.) containing a 2 mm IsoporeTM
membrane lter (Millipore, Massachusetts, USA), to remove all oocyst
shells and unexcysted oocysts, leaving a pure suspension of sporozoites. Following ltration, samples were checked by microscopy
to ensure that they were oocyst-free and sporozoites were enumerated using a haemocytometer. As a control, intact but heatinactivated puried sporozoites (incubated at 70 C for 30 min) were
also processed for both cell-free and HCT-8 cultivation.

2.4. Cell-free culture

Excysted C. parvum oocysts (1 mL containing 11.5 106 organisms) were placed into 75 cm2 culture asks containing 20 mL of
cell free maintenance media (100 mL RPMI-1640), as previously described by Hijjawi et al. (2004), with the exception that the layer
of coagulated newborn calf serum under the culture medium was
not included. Cultures were incubated at 37 C, 5% CO2. In order to
characterise life cycle stages of Cryptosporidium in cell free culture
at different time points by SEM, 1 mL samples were taken at 2 h,
8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h, 146 h and 170 h.

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

ARTICLE IN PRESS
R. Yang et al./Experimental Parasitology (2015)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66

2.5. HCT-8 cell culture

HCT-8 cells were grown in RPMI-1640, 10% FCS in 75 cm2 culture
asks and seeded 24 h prior to infection to allow them to reach monolayer as previously described by Hijjawi et al. (2002). HCT-8 cells
were infected by removing the existing media and adding 1 mL of
Cryptosporidium maintenance medium containing either 8 104 pretreated non-ltered oocysts or ltered sporozoites (through a 0.22 m
sterile lter) to each well and then incubated at 37 C with 5% CO2.
2.6. Cell-free culture for gene expression studies
Cell-free cultures were set up at the same time as the HCT-8 cell
cultures. One millilitre of Cryptosporidium maintenance medium containing either 8 104 pre-treated non-ltered oocysts or ltered
sporozoites (0.22 m) to each well was then incubated at 37 C incubator with 5% CO2.
2.7. Light microscopy
Cultures were examined regularly using an inverted light microscope (Olympus IMT-2) at magnications ranging from 100 to
400. Cultures were screened using this microscope to check for the
presence of life cycle stages and also to check for evidence of
contamination.

volumes. The following loci were amplied; the 18S ribosomal RNA
(rRNA) gene using primers described by Morgan et al. (1997), the
Cryptosporidium oocyst wall protein (COWP) gene using primers designed by Templeton et al. (2004), the sporozoite surface antigen
protein (cp15) gene using primers designed by Jenkins and Fayer
(1995) and the glycoprotein 900 (gp900) gene using primers designed by Barnes et al. (1998). Each 15 L PCR mixture contained
3 L of OneStep RT-PCR Buffer (Qiagen, Victoria, Australia), 1 L dNTP
mix (2 mM each), 2 units of OneStep RT-PCR Enzyme Mix (Qiagen,
Victoria, Australia), 1 unit of RNase inhibitor, 1 L of Syto 9 (2 M
nal concentration), 0.2 M each of forward and reverse primers
and 1 L of template RNA. The PCR cycling conditions consisted of
a reverse transcription stage at 95 C for 30 min and followed by an
initial PCR activation step of 15 min at 95 C then 40 cycles of 95 C
for 30 s, 58 C for 30 s and 72 C for 1 min. For calibration of the standard curve, the cDNA of the 18S rRNA, COWP, cp15 and cp900 loci
were quantied using droplet digital PCR (ddPCR) using a QX100TM
droplet digital PCR system (Bio-Rad) according to the manufacturers instructions. Ten-fold dilutions, from 50,000 to 5 copies, were
made as standards for RT-PCR, which was performed on the RotorGene
Q Real-time PCR machine (Qiagen, Victoria, Australia). The transcript copies for each of the 4 loci generated using RT-PCR were
calculated using RotorGene Q software. The amount of signal for
COWP, cp15 and cp900 was normalized to 18S rRNA for each time
period as previously described (Abrahamsen and Schroeder, 1999;
Mauzy et al., 2012; Schroeder et al., 1998).

