REPRODUCTION OF THE CYCLE OF COCCIDIA
EIMERIA ACERVULINA (TYZZER, 1929) IN CELL
CULTURES OF CHICKEN KIDNEYS
Muriel Naciri-Bontemps
To cite this version:
Muriel Naciri-Bontemps. REPRODUCTION OF THE CYCLE OF COCCIDIA EIMERIA
ACERVULINA (TYZZER, 1929) IN CELL CULTURES OF CHICKEN KIDNEYS. Annales
de Recherches Vétérinaires, INRA Editions, 1976, 7 (3), pp.223-230. <hal-00900893>
HAL Id: hal-00900893
https://hal.archives-ouvertes.fr/hal-00900893
Submitted on 1 Jan 1976
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REPRODUCTION OF THE CYCLE
OF COCCIDIA EIMERIA ACERVULINA (TYZZER,
IN CELL CULTURES OF CHICKEN KIDNEYS
1929)
Muriel NACIRI-BONTEMPS
Laboratoire de Parasitologie, 1. N. R. A..
Centre de Recherches de Tours,
Nouzilly, 37380 Monnaie
SUMMARY
The cycle of Eimeria acervuldna was grown in primary culture of cells of three-week-oldchicken kidneys. EHT medium (Eagle, Hydrolysate of Lactalbumine, Bacto-Tryptose phosphate)
allowed the development of this coccidium.
44 hours after infestation of the cells with sporozoites, the first schizonts appeared. The
merozoites of first generation were released after 54 hours. They invaded the neighbouring cells
and developed in them into schizonts of second generation mature after 68 hours. After 72 and
3 hours, a third and a fourth generation of schizonts were noticed.
g
Merozoites IV create the Gamogony. To obtain the gamogony in vitro, we inoculated the
ultures with merozoites IV recovered from axenic animals. The oocysts were released into the
’
45 hours after inoculation.
nedium
1.
-
INTRODUCTION
These last ten years, several authors have been able to reproduce the cycle of
coccidia, either in embryos or in cell cultures. It was rather easy
ia acervulina and Eimeria !naxima. In
y
ia, except for Eime
y
Eime
to
not
obtain
it
was
embryos,
development after inoculations of sporopossible
zoites of Eimeria acervulina (LONG, i966 ; SHIBALOVA, 1969, 1970, 1972). Recently,
ia mivati is only a variety of E. acervulina
y
LONG )
1973 has considered that Eime
(
TROUT
and that it is the only one capable of multiplying in embryos. In cell cultures, S
of
of
E.
acervulina
to
the
obtained
a
et al. 6
only
beginning
development
stage
19
(
)
5
of immature first generation schizonts. ITAGA! et al. )
1974 also observed the pene(
tration of the sporozoites without further development.
!rtain species of
for the chicken’s
The present importance of intestinal species and especially of E. acervulina
incited us to return to this problem. These cultures are indeed of great importance
for pharmacological or immunological studies. To avoid the problems of cell division
linked to the duration of the parasitic cycle, we tried to obtain the asexual and sexual
stages separately, starting either from the sporozoite or the merozoite obtained in
axenic animals.
II.
-
MATERIALS
.
1
-
AND METHODS
Cell cultures
cultures of chicken kidneys are used. They are obtained from the trypsinized
of three week old animals. The cells are suspended in PBS and their number is estimated
o cells/ml
with Thoma cell. The cultures are established on coverslips in Petri dishes from 700 00
or 7 X I
6 per dix. The growth medium contains 5 p. 100 hydrolysate of lactalbumine, 75
o
,6 p. Too
deionized water, 8,
4 p. 100 Earle (X 10
), 5 p. Too acto-tryptose phosphate, 5 p. Too calf serum,
0 UI per ml Penicillin and 100 ¡
10
g per ml Streptomycin.
L
The pH is adjusted to 7
°C
2 with sodium bicarbonate. The Petri dishes are placed at 37
,
in an atmophere of 5 p. 100 C0
. The variations of the pH are estimated with phenol red.
2
Primary cell
kidneys
.
2
.r.
2
-
-
The
parasite
rJbtaining the parasitic stvai?!.
was purified by inoculation of four-week
old-chickens with a single oocyst isolated by micro-manipulation. Four days later, the oocysts are recovered in the duodenum.
We used two infective germs :
The strain
-
-
.
2
the sporozoite
the merozoite
-
Obtaining
the
sporozoites.
The oocyst sterilization is achieved by passage in sodium hypochlorite (NaOCl 45
°, 8 p. ioo) for
half an hour. The sporocysts are released by grinding. Excystation is obtained by placing the sporocysts in an agitated medium of trypsin pig bile, at pH 7
,6, at 41
°C. The released sporozoites are
suspended in PBS and purified by passage in a leuko-pak column containing synthetic fibres
O
B
E
T
MPS
N P. YvoR>:,
(Muriel ,
1974).
