PARINT-01501; No of Pages 4

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Parasitology International

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First description of an in vitro culture system for Eimeria ovinoidalis

macromeront formation in primary host endothelial cells
Tessa Carrau a,b, Liliana Machado Ribeiro Silva a,⁎, David Pérez b, Rocio Ruiz de Ybáñez b,
Anja Taubert a, Carlos Hermosilla a
a
Institute of Parasitology, Justus Liebig University Giessen, BFS, Schubertstr. 81, 35392 Giessen, Germany
b
Parasitología, Departamento de Sanidad Animal, Facultad de Veterinaria, Campus de Excelencia Internacional Regional “Campus Mare Nostrum”, Universidad de Murcia, 30100 Espinardo, Mur-
cia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine coccidiosis. As
Received 22 December 2015 one of the most pathogenic species in sheep, the principal clinical sign is profuse diarrhoea in young animals,
Received in revised form 5 April 2016 which leads to important economic losses in the ovine industry. We here aimed to establish an in vitro culture
Accepted 3 May 2016
system for the development of E. ovinoidalis macromeronts, as no suitable systems are currently available for
Available online xxxx
any ovine Eimeria species. Faecal samples containing more than 90% of E. ovinoidalis oocysts were collected
Keywords:
from naturally infected lambs and ewes in Murcia Region (Spain). E. ovinoidalis oocysts were collected, left to
Eimeria ovinoidalis sporulate in potassium dichromate and stored at 4 °C until further studies were conducted. Moreover, a suitable
In vitro culture system excystation protocol was effectively established, resulting in the release of viable sporozoites, which were
Ovine allowed to infect primary bovine umbilical vein endothelial cells (BUVEC) and permanent bovine colonic epithe-
BUVEC lial cells (BCEC). In vitro first merogony was successfully accomplished exclusively in BUVEC leading to
First merogony macromeront formation (up to 100 μm) and the release of fully developed and viable merozoites I stages.
Given that we were able to establish a suitable in vitro system for the first merogony of such pathogenic Eimeria
species in sheep, advances might be further made not only on studies regarding the control of ovine coccidiosis,
such as drug screenings, but also on the better understanding of molecular parasite–host cell interactions as al-
ready demonstrated for other ruminant Eimeria species.
© 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Eimeria ovinoidalis is a monoxenous apicomplexan parasite of the
genus Eimeria considered one of the most pathogenic species within
the sheep industry. Sheep coccidiosis due to E. ovinoidalis infections
have been reported worldwide, causing severe typhlocolitis mainly in
young lambs [1]. In vivo, the first-generation macromeronts of E.
ovinoidalis can reach sizes of up to 300 μm within 9 days post-infection
(p.i.) in host epithelial cells of the small intestine and thereby releasing
N120.000 merozoites I. Hereinafter, a rather fast second merogony
occur (3 days) in host epithelial cells in the crypts of the large intestine,
and resulting merozoites II then have to undergo the sexual gamogony
again in the same host cells type [2].
E. ovinoidalis coccidiosis become a substantial livestock farming
problem, especially with increased stocking densities and reduced
⁎ Corresponding author.
E-mail address: Liliana.Silva@vetmed.uni-giessen.de (L.M.R. Silva).
availability of pasture for sheep [2]. The principal symptom observed
is profuse diarrhoea related to the loss of gut absorptive capacity com-
bined with watery and mucous faeces, followed by dehydration and
consequent weight loss [2].
Currently, the global sheep population is around one billion animals,
in which Europe and Central Asia share 12% of the world's ovine popu-
lation. Moreover, Mediterranean basin countries have been the leaders
of small ruminant production in Europe. As such, Spain rears in total
16.5 million head of sheep [3].
Unfortunately, there is no currently available in vitro system for the
development of any sheep Eimeria spp. (e.g. E. ovinoidalis, E. bakuensis
and E. crandalis), even though suitable in vitro systems have been suc-
cessfully described for other Eimeria species [4–7].
We therefore aimed to establish an in vitro culture system for E.
ovinoidalis macromeront formation, using primary host endothelial-
and permanent host epithelial-cells for consequent production of viable
merozoites I, which could be used in future studies concerning basic re-
search on molecular parasite–host cell interactions [4,8].