2.8. SEM sample preparation

2.10. Statistical analysis
At each time point (2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h,
146 h and 170 h), media containing life-cycle stages in culture asks
were mixed by pipetting to lift any stages that were stuck to the
ask and then 2 mL aliquots was removed and centrifuged at 2000 g
for 5 minutes to pellet the life cycle stages. The supernatant was
removed and 500 L of 5% glutaraldehyde in phosphate buffer was
added to the pellet and stored at 4 C for 2 h or overnight.
The sample was centrifuged at 2000 g for 4 minutes. The supernatant was removed and the pellet was washed in 1 mL of
phosphate buffer, centrifuged again at 2000 g for 4 min and the
supernatant was removed. Five hundred microlitres of 1% osmium
tetroxide in phosphate buffer was added to the sample and incubated at 4 C for 1 hour. The sample was centrifuged at 2000 g for
4 min and the supernatant was removed. One millilitre of phosphate buffer was added to the sample and incubated at room
temperature for 15 min. The sample was again centrifuged at 2000 g
for 4 min and the supernatant was removed. One millilitre of 30%
ethanol was added to the sample, incubated at room temperature
for 15 min and centrifuged at 2000 g for 4 min. The process was
repeated with 50%, 70%, 90% and 100% ethanol. The sample was suspended in 100 L of 100% ethanol and was placed on a SEM stub
and allowed to air-dry overnight.
The SEM stub was coated with gold and was examined using the
Zeiss 1555 scanning electron microscope at the Centre for Microscopy and Characterisation Analysis at the University of Western
Australia.
2.9. Comparison of gene expression in HCT-8 versus cell-free
cultures
RNA extraction of HCT-8 and cell-free culture samples were performed using a Total RNA Isolation System (Promega, Corporate Park,
Sydney, Australia). The protocol was followed according to the manufacturers instructions with the exception that at the end of the
RNA extraction, 2 L of DNase I was added to all samples and incubated at 37 C for 30 min followed by the addition of 1 L of 500 mM
EDTA incubated at 65 C for 15 min. Reverse transcription (RT)
quantitative PCRs (qPCRs) were performed in triplicate in 15 L

Statistical analysis including calculation of standard deviation

and p-values (Students t-test) to compare transcript copies obtained from cell culture and cell-free culture and from nonltered oocysts versus ltered sporozoites was performed using the
Microsoft Excel software package.
3. Results
3.1. Scanning electron microscopy
Observations by electron microscopy of ltered sporozoites and
non-ltered oocyst samples from various time points showed the
presence of sporozoites, trophozoites and type I merozoite stages.
Sporozoites in cell-free culture were elongated and had dened apical
regions (Fig. 1A). Sporozoites measured between 3.25 0.5 m and
3.6 0.6 m. Trophozoites in cell-free culture were observed at 24 h.
Trophozoites were either spindle shaped or rounded and appeared in grape-like clusters (Fig. 1B). Trophozoites measured
11.1 m in size. Type I merozoites were observed as early as 22 h
in cell-free culture. Type I merozoites were rod-shaped with dened
apical regions (Fig. 1C) and were similar to those observed in cell
culture. Type I merozoites measured 0.70.9 m 1.541.75 m in
cell-free culture. Type I merozoites in cell-free cultures were present
in grape-like clusters similar to trophozoites (Fig. 1D).
3.2. Analysis of gene expression in both cell culture and cell-free
culture
The detection limit for the RT-qPCR assays used was 5 cDNA
copies for each of the four loci used.
3.2.1. 18S rRNA
Amplication of 18S rRNA revealed relatively constant expression between 2 h and 26 h for both cell culture and cell-free culture
but expression increased at 50 h and remained high (Fig. 2). Expression in cell-free cultures peaked at 146 h and was lower than
18S rRNA expression in cell culture. Gene expression between ltered

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132

ARTICLE IN PRESS
R. Yang et al./Experimental Parasitology (2015)

Mer

Mer
2 m

S
T
Mer
Mer
1
2
3
4

Fig. 1. Electron micrograph of (A) sporozoites at 0 h post-inoculation from a non-ltered oocyst sample in cell-free culture. (B) Trophozoites and type I merozoites with
apical regions at 46 h post-inoculation from ltered sporozoite samples in cell-free culture, (C) type I merozoites at 22 h post-inoculation from a non-ltered oocyst sample
in cell-free culture and (D) sporozoites, trophozoites and type I merozoites at 46 h post-inoculation from a ltered sporozoite sample in cell-free culture. Scale bar: 2 m.
S = Sporozoites, T = trophozoites and type 1 merozoites.

