Chabaud, A.G.

Deharo, Eric

Gautret, P.

Ginsburg, H.

Landau, L.

Horizon / Pleins textes

PLASMODIUM VINCKEI VINCKEI, P. ~ LENTUM AND P. YOEUI YOEUI :
CHRONOBIOLOGY OF THE ASEXUAL CYCLE IN THE BLOOD
GAUfRET P.·, DEHARO E.·, CHABAUD A.G.·, GINSBURG H.·· and LANDAU L·
,
,
. .
'.
Summary:
The biological rhythms of Plasmodium vinckei vinckei, P. v. lentum
and P. yoelii yoelii i.e. synchronicity, duration of the erythrocytic
cycle, timing of the schizogony and of the penetration of mero-
zoites into red blood cells, were studied in the Swiss white mouse.
Two different methods of synchronisation were used: the freezing-
thawing of parasitized blood and the inoculation of a single para-
sitic stage, separated from the other stages by centrifugation
through a Percoll®Glucose gradient. The duration of the schizogo-
nic cycle of P. v. vinckei and P. v. lentum, two highly synchronous
subspecies, was 24 hours. With P. v. vinckei the timing of the
schizogony was independent of the circadian rhythm of the host
and was set by the time of inoculation. With P. v. lentum the
timing of the schizogony and merozoites penetration into red
blood cells depended both, on the hasts rhythm and the lime of
inoculation of frozen-thawed blood: schizogany occurred at
18:00 if the inoculum was injected at 06:00 or 12:00, and at
06:00 if injected at 18:00 or 00:01. P. y. yoelii a naturally asyn-
chronous parasite was synchronized by means of a Percoll®-
Glucose gradient. The duration of its intraerythrocytic cycle was
found to be 18 hours, similar to that of the other subspecies of P.
yoelii.
KEY WORDS : Plasmodium vinckei vinckei. Plasmodium vinckei len/um.
Plasmodium yoeli; yoelii. duration 011he erythrocytic cycle. timing of the ery-
throcytic cycle. synchronicity. biologicol rhythms.
INTRODUO"ION
T he rodent malarias have been used as modelsfor studying various aspects of the biology ofmalaria parasites for more than 40 years. Recent
work (Montalvo et al., 1988, Cambie et al., 1990,
Landau et al., 1990) has shown that chronobiological
data are of great importance because the biological
rhythm of the rodent parasites may vary from one spe-
cies to the other, or even from one subspecies or strain
to the other. The consequence of these notions is that,
in many cases, an accurate interpretation of experimen-
tal results cannot be achieved without taking into
account these chronobiological data. In this work, we
• Laboraloire de Biologie Parasilaire, associe au CNRS (URA 114), et
Labordloire de Protozoologie el Parasitologie comparee (EPHE),
Museum Nalional d'Histoire Nalurelle, 61 rue BulTon, 75231 Paris
cedex 05, France.
•• Department of Biological Chemislry, The Hebrew Universiry of
Jerusalem, Jerusalem, Israel.
Parasile, 1994, 1,235-239
Resume -' CIlRO:,\OBlOLOGIE PL' CYCLE SCHIZOGONlQCE ERYrnROCY-
TAIRE PE PI.ASAIODJI:II 17.\CKEJ 17.\CKEJ, P. I'. U-~\7VM ET P. IOEUlIOEllI.
Etude des rythmes biologiques de 10 schizogonie erythrocytaire de
Plasmodium vinckei vinckei, P. v. lentum et P. yoelii yoelii.
Les caracleres etudies sont le synchronisme plus ou moins marque
des differents stades, 10 durre des cycles, les horaires des schizogo-
nies et 10 chronologie de 10 penetration des merozoiles dons les
hematies.
Deux techniques de synchronisation ont ete utilisees: f'inoculation de
sang infectant congele puis brutalement decongele ou bien f'inocv-
lotion d'un seul stade {Xlrasitaire, se{Xlre des autres stades {Xlr cen-
trifugation sur gradient de Percoll- glucose.