http://dx.doi.org/10.1016/j.parint.2016.05.003
1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: T. Carrau, et al., First description of an in vitro culture system for Eimeria ovinoidalis macromeront formation in primary
host endothelial cells, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.05.003
2 T. Carrau et al. / Parasitology International xxx (2016) xxx–xxx
2. Material and methods
E. ovinoidalis oocysts used for the in vitro experiments were isolated
from naturally infected sheep. Briefly, faecal samples collected directly
from the rectum of naturally infected lambs and ewes, from Murcia Re-
gion, Spain, were assessed with a modified McMaster technique for de-
termination of the number of Eimeria oocysts per gram of faeces (OPG).
Eimeria species identification was based on morphometric characteris-
tics [9] after sporulation in 2% (w/v) potassium dichromate for
12 days, at room temperature (RT), with aeration every 2 to 4 h by stir-
ring the oocysts mixture.
Samples containing more than 90% of E. ovinoidalis oocysts were
stored in a 2% (w/v) potassium dichromate solution at 4 °C until further
use [4].
For isolation of viable E. ovinoidalis sporozoites [9], sporulated E.
ovinoidalis oocysts stock solution was added to 4% (v/v) sodium hy-
pochlorite solution and magnetically stirred on ice for 20 min. After
vortex for 20 s, oocyst solution was centrifuged (300 × g, 5 min) and
supernatant was mixed with bi-distilled water (1:1). Pelleted oo-
cyst solution (400 × g, 10 min) was then layered in Percoll™ (GE
Healthcare, UK) 60% gradients and centrifuged for 20 min at
400 × g. After centrifugation, oocyst bands were collected and fil-
tered through a 15 μm pore size PluriSelect ® filter system. After-
wards, oocysts were washed (600 × g, 20 min) in cell culture
medium M199 (Gibco) supplemented with 1% (v/v) penicillin
(500 U/ml; Sigma-Aldrich) and streptomycin (500 μg/ml; Sigma-Al-
drich) (PS). Then, oocysts were suspended in sterile 0.02 M L-cyste-
ine/0.2 M NaHCO 3 (Merck) solution and incubated in a 100% CO 2
atmosphere (37 °C, 20 h). Thereafter, oocysts were re-suspended
in the excystation medium: Hank's balanced salt solution (HBSS,
Gibco) containing 0.4% (w/v) trypsin (Sigma) and 8% (v/v) sterile
filtered bovine bile (due to the lack of ovine bile availability), and
incubated up to 4 h (37 °C, 5% CO 2 atmosphere). Every hour,
excystation progress was checked and the number of released spo-
rozoites was estimated. Freshly egressed sporozoites were then
washed twice (600 × g, 15 min) and suspended in tissue culture me-
dium until further use.
Ruminant primary endothelial cells were used as host cells for E.
ovinoidalis macromeront formation and production of viable mero-
zoite I stages. Briefly, primary bovine umbilical vein endothelial
cells (BUVEC) were isolated [10] and cultured in endothelial cells
growth medium (ECGM, PromoCell). Medium was changed with
modified ECGM (ModECGM, ECGM mixed with 70% (v/v) M199,
10% FCS and 1% PS) every 2–3 days until confluent BUVEC mono-
layers (n = 2) were infected with 1.5 × 103 freshly excysted sporozo-
ites. Culture medium was changed at 24 h p.i. and then successively
every 2 days. In order to keep up with E. ovinoidalis macromeront de-
velopment and merozoites I release, infected cells were investigated
daily until 22 days p.i.
For comparative purposes, permanent epithelial cells were also
infected with freshly excysted sporozoites. Primary bovine colonic
epithelial cells (BCEC) [11] were maintained in Iscove's modified
Dulbecco culture medium (IMEM, Gibco) supplemented with 1%
PS, 10% FCS and 2% L -glutamine (all from Sigma-Aldrich). Culture
medium was changed every 2–3 days until confluent BCEC mono-
layers were infected. Culture medium was changed at 24 h p.i. and
then successively every 2 days. Infected host cells were daily inves-
tigated until 22 days p.i.
E. ovinoidalis macromeront formation in host cells is an energy- and
nutrient-demanding process. In order to improve the parasite energy
supply in vitro, the supplementation of cell culture media with 10 mM
glucose [D(+)-glucose anhydrous; Roth] was additionally carried out
from 7 days p.i. onwards. Size of macromeronts was measured and
total merozoite I production was estimated after the rupture of E.
ovinoidalis-macromeront maturation in glucose-treated and untreated
controls.