5
6
7
8
9

sporozoites and non-ltered oocysts appeared to be similar in both

types of cultures. All RT-qPCR assays were repeated at least twice
with different cDNA preparations for all time points and yielded
highly reproducible results.

10
11
12
13
14
15
16
17
18
19

3.2.2. CP15 gene

RT-qPCR analysis of cp15 showed that gene expression peaked
at 74 h for cell culture and 98 h for cell-free culture. Overall nonltered oocyst samples had more gene copies than ltered sporozoites,
(i.e. 189 and 120 gene copies were detected from non-ltered oocysts
and ltered sporozoites respectively in cell culture at 74 hours
whereas 65 and 48 gene copies were detected from non-ltered
oocysts and ltered sporozoites respectively in cell-free culture at
75 h (Fig. 3). This difference however, was not signicant (P < 0.05).

20
21
22
23
24
25
26
27

3.2.3. gp900 gene

gp900 expression between cell culture and cell-free culture was
similar. In cell culture gp900 expression peaked at 74 and 98 h and
in cell-free culture gene expression peaked at 74 h (Fig. 4). Transcript copies in non-ltered oocyst samples were higher than ltered
sporozoite samples for both cell culture and cell-free culture and
this difference was signicant (P < 0.05).

3.2.4. COWP gene

COWP expression in cell culture peaked at 50 h with a mean of
4016 gene copies detected whereas in cell-free culture, COWP expression was very low and peaked at 122 h (mean of 52 copies
detected) (Fig. 5). This difference was signicant (P < 0.05).

28
29
30
31
32
33

4. Discussion
In the present study, scanning electron microscopy was used to
analyse life cycle stages of C. parvum in cell-free culture. Observations of the SEM images revealed the presence of sporozoites,
trophozoites and type I merozoites. These stages were distinguished by their morphology and size and were similar to SEM
images of these life cycle stages from cell culture by Borowski et al.
(2010).
In cell free culture, sporozoites had an elongated shape and
dened apical regions and measured 3.25 0.5 m3.6 0.6 m.
Trophozoites in cell-free culture were observed at 24 h, which is
consistent with ndings in cell culture (Borowski et al., 2010). Trophozoites were either rounded or spindle shaped. In comparison
to trophozoites in cell culture, trophozoites in cell-free culture were
smaller in size and measured 11.1 m compared to <12.5 m
for cell culture (Borowski et al., 2010). Type I merozoites were

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

ARTICLE IN PRESS
R. Yang et al./Experimental Parasitology (2015)

Number of gene transcripts amplified

18S rRNA expression

4000

Cell culture

4000

3500

3500

3000

3000

2500

2500

2000

2000

1500

1500

1000

1000

500

500

Cell Free

Time (hours)
1

Fig. 2. 18S rRNA expression in (A) cell culture and (B) cell-free cultures for non-ltered oocysts, ltered sporozoites and control samples at 2, 8, 14, 26, 50, 74, 98 and 122 h.

Number of gene transcripts amplified

cp15 Expression
Cell culture

Cell Free

3000
600
2500
500
2000
400
1500
300
1000
200
500

100

Time (hours)
3

Fig. 3. cp15 Expression in (A) cell culture and (B) cell-free culture for non-ltered oocysts, ltered sporozoites and control samples at 2, 8, 14, 26, 50, 74, 98 and 122 h.

4
5
6
7
8
9

observed as early as 22 h in cell-free culture which is consistent

with observations in cell culture (Borowski et al., 2010). Type I merozoites were rod-shaped with dened apical regions and measured
0.70.9 m 1.541.75 m in cell-free culture. In cell culture however,
type I merozoites were 0.4 1 m in size (Borowski et al., 2010).

These differences in size between life-cycle stages in cell-culture

and cell-free may have been due to shrinkage during processing.
The time points at which the life cycle stages were identied in
cell-free culture were similar to observations made in cell culture
(Borowski et al., 2010). This is the rst time these life cycle stages

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

10
11
12
13
14

ARTICLE IN PRESS
R. Yang et al./Experimental Parasitology (2015)

gp900 Expression
Number of gene transcripts amplified

Cell culture

Cell Free

300

300

250

250

200

200

150

150

100

100

50

50

Time (hours)
1

Fig. 4. gp900 expression in (A) cell culture and (B) cell-free for non-ltered oocysts, ltered sporozoites and control samples at 2, 8, 14, 26, 50, 74, 98 and 122 h.