La durre du cycle des deux sous-espiK:es tres synchrones
Plasmodium v. vinckei et P. v. lentum est de 24 heures,
Avec P.v. vinckei, f'horaire de 10 schizogonie est independont du
rythme circadien de f'hote et est determine {Xlr f'heure de f'inocula-
tion. Avec P v. lentum, f'horaire de la schizogonie et de 10 penetra-
tion des merozoiles dans les hemoties depend ci la fois du rythme
circadien de f'hote et de f'heure d'inocufation du song decongele:
10 schizogonie survient ci 18 heures si f'inoculotion est faite ci 6
heures, ou ci midi et ci 6 heures si f'inoculotion est faite ci 18 heures
ou ci minuit. La sous-espiK:e tres osynchrone P y. yoelii a ete syn-
chronisre {Xlr 10 technique du gradient Percoll- glucose La durre du
cycle est de I8 heures. identique ci celle des autres saus-espiK:es de
P yoelii
MOJS CI.ES: Plasmodium vinckei vinckei. P. v, lenlum. P yoeIii yoelii. Durre du
cycle erylhrocytaire. Horoire du cycle erythrocytoire. synchronisme. rythmes bio-
fogiques.
have investigated the duration of the asexual erythrocy-
tic cycle and the "timing niche" (Cambie et al., 1990) of
2 subspecies of P. vinckei and one of P. yoelii. Two
methods of synchronization have been employed
according to the strain: 1) P. vinckei : freezing-thawing
of infected blood which increases the natural synchro-
nicity of the strain (Montalvo et aI., 1988); 2) P. yoeli :
centrifugation through a Percoll®-Glucose gradient
(Deharo et aI., 1994) which results in a synchronous
infection for at least 2 schizogonic cycles.
MAJERIAL AND METHODS
STRAINS
- Plasmodium vinckei vinckei Rodhain, 1952, (67) a
synchronous parasite from Za·ire.
- Plasmodium vinckei lentum Landau, Michel, Adam
and Boulard, 1970, 094 ZZ L) a synchronous parasite
from the Congo Republic.
- Plasmodium yoelii yoelii Landau and Killick- RESULTS
Kendrick, 1966, (265 BY), an asynchronous parasite ---------------------
from the Centralafrican Republic. p. V. VlNCKEI
60
METHODS OF SYNCHRONIZATION
- Freezing-thawing of injected blood: Rapid freezing
and thawing before injecting infected blood from a
donor to a recipient mouse was shown (Montalvo
Alvarez et al., 1988) to destroy most intra-cellular
parasites and to allow in the recipient mouse an
infection initiated by the only survi\'ing stage, the free
merozoite. The method has since been used for stu-
dies on chronotherapy with P. v. petteri (Cambie et
al., 1991, Caillard et al., 1992 and 1993). In the pre-
sent study the method was employed to determine
the relationships between the time of inoculation and
the time of schizogony of P. v. villckei and P. v. len-
turn. In all experiments, mice were inoculated intra-
peritoneally with the same amount of blood from a
frozen aliquoted stock. It is impossible to determine
the number of merozoites inoculated by this tech-
nique but all mice, in a given experiment, received
the same inoculum.
- Percoll®-Glucose gradient: the technique was devi-
sed (Deharo et al., 1994) for synchronizing P. yoelii.
Parasitized blood (P. y. yoelii) was centrifuged
through a discontinous gradient of Percoll®-Glucose
and separated into two main layers: a layer contai-
ning old parasites : Mid-term trophozoite (MT); Old
trophozoite (aT); Schizont (S) and a layer containing
only Rings (R) and Young trophozoites (YT). The lat-
ter was inoculated intravenously into mice in which a
patent, synchronous infection, evoh'ed over the first
two schizogonic cycles.
COURSE OF PARASITEMIAS
Male Swiss outbred mice (Iffa Credo, France) were
used for the follow-up of the infection. Thin smears
were made from tail blood, fixed in methanol and
stained by Giemsa stain. Parasitemias were evaluated
by examining 2000 red blood cells. The parasitic pat-
tern can be expressed as the relative proportion of
each stage: Rs, YTs, MTs, OTs and Ss according to
the classification by Cambie et at.. 1991. The evolu-
tion of the parasitemia and the parasitic pattern was
followed in P. v. vinckei from day -! and in P. v. len-
turn from day 8 post inoculation. ""hen parasitemias
reached 1 % or more. In P. y. yoelii. infections were
immediately patent. Blood smears ""ere performed at
3 or 6 hours interval during 48 or 54 hours and all
experiments were stopped before the parasitic crisis,
as described by Bastien et al. (986).