.
3
2.
-
Obtaining
the merazoites.
They are obtained in four-week-old axenic chickens. The animals are given 500 000 oocysts,
sterilized by successive passage in sodium hypochlorite 45
.
° at 10 p. 100 and in formol at 10 p. 100
The duodenal contents are recovered 8
9 hours later and placed in a trypsin solution at
25 p. Too. After digestion, the cell debris are eliminated by passage in the leuko-pak column of
,
0
synthetic fibres. We thus obtain numerous clean and sterile merozoites.
.
3
1
.
3
-
fnoculatiox
-
of
Irtoeudation of the
the
cell cultures and maintenance
sporozoites.
After 4
8 hours, the cells form a 25 p. 100 confluent cellular monolayer. It is then inoculated
with the sporozoites. The inoculation medium is similar to the growth medium and contains
150
ooo sporozoites of E, acervulina per ml. The Petri dishes are placed at 4
C and 4 hours later
o
the inoculum is removed and replaced by a maintenance medium containing only 2 p. 100 bactotryptose phosphate and z p. 100 calf serum. Every 24 hours, the medium is changed to maintain
the pH at 7
.
2
,
2
.
3
-
Inoculation
of the
The inoculum contains
20
I
mevozoites.
million merozoites per ml medium. It is left
on
the cell
layer during
hours, then the medium is changed daily.
.
4
Samples
they
are
are
taken every
stained with
12
-
Fixation and
hours. The cells
are
staining
fixed with Acetic-Methanol
V
I
(
V
3
),
/
then
May-Grunwald-Giemsa.
III
i.
- RESULTAT9
- Sehizogony
of thesporozoites into the cells.
The microscopic examination of a dish after inoculation of the sporozoites of
E. acervulina, without any staining, enables one to note mobile parasites above the
cells. These parasites show a rather indistinct central nucleus, a very clear refringent
posterior globule and sometimes a small refringent anterior globule. They penetrate
the cells the first minutes after inoculation and they invariably settle near the nucleus
.
I
-
Penetration
of the host-cell.
After fixation and staining with May-Grunwald Giemsa, the intracellular
sporozoites can be distinguished from those which have not yet penetrated the cells
by the presence of a peripheral vacuole in the cytoplasm of the host-cell (Pl. I,
ph. z). They measure about 7 to 10 !./2 to 3 !1- and are easily recognizable thanks to
their eosinophile refringent globule that will persist during the total duration of the
first schizogony.
=.2,
-
T!O!/KM’<M’<gS.
From the 20
th to the 3
oth hour, the intracellular sporozoites develop into trophoare very similar to sporozoites but they are much larger, particularly
about their median parts. Their nuclei have indeed become bigger and their posterior
;They
zoites
refringent globules more spherical.
They measure about 7
,8 ,6
1-. This shape is predominant
k/3 !
t
’
at 30 hours . At
almost
observed
:
some
be
are
totally spherical
44 hours, 2 types of trophozoites
9 S
,
1-/6, others show a cytoplasmic outgrowth
!
-;
!1
) and measure about 7
(Pl. I, ph. 2
that often contains the refringent globule and they look like the trophozoites (B
GAK
A
IT
OR and IN
D
N
I, 68)
R
V>;TT>~
G
19 or type III described by I
(
type) described by A
et al. 74).
sg
(
can
.
3
i.
-
First
generation schizonts.
2 days after inoculation, numerous first generation schizonts are present in the
cultures. The nucleus of the trophozoite divides and produces a young binucleate
first generation schizont that measures about 9 !t/6 !
1-. Then the size of the schizont
nuclei
increase.
The
and the number of.
refringent globule, smaller than a nucleus,
clear.
This
but
becomes
less
still
visible
is
globule, characteristic of the ist generation,
as
as
soon
the
ist
generation merozoites start appearing. The immature
disappears
ist generation schizonts are very polymorphous. The sizes are therefore variable.
The schizont (Pl. II, ph. 7
,6 fL/IOA !,.
) is slighly oblong and measures about 15
is over, the cytoplasm surrounds each nucleus and the
the
nuclear
division
When
merozoites appear. At 5
4 hours, numerous mature ist generation schizonts can be
6 ist
observed. They measure 10 to 19 fL/15 to i8 !, and contain, on an average, 1
to
to
2
a
around
residual
merozoites
5
turning
body.
measuring 4
!,
{!1,5
generation
.4.
1
-
Second
generation
schizonts.