Please cite this article as: T. Carrau, et al., First description of an in vitro culture system for Eimeria ovinoidalis macromeront formation in primary
host endothelial cells, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.05.003
3. Results
Field collected E. ovinoidalis sporulated oocysts presented an ovoid
shape and were slightly flattened at the micropyle end. The oocysts
consisted of two well-formed oocyst wall layers. The outer oocyst wall
was smooth and colourless and the inner wall layer was yellowish. Mea-
surements of oocysts revealed a medium length of 19.7 to 31.3 μm
(24.1 ± 2.2) and a medium width of 15.4 to 23.7 μm (19.2 ± 1.6), ac-
cording to the morphology of this species [9]. The achieved E. ovinoidalis
sporogony varied from 7 to 12 days at RT and constant O2 supply.
Viable and active sporozoites were successfully obtained from spor-
ulated E. ovinoidalis oocysts. After 45 min of oocyst incubation in
excystation medium, sporozoites started to show dynamic movements
within sporocysts and after 45 min of incubation, the first free-released
sporozoites were visibly moving within the oocysts circumplasm (sup-
plementary data: movie). Then, circumplasm-located sporozoites were
able to egress through the oocyst micropyle. The excystation process
continued up to 4 h, as approximately 40% of the sporozoites were re-
leased from sporulated oocysts despite the use of bovine bile. Further-
more, freshly released sporozoites showed typical movements such as
gliding motility and contractility, evidencing their viability.
BUVEC and BCEC confluent monolayers were exposed to freshly
egressed E. ovinoidalis sporozoites to allow parasite invasion and further
intracellular development. After 120 min of infection, most of the sporo-
zoites had completed host cell invasion in both cell lines, and were
found close to the host cell nucleus. Occasional sporozoite egress was
observed from day 2 p.i. onwards until 22 days p.i.
At 24 h p.i., the morphology of intracellular sporozoites changed, be-
coming shorter and thicker than extracellular sporozoites (Fig. 1A), and
thereby initiating the development into trophozoite stages. Moreover,
during E. ovinoidalis intracellular development, morphological changes
of the host endothelial cell nucleus were observed: from a usually
dark-spotted heterochromatine type to a “fried egg-shaped”
euchromatine nucleus type [12,13].
Despite the ability of sporozoites to invade both cell lines, no further
intracellular development in BCEC cells was observed. In contrast, in
BUVEC, while some sporozoites persisted unaltered until the end of
the observation period (22 days p.i.), others clearly rounded up and
transformed into well-defined trophozoite stages (Fig. 1B). Subsequent-
ly, E. ovinoidalis trophozoites developed into immature meronts I
starting from 14 to 17 days after sporozoite infection (Fig. 1C,D). Some
trophozoites delayed or even completely stopped their further develop-
ment. Regarding macromeront formation, not all immature meronts I
stages developed into fully mature macromeronts (Fig. 1D). Therefore,
at the end of the observation period, a rather unsynchronous
macromeront development was observed (Fig. 1D,E). Complete matu-
ration of E. ovinoidalis macromeronts was achieved at 17–22 days p.i.,
showing rosette-like structures (Fig. 1E) clearly corresponding to mor-
phological features of in vivo occurring macromeront stages [14]. At
18–22 days p.i., rupture of mature macromeronts was observed with re-
lease of motile and viable merozoites I (Fig. 1F, arrows), but also rupture
of macromeronts, releasing a mixture of completely and incompletely
developed merozoites I was detected.
4. Discussion
E. ovinoidalis is one of the most pathogenic species in ovine coccidi-
osis due to its massive replication capacity within the first merogony in
host epithelial cells [15]. In this work, we accomplished to isolate an al-
most pure (90%) E. ovinoidalis field strain from Spain.
Until now, an appropriate ovine Eimeria in vitro culture system is still
missed in the literature, despite the fact that more data are available on
suitable in vitro systems for other related ruminant species with
macromeront formation [8,9,14]. Therefore, the aim of this study was
to establish a suitable in vitro system for E. ovinoidalis macromeront de-
velopment. This new in vitro system will be a useful tool to study early
 T. Carrau et al. / Parasitology International xxx (2016) xxx–xxx 3