Two batches of 3 mice each were inoculated with
frozen-thawed infected blood, one at 12:00, the other
at 00:01. The evolution of the parasitic pattern can be
followed in the curves of Fig. 1. The peak of the
stages contained from 60 to 70 % of that stage. When
the inoculation was performed at 12:00, Rs peaked at
12:00, YTs at 18:00, MTs at 00:01, OTs at 06:00. When
the inoculation was performed at 00:01, Rs peaked at
00:01, YTs at 06:00, MTs at 12:00, OTs at 18:00. The
interval between peaks was consistently 24 hours.
In conclusion, the duration of the schizogonic cycle is
24 hours; the timing of the schizogony is independent
of the circadian rhythm of the host and depends only
on the time of inoculation. Schizogony occurred 24
hours post-inoculation and this indicates that the
penetration of merozoites inoculated with the frozen
blood is immediate.
100 STAGES (%)
R
BO'
Y.T
HO
,A
40 / \: i
20. ~J .. "
'-.
100 .-
M.T
BO
60 j\40
20 V~
.--
100
O.T
HO-
60 \
40 \J20 ~.
0
12 IH 0 6 12 IH 0 (, 12 IH
TIME (hI'S)
Fig. 1 - Follow-up or lhe parasilic pallern (mean percentages or
each slage) or P. v. l'inckei. Time of inoculalion: 12:00 null line)
and 00:01 (inlermillenl line).
,
i
1
r
236--------------- Parasile, 1994, 1, 235-239
,P. V. LENTUM
Four batches of three mice each were inoculated with
frozen-thawed infected blood, at different times:
06:00, 12:00, 00:01 and 18:00. The evolution of the
parasitic pattern is represented in the curves of Fig. 2.
Peaks of stages, except rings, reached from 60 to
70 % of that stage. Peaks of Rs did not exceed 34% of
Rs due to the fact that the duration of the stage is 3
hours only and that blood smears were performed
every 6 hours. In mice inoculated at 18:00 and 00:01,
Rs peaked at 06:00, YTs at 12:00, MTs at 18:00, OTs
at 00:01. In mice inoculated at 06:00 and 12:00, Rs
peaked at 18:00, YTs at 00:01, MTs at 06:00 and OTs
at 12:00. The curves showing the evolution of stages
in the mice inoculated at 06:00 are mean values for
two mice; the third one in the batch reached crisis
time before the end of the experiment and was dis-
carded. The interval between peaks was consistently
24 hours.
In conclusion, P. v. fenturn has a synchronous deve-
lopment, the duration of a schizogonic cycle is 24
hours, the timing of the schizogony depends on the
time of inoculation and also on the circadian rhythm
of the host. When mice were inoculated at 06:00 or
12:00, schizogony occurred around 18:00, while it
occurred around 06:00 in mice inoculated at 00:01 or
18:00.
P. Y. YOELll
Four mice were inoculated intravenously at 13:00
with Rs and YTs concentrated in a layer of the
Percoll®-Glucose gradient. The parasitemia and para-
sitic pattern were e\'aluated at 3 hours intervals
during the 54 hours post-inoculation and results are
plotted in Table 1. The stage composition of the ino-
culum injected at 13:00 was 63 % Rs and 37 % YTs. In
Fig. 3, the curve represents the evolution of the per-
centages of Rs during the 54 hours follow-up. The
peaks of Rs occurred at 13:00 (time of inoculation), at
07:00 on Day 1, and at 01:00 on Day 2. They were
separated by an intef\'al of 18 hours. Other stages'
peaks, as can be seen in Table I are also 18 hours
apart. However, the height of the peaks tended to
decrease as the infection became less synchronous
(after the second cycle), as shown in table I where it
can be seen that, 54 hours post-inoculation, all stages
could be found.