Number of gene transcripts amplified

COWP Expression
Cell culture

Cell Free
200

30000

180
25000

160
140

20000
120
100

15000

80
10000

60
40

5000
20
0

Time (hours)
3

Fig. 5. COWP expression in (A) cell culture and (B) cell-free culture for non-ltered oocysts, ltered sporozoites and control samples at 2, 8, 14, 26, 50, 74, 98 and 122 h.

4
5
6
7
8
9
10
11
12
13

from cell-free culture have been identied by SEM and provides

further support for the ability of Cryptosporidium to proceed through
its life cycle in cell-free culture.
Other life cycle stages were not identied in cell free cultures
but this may be due to air-drying method used to dry the SEM
samples. When samples are air-dried, surface tension occurring
during evaporation of dehydrating uids such as alcohol can cause
substantial and irreversible deformation of structures (Buravkov
et al., 2011). Therefore small and fragile life cycle stages such as

microgamonts may not be identied because the air drying method

could have caused them to collapse.
An alternative drying method that could be used instead of air
drying is critical-point drying. Critical point drying involves the replacement of the organic dehydration uid in the specimen with
liquid carbon dioxide and subsequent conversion of the CO2 into
gas. After the gaseous CO2 is released the specimen is dried without
having passed through any phase boundary thus reducing the
amount of shrinkage (Holgate and Webb, 2003).

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

14
15
16
17
18
19
20
21
22

ARTICLE IN PRESS
R. Yang et al./Experimental Parasitology (2015)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66

In the present study, critical point drying was also attempted to

dry C. parvum samples however, results showed that life cycle stages
appeared to be more degraded and in most cases no specimen was
seen, suggesting that the life cycle may have been washed away in
the critical point drying chamber. For critical point drying, samples
are required to be xed onto poly-L-lysine coated cover slips
(Borowski et al., 2010). Therefore it is possible that the samples may
not have been adequately xed onto the poly-L-lysine coated cover
slips and lost during processing.
Unlike cell culture, where host cells were grown on cover slips
and infected with C. parvum, it was dicult to harvest the life cycle
stages in cell-free culture, as they were free-oating in cell-free
media. Therefore the processing method involved many mechanically disruptive steps such as centrifugation and removal of
supernatant using a pipette, all of which would have resulted in loss
of sample.
For future SEM studies on C. parvum in cell-free culture, less invasive methods such as freeze-drying or cryo-SEM could be utilised
for best results. In freeze-drying, wet samples are fast frozen, transferred to a freeze-drying unit where a low temperature (80 C to
90 C) is maintained and the ice is sublimed slowly under vacuum.
This method has been shown to retain microstructure features of
specimens (Holgate and Webb, 2003).
This is the rst study to compare gene expression patterns of
C. parvum in cell culture and cell-free culture. The genes used in the
present study were chosen to provide a comparison with a previous study, which examined gene expression in cell culture (Jakobi
and Petry, 2006). The cp15 gene is expressed by the infective sporozoite and merozoite stages (Jenkins and Fayer, 1995) and is involved
in the invasion and/or the host immune response to infection (Moss
et al., 1998; Sturbaum et al., 2003; Tilley and Upton, 1994). The gp900
gene is a microneme protein involved in invasion that has been deQ3 tected in both sporozoites and merozoites (Bonnin et al., 2001;
Cevallos et al., 2000; Petersen et al., 1992). The COWP gene has been
localised in the wall-forming bodies of early and late macrogametes and the inner layer of the oocyst wall (Spano et al., 1997).
Amplication of 18S rRNA was used as an indicator of total transcriptional activity.
Overall, similar results were obtained in the present study for
HCT-8 cell cultures, compared to a previous study by Jakobi and Petry
(2006) for gene expression studies of cp15, gp900 and COWP, with
some differences. Gene expression, however, was delayed for CP15
and COWP in cell-free culture and the number of gene copies for
all genes tested was lower in cell-free culture compared to cell
culture. In the study by Jakobi and Petry (2006), 18S rRNA expression increased between 2 h and 8 h p.i., was constant between 8 h
and 26 h p.i. and declined thereafter. In another study by Abrahamsen
and Schroeder (1999), on C. parvum infected HCT-8 cells, 18S rRNA
levels steadily increased over time (648 h), were maximal at 48 h
and were substantially decreased at 72 h. In the present study, 18S
rRNA expression in both cell culture and cell-free was relatively constant between 2 h and 26 h but expression increased at 50 h and
remained high.
For cp15, Jakobi and Petry (2006) reported that expression in
HCT-8 cell cultures was detected at 2 h p.i. and peaked at 26 h.
However, in the present study, cp15 expression was detected at 8 h
and peaked at 74 h in cell culture and in cell-free culture peaked
at 98 h. Expression of cp15 in cell-free culture was nearly three times
the level of expression in cell free. gp900 expression was detected
at 14 h and peaked at 14 h and 26 h in the study by Jakobi and Petry
(2006), whereas in the present study, gp900 was also detected at
14 h however gene expression peaked at 74 h and 98 h in cell culture
and at 74 h for cell-free culture. For COWP, the study by Jakobi and
Petry (2006) showed that COWP expression was detected and peaked
at 50 h and declined thereafter. Similarly, the study by Abrahamsen
and Schroeder (1999) also reported that COWP mRNA levels in HCT-8