From 54 hours, a few ist generation merozoites released from the schizonts
be observed. They are mobile but they do not move much over the surface of
nd generation trophozoites that
the culture, invading the neighbouring cells to give 2
develop into schizonts (Pl. II, ph. io). These schizonts are smaller than ist generation
schizonts. They are usually spherical and measure about 12 [L/
io !,. When they are
merozoites
on an average.
68
contain
10
n
2
d
after
mature,
hours, they
generation
There is no residual body (Pl. II, ph. II
).
can
generation schixonts.
nd generation merozoites have the same size that the ist generation meroThe 2
zoites. They penetrate the cells and develop in them into 3
rd generation schizonts
about 19 [A/15
rd generation merozoites
. which, when mature, contain about 6 3
V
nd generation schizonts, 6 to 8 long. After 73 hours, these
larger than the ist or 2
merozoites are released into the medium (Pl. II, ph. 12
).
.5.
1
.6.
1
-
-
Third
Fourth
generation
schizonts.
From 73 hours, we observe nests of 4
th generation schizonts (Pl. III, ph. 13
)
that can reach very big sizes, 5
i!,/20 !. (Pl. III, ph. 14
).Some schizonts have compartmented cytoplasm and can contain 4
th generation merozoites 9
fL to 10
fL long that
will give gamogony. But it is not rare, even at II9 hours, to meet mature 4
th generation schizonts.
.
2
-
Gamogorcy
The inoculation of our cell cultures with fourth generation merozoites, recovered
hours after infestation of axenic chickens, enabled us to obtain the gamogony of
E. aceruulina. These merozoites penetrate the cells rapidly and develop in them into
trophozoites that develop into either male microgamonts or female macrogamonts.
9
8
1
.
2
-
ogametogenesis.
y
Mic
Until 21 hours, it is not possible to distinguish the trophozoite (Pl. II, ph. 10
)
that will give a microgametocyte, from the trophozoite that will develop into macro8 hours, the young microgametocytes are identified by
gametocyte. From 21 to 2
the presence of a great number of small masses of nuclear material scattered in the
cytoplasm. These masses, more or less spherical or ovoid, come from the intensive
division of the nucleus of the trophozoite. As the microgametocyte matures, they
become oblong and finally comma-shaped. Then they progressively separate from
the central cytoplasmic mass and appear at the periphery (Pl. IV, ph. 17
).
From 2
8 to 45 hours, numerous mature microgamonts, of variable sizes, with
8 V/12 !t (Pl. IV, ph. 1
sometimes indistinct walls, measuring about 1
8), contain
microgametes which are too numerous to be counted. These microgametes are releaperipheral vacuole and then released into the cell.
measure
2 to 2
They
,5 li
, are mobile and move thanks to their flagella. The
are
visible
under
the optical microscope. The microgametes fecundate
hardly
flagella
the macrogamonts that develop almost simultaneously in the neighbouring cells.
sed into the
.
2
2.
-
Macrogametogenesis.
Unlike in microgametogenesis, the nucleus of the sporozite does not divide
but on the contrary increases in breadth and, after staining with May-GrunwaldGiemsa, appears like a large light patch with a very dark slightly eccentric nucleole
(Pl. IV, ph. 19).
The young macrogametocyte is often egg-shaped. It measures 13 !,!7,8 !, at
hours and contains a fine granulous cytoplasm. Then, as it grows old, the nucleus
becomes spherical and its volume decreases whereas the nucleole becomes bigger
and sometimes spreads over the whole nuclear space. After 2
8 hours, we can observe
old macrogametes whose cytoplasm is full of big plastinoid granules that appear
like vacuoles at the periphery (Pl. IV, ph. 20
). At this stage, they can be fecundated.
After the fecundation, a zygote appears. The nucleus of the macrogamete
becomes less clear and disappears. The cytoplasmic inclusions gather at the periphery and help to the forming of the shell of the oocyst. The cytoplasm retracts
and becomes spherical, forming a cytoplasmic mass which is still visible through
the thin and transparent shell of the young oocyst. Then this shell becomes thicker
and opaque and it can no longer be stained with May-Grunwald-Giemsa (PI. IV,
ph. 2
z), At about 45 hours, the cell release the oocyst thus formed into the medium.
It measures about 1
6 to 1
8 !./r3 to 15 [1
..
21
IV.
-
DISCUSSION AND CONCLUSION
If we compare the cycle obtained in cell cultures to the normal development
of E. acervulina in the chicken (V!’r’r!xr.mtG and ,
ORAN 19
D
66), we note they are
very similar.