Fig. 1. Infection and in vitro development of Eimeria ovinoidalis in BUVEC. Confluent BUVEC were infected with freshly excysted E. ovinoidalis sporozoites, and macromeront development
was monitored until 22 days p.i.: (A) At 1 day p.i., intracellular sporozoites (arrows) presented morphology changes becoming shorter and thicker than extracellular sporozoites; (B)
trophozoites (arrows) were observed from 7 days p.i. onwards; (C) immature macromeront formation was observed at 14 days p.i., and (D) at 18 days p.i.; (E) at 17 days p.i., mature
meronts with fully developed merozoites I were witnessed; (F) free released merozoites I (arrows) were detected from 18 days p.i.

molecular host cell–parasite interactions as successfully reported for re-
lated ruminant Eimeria species [7,8,13,16].
During the excystation process and in contrast to what occurs during
Eimeria bovis (cattle) or Eimeria ninakohlyakimovae (goat) excystation
in vitro, E. ovinoidalis sporozoites egressed through the micropyle after
being released into the oocyst circumplasm, as observed in Eimeria
arloingi [7]. Excystation resulted in the release of viable and motile spo-
rozoites, which were able to actively infect host endothelial cells and to
continue further obligate intracellular development.
In vivo, severe E. ovinoidalis infection in lambs causes lesions of the
follicle-associated epithelium and thereby atrophy of the ileal Peyer's
patch follicles [17]. However, E. ovinoidalis sporozoites failed to develop
in epithelial cells, but were able to undergo successful intracellular
macromeront formation in endothelial cells, releasing fully developed
merozoites I at 18–22 days p.i., as reported for other permanent cell
lines infected with other Eimeira species [7]. In disagreement with our
findings, best in vitro development of caprine E. ninakohlyakimovae
macromeronts was demonstrated in permanent BCEC, followed by pri-
mary BUVEC cultures [6].
Sporozoite egression was constantly observed to occur until the end
of the experimental period [6,7]. We postulate that egressed E.
ovinoidalis sporozoites might have been able to reinvade more suitable
host endothelial cells. Apicomplexan sporozoite egress is usually associ-
ated with the need of sporozoites to reach their final specific host cells,
thereby transmigrating through host cells by breaching the plasma
membrane without forming a parasitophorous vacuole [10,18]. None-
theless, in vivo Eimeria sporozoites can remain in dormozoite/
hynozoite-like stages for an indefinite time [19]. Moreover, intracellular
stages of E. ovinoidalis (sporozoites, trophozoites, macromeronts) did
not further develop synchronously in vitro. Therefore, intracellular E.

Please cite this article as: T. Carrau, et al., First description of an in vitro culture system for Eimeria ovinoidalis macromeront formation in primary
host endothelial cells, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.05.003
4 T. Carrau et al. / Parasitology International xxx (2016) xxx–xxx
ovinoidalis macromeront development exhibited different sizes and
morphologies, and even diverse degrees of maturation.
Improvement of in vitro culture of E. ovinoidalis with supplementa-
tion of culture medium with glucose was performed, proving the impor-
tance of carbohydrates for apicomplexan parasite growth in vitro [20],
and for the increment of Eimeria spp. merozoites I production.
Overall, in vitro E. ovinoidalis macromeront formation was successful.
Hereby, we gained access to 3 different stages of the same parasite spe-
cies, i.e. sporozoites, trophozoites and merozoites I, representing a valu-
able tool for the replacement of animal experiments concerning
pharmaceutical screenings against these particular parasitic stages, as
well as for basic research applications such as genome-wide tran-
scriptome [12].
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.parint.2016.05.003.
Acknowledgments
The authors would like to thank the Murcian farmers for their collab-
oration in sample collection and to Brigitte Hofmann and Dr. Christin
Ritter (Institute of Parasitology, JLU Giessen) for their help in cell culture
maintenance and parasite excystation protocols.
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