cell cultures were nearly undetectable from 6 to 24 h and dramatically increased at 48 h although in that study, COWP expression
remained elevated at 72 h. In the current study, COWP expression
in cell culture was also detected at 50 h and peaked at 50 h and 74 h
(which is consistent with expression of the COWP gene exclusively by macrogametes (Spano et al., 1997) . In cell-free culture
however, COWP expression was very low (052 gene copies) and
peaked at 122 h. This suggests that the development of new oocysts
in cell-free culture may be delayed compared to cell culture.
A recent study conducted a genome-wide transcriptome analysis of C. parvum infected HCT-8 cells over a 72 hr period (Mauzy
et al., 2012). Quantitative-PCR (qPCR) for 3302 genes (87% of the
protein coding genes) indicated that each gene has detectable transcription in at least one time point assessed (Mauzy et al., 2012).
Further studies, which involve a wider range of genes, should be
conducted to better understand the expression of Cryptosporidium
genes in cell-free culture.
The present study investigated for the rst time gene expression of C. parvum in cell-free culture and compared it with gene
expression in cell culture. Results showed that gene expression patterns between cell culture and cell-free culture were similar but
delayed in cell-free culture. This study also analysed for the rst time
life cycle stages of C. parvum in cell-free culture. Sporozoites, trophozoites and type I merozoites were identied. Together, these
initial data provide further support for the ability of Cryptosporidium
to proceed through its life cycle stages although further studies are
required.