Indeed, the four schizogonies occur in the same lengths of time as in vivo. The
schizonts are usually larger in vitro but, on an average, they contain the same number
of merozoites, except for the 4
th generation schizonts which are much bigger and
a
hundred
merozoites
instead of 32
.
produce
takes
also
Gamogony
place in 40 hours. The gamonts and the oocysts obtained
have the same size that those met in vivo. After 45 hours, no parasite can be seen in
vulina in cell cultures.
y
our cells. It is therefore possible to reproduce the cycle of E. ace
It is not easy to recognize the different stages ; an important number of schizogonies
8 hours (from 40 hours after
4 generations) appear indeed in a little more than 4
(
inoculation to 95 hours).
Moreover, it is not rare
st generation schizonts
to meet at the same time young 1
schizonts.
with their refringent globules and 3
In vitro, we also find
rd generation
these different generations present at the same time, just as they can be found
in the animal. This contrasts with the development of E. te!aella. Each generation
of E. tenella occurs indeed slower and is more distinct.
The conditions of culture and the medium have a great effect on the development of the parasite in the cells. In an EHT medium, in a CO, enriched atmosphere,
B type trophozoites can be observed (Doxnrr and ,
RLING 19
E
TT
VE
68) (Pl. III and
IV). They develop in a normal way into schizonts which, once mature, keep their
cytoplasmic outgrowth (Pl. I, ph. 6). The first generation schizonts produced by
these B type schizonts are identical to those produced by the normal schizonts
(A type). Yet, no B type schizont can be found in cultures made in EHT + Hepes (
).
1
Besides, in trials carried out with Eagle-Tris -f- io p. 100 calf serum we could
not obtain a development. Yet, when we used foetal calf serum we could
observe a few ist generation schizonts. Their development was delayed and the
schizonts did not reach maturity. This shows the importance of the culture medium
and explains perhaps why other authors failed to obtain development.
Reçu pour pubdication
en
mai 1976.
RÉSUMÉ
.
REPRODUCTION DU
(TYZZER, 1929)
CYCLE DE LA COCCIDIE ElME
A
Rf
ACERVULINA
SUR CULTURES DE CELLULES DE REIN DE POULET
Le cycle d’Eimeria acervulina est réalisé sur culture primaire de cellules de rein de poulet
de trois semaines. Le milieu qui nous a permis d’obtenir le développement de cette coccidie
est le milieu EHT (Eagle, Hydrolysat de lactalbumine, Bacto-Tryptose phosphate).
Après infestation des cellules (temps o) avec des sporozoïtes, les premiers schizontes apparaissent vers 44 heures. Les mérozoïtes de r
re génération sont libérés vers 54 heures. Ils envahissent
les cellules voisines et s’y développent, donnant des schizontes de 2e génération mûrs vers 68
heures. A partir de 72 heures et 93 heures se déroulent respectivement une 3e et une
e génération
4
de schizontes.
Les mérozoïtes IV donnent naissance à la Gamogonie. Pour obtenir celle-ci in vitro, nous
avons inoculé les cultures avec des mérozoïtes IV récupérés sur animaux axéniques. Les oocystes
sont libérés dans le milieu 45 heures après l’inoculation.
RÉFÉRENCES BIBLIOGRAPHIQUES
BorrTEMrs M., Y
VORE P., 1974
. Technique de purification de suspensions de sporozoites d’Eimeria
sur colonne de fibres synthétiques.
Ann. Rech, vét., 5, rog-rr
.
3
ETTERLING S. M., 19
ORAN D. J., V
D
. Survival and development of Eimeria meleagrimitis Tyzzer
5
6
1929 in bovine kidney and turkey intestine ceH cultures. J. Protozool., 15, 0
6c8
9
7
z
.
IRAY N., U
U
SUBOK
T
R
A M., O
TSUKI K., 1974
AK K., AMA
G
A
IT
I
. Development of Eimeria tenella
E. brunetti and E. acervulina in cell cultures. Jap. J. Vet. Sci., 36, 4
67.
2
8
LONG 19
of
n
eriaEi
in avian embryos. 575
P. L., The growth of some species 1
66.
J, Parasitol.,
8
5
I.
56,
LONG 1973
P. L., Studies on the relationship between E. acervulina and E. mivati. Parasitology,
.
67,
-155.
143
SxIaAI,ovA T. A., Koxol,nv A.
OBCHAK 1. A., 19
M., S
. Cultivation of chicken coccidia in chick
9
6
embryos (in Russian). l’eterinariya, Moskva, 11, 68,.
7
r.ovA T. A., rg
SA
SxI
o. Cultivation of the endogenous stages of chicken coccidia in embryos and tissue
7
cultures. J. Parasitol.,56 (
, sect. II, part. I), 315-3i6!
4
)
the
use
Acide 4
[(hydroxy-2
piperaziiiyl- i -éthyl)
j -2 -éthane
of C0
2 is not necessary
sulfonique C
8 H&dquo; N20!S
’
P.M. : 23
.
30
8,
Thus,