67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94

References
Abrahamsen, M.S., Schroeder, A.A., 1999. Characterization of intracellular
Cryptosporidium parvum gene expression. Mol. Biochem. Parasitol. 104 (1),
141146.
Aldeyarbi, H.M., Karanis, P., 2014. Electron microscopic study of in vitro axenic
development of Cryptosporidium parvum. Fifth International Giardia and
Cryptosporidium Conference. Uppsala, Sweden. May 2730, p.64.
Baldursson, S., Karanis, P., 2011. Waterborne transmission of protozoan parasites:
review of worldwide outbreaks an update 20042010. Water Res. 45 (20),
66036614.
Barnes, D.A., Bonnin, A., Huang, J.X., Gousset, L., Wu, J., Gut, J., et al., 1998. A novel
multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates
invasion. Mol. Biochem. Parasitol. 96, 93110.
Barta, J.R., Thompson, R.C., 2006. What is Cryptosporidium? Reappraising its biology
and phylogenetic anities. Trends Parasitol. 22, 463468.
Bonnin, A., Ojcius, D.M., Souque, P., Barnes, D.A., Doyle, P.S., Gut, J., et al., 2001.
Characterization of a monoclonal antibody reacting with antigen-4 domain of
gp900 in Cryptosporidium parvum invasive stages. Parasitol. Res. 87, 589592.
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, 1326.
Boxell, A., Hijjawi, N., Monis, P., Ryan, U., 2008. Comparison of various staining
methods for the detection of Cryptosporidium in cell-free culture. Exp. Parasitol.
120, 6772.
Bull, S., Chalmers, R., Sturdee, A.P., Curry, A., Kennaugh, J., 1998. Cross reaction of
an anti-Cryptosporidium monoclonal antibody with sporocysts of Monocystis
species. Vet. Parasitol. 77, 195197.
Buravkov, S.V., Chernikov, V.P., Buravkova, L.B., 2011. Simple method of specimen
preparation for scanning electron microscopy. Bull. Exp. Biol. Med. 151, 378382.
Carreno, R.A., Martin, D.S., Barta, J.R., 1999. Cryptosporidium is more closely related
to gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan
parasites inferred using small-subunit ribosomal RNA gene sequences. Parasitol.
Res. 85, 899904.
Cevallos, A.M., Bhat, N., Verdon, R., Hamer, D.H., Stein, B., Tzipori, S., et al., 2000.
Mediation of Cryptosporidium parvum infection in vitro by mucin-like
glycoproteins dened by a neutralizing monoclonal antibody. Infect. Immun. 68,
51675175.
Chappell, C.L., Okhuysen, P.C., Langer-Curry, R., Widmer, G., Akiyoshi, D.E., Tanriverdi,
S., et al., 2006. Cryptosporidium hominis: experimental challenge of healthy adults.
Am. J. Trop. Med. Hyg. 75, 851857.
Edwards, H., Thompson, R.C., Koh, W.H., Clode, P.L., 2012. Labeling surface epitopes
to identify Cryptosporidium life stages using a scanning electron microscopy-based
immunogold approach. Mol. Cell. Probes 26 (1), 2128.
Gargala, G., 2008. Drug treatment and novel drug target against Cryptosporidium.
Parasite 15, 275281.
Girouard, D., Gallant, J., Akiyoshi, D.E., Nunnari, J., Tzipori, S., 2006. Failure to propagate
Cryptosporidium spp. in cell-free culture. J. Parasitol. 92 (2), 399400.

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142

ARTICLE IN PRESS
8

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39

R. Yang et al./Experimental Parasitology (2015)

Hijjawi, N., 2010. Cryptosporidium: new developments in cell culture. Exp. Parasitol.
124, 5460.
Hijjawi, N., Estcourt, A., Yang, R., Monis, P., Ryan, U., 2010. Complete development
and multiplication of Cryptosporidium hominis in cell-free culture. Vet. Parasitol.
169, 2936.
Hijjawi, N.S., Meloni, B.P., Ryan, U.M., Olson, M.E., Thompson, R.C.A., 2002. Successful
in vitro cultivation of Cryptosporidium andersoni: evidence for the existence of
novel extracellular stages in the life cycle and implications for the classication
of Cryptosporidium. Int. J. Parasitol. 32, 17191726.
Hijjawi, N.S., Meloni, B.P., Nganzo, M., Ryan, U., Olson, M.E., Cox, P.T., et al., 2004.
Complete development of Cryptosporidium parvum in host cell-free culture. Int.
J. Parasitol. 34, 769777.
Holgate, J.H., Webb, J., 2003. Microscopy: transmission electron microscopy. In:
Benjamin, C. (Ed.), Encyclopedia of Food Sciences and Nutrition, second ed.
Academic Press., Oxford, pp. 39283934.
Huang, L., Zhu, H., Zhang, S., Wang, R., Liu, L., Jian, F., et al., 2014. An in vitro model
of infection of chicken embryos by Cryptosporidium baileyi. Exp. Parasitol. 147,
4147.
Jakobi, V., Petry, F., 2006. Differential expression of Cryptosporidium parvum genes
encoding sporozoite surface antigens in infected HCT-8 host cells. Microbes Infect.
8, 21862194.
Jenkins, M.C., Fayer, R., 1995. Cloning and expression of cDNA encoding an antigenic
Cryptosporidium parvum protein. Mol. Biochem. Parasitol. 71, 4952.
Karanis, P., Kimura, A., Nagasawa, H., Igarashi, I., Suzuki, N., 2008. Observations on
Cryptosporidium life cycle stages during excystation. J. Parasitol. 94, 298
300.
Kartashev, V.V., Ambalov, I., Beketova, E.V., Ponomarenko, I., Malysheva, M.I., Bykov,
S.A., 2009. Possibilities of culturing Cryptosporidia in the cell-free medium. Med.
Parazitol. (Mosk) 4, 1920.
Koh, W., Clode, P.L., Monis, P., Thompson, R.C., 2013. Multiplication of the waterborne
pathogen Cryptosporidium parvum in an aquatic biolm system. Parasit. Vectors.
6, 270.
Leander, B.S., Clopton, R.E., Keeling, P.J., 2003. Phylogeny of gregarines (Apicomplexa)
as inferred from small-subunit rDNA and beta-tubulin. Int. J. Syst. Evol. Microbiol.
53, 345354.
Leitch, G.J., He, Q., 2011. Cryptosporidiosis-an overview. J. Biomed. Res. 25, 116.
Mauzy, M.J., Enomoto, S., Lancto, C.A., Abrahamsen, M.S., Rutherford, M.S., 2012. The
Cryptosporidium parvum transcriptome during in vitro development. PLoS ONE
7 (3), e31715.

Meloni, B.P., Thompson, R.C.A., 1996. Simplied methods for obtaining puried oocysts
from mice and for growing Cryptosporidium parvum in vitro. J. Parasitol. 82,
757762.
Morgan, U.M., Constantine, C.C., Forbes, D.A., Thompson, R.C.A., 1997. Differentiation
between human and animal isolates of Cryptosporidium parvum using rDNA
sequencing and direct PCR analysis. J. Parasitol. 83, 825830.
Moss, D.M., Chappell, C.L., Okhuysen, P.C., DuPont, H.L., Arrowood, M.J., Hightower,
A.W., et al., 1998. The antibody response to 27-, 17-, and 15-kDa Cryptosporidium
antigens following experimental infection in humans. J. Infect. Dis. 178, 827e833.
OHara, S.P., Chen, X.M., 2011. The cell biology of Cryptosporidium infection. Microbes
Infect. 13, 721730.
Petersen, C., Gut, J., Doyle, P.S., Crabb, J.H., Nelson, R.G., Leech, J.H., 1992.
Characterization of a > 900,000-M(r) Cryptosporidium parvum sporozoite
glycoprotein recognized by protective hyperimmune bovine colostral
immunoglobulin. Infect. Immun. 60, 51325138.
Rosales, M.J., Cordn, G.P., Moreno, M.S., Snchez, C.M., Mascar, C., 2005. Extracellular
like-gregarine stages of Cryptosporidium parvum. Acta Trop. 95, 7478.
Rossignol, J.F., 2010. Cryptosporidium and Giardia: treatment options and prospects
for new drugs. Exp. Parasitol. 124, 4553.
Schroeder, A.A., Brown, A.M., Abrahamsen, M.S., 1998. Identication and cloning of
a developmentally regulated Cryptosporidium parvum gene by differential mRNA
display PCR. Gene 216, 327334.
Sharling, L., Liu, X., Gollapalli, D.R., Maurya, S.K., Hedstrom, L., Striepen, B., 2010. A
screening pipeline for antiparasitic agents targeting Cryptosporidium inosine
monophosphate dehydrogenase. PloS Negl. Trop. Dis. 4, e794.
Spano, R., Purl, C., Ranucci, L., Putignani, L., Crisanti, A., 1997. Cloning of the entire
COWP gene of Cryptosporidium parvum and ultrastructural localization of the
protein during sexual stage development. Parasitology 114, 427437.
Sturbaum, G.D., Jost, B.H., Sterling, C.R., 2003. Nucleotide changes within three
Cryptosporidium parvum surface protein encoding genes differentiate genotype
I from genotype II isolates. Mol. Biochem. Parasitol. 128, 8790.
Templeton, T.J., Lancto, C.A., Vigdorovich, V., Liu, C., London, N.R., Hadsall, K., et al.,
2004. The Cryptosporidium oocyst wall protein is a member of a multigene family
and has a homolog in Toxoplasma. Infect. Immun. 72, 980987.
Tilley, M., Upton, S.J., 1994. Both CP15 and CP25 are left as trails behind gliding
sporozoites of Cryptosporidium parvum (Apicomplexa). FEMS Microbiol. Lett. 120,
275279.
Zhang, L., Sheoran, A.S., Widmer, G., 2009. Cryptosporidium parvum DNA replication
in cell-free culture. J. Parasitol. 955, 12391242.

Please cite this article in press as: R. Yang, Y. Elankumaran, N. Hijjawi, U. Ryan, Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression
studies, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.002

40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78