In conclusion, the duration of a schizogonic cycle of
P. y. yoefii is 18 hours: the parasite remains synchro-
nous during the first t\,"o cycles; afterwards particular
stages do not predominate so evidently.
..
lOO STAGES (%)
R
80'
60-
40-
20 - - . •
lOO--~~
Y.T
80-
80
: ... ,\. ..••.. \
20 ~ ,.~.
100---
O.T
80·
- . -
: '\ :~,0>:"./\ I.. '\ /'
20 '\ ) "',. " \) ,
V ' 'v'
o
12 18 0 6 12 III 0 6 12 18
TIME (h,,,)
lOO STAGES (%)
R
80
60
40
I:----C~~-
Y.T
80
100---.--"-
M.T
80
lOO I
O.T
80
60 ~ . : '/\' .
\2•. + /~.
:: ·V.~L'
o
o 6 12 III 0 6 12 18 0
TIME (h,,)
Fig. 2 - Follow-up of the parasitic pattern (mean
percentages of each stage) of P. u. lenturn; left:
Time of inoculation 12:00 (full line) and 18:00
(dashed line). right: Time of inoculation: 00:01
(full line) and 06:00 (dashed line).
I-'P~ara~Sit~e:.,::199~4,:..-1!.:.,~2~3~5'':23~9~ ••••••••••• _
- ?'l.7
RINGS (%)
100
80
60
40
20
o
13 16 19 22 4 7 10 13 16 19 22 4 7 10 13 16 19
TIME (hrs)
Fig. 3 - Follow-up of the rings percentage of P. y. yoelii.
TIME PARASITEMlA R Y.T M.T G.T S I
(Ius) % ± SO % ± SO % ± SO % ± SO % ± SO % ± SO
13:00 0.1 0 62.7 5.8 37.3 5.8 0 0 0 0 0 0
16:00 0.1 0 17.3 8.7 70.7 12.5 12 9.6 0 0 0 0
19:00 0.1 0 3.7 2.5 26.7 5.8 67 3 2.7 3.1 0.7 1.2
22:00 0.1 0 0 0 8.7 2.3 63.3 12.7 27.3 15.4 0.7 1.2
01:00 0.2 0.1 0 0 3.3 3.1 23.7 5.5 71.7 10.4 1.3 2.3
04:00 0.2 0.1 4.3 0.6 1.7 2.9 12 6.1 61.3 10.3 20.7 4.5
07:00 0.3 0.1 48 5.6 21.7 1.5 3 0 16 8.5 11.3 4.7
10:00 0.4 0.2 29.7 10.1 57.7 4 7.3 5.5 2.7 3.8 2.7 2.3
13:00 0.5 0.2 4.7 1.2 31 4.6 57.3 2.3 5 1.7 2 1.7
16:00 0.5 0 0 0 13.3 4.2 64 6 22 8.7 0.7 1.2
19:00 0.7 0.2 0 0 3.7 0.6 23.7 7.8 72.7 9 3.3 4.2
22:00 0.7 0.2 13.3 8.1 10 7.2 10.7 5.9 55 6.2 10.3 3.8
01:00 1.5 0.3 46 3.5 22 10.6 7 3.6 15 4.6 10 7.21
04:00 2.8 1.9 19 9.9 52.7 7.8 11.3 1.2 7.7 3.8 9.3 2.3
07:00 2.9 1.8 4.7 3.1 34 6.9 47.7 0.6 12 2 1.7 2.1
10:00 1.9 0.5 2 3.5 13.7 10 54.7 4.2 27.7 7.4 2 3.5
13:00 1.9 0.7 4.7 5 9 2.7 27.3 7.1 52.3 5.1 6.7 5.8
16:00 2 0.6 9.3 5.1 20 4.6 19 7 44.7 10 7 3.6
19:00 2.6 1 37 6.2 21.3 6 14.3 4 22 10.6 5 5
Table 1 - Follow-up of the parasitemia and parasitic pattern (percentage of each stage) of P. y. yoelii.
_ Parasite, 1994, 1,235-239238---------------i f---------------
•
I
DISCUSSION ACKNOWLEDGMENTS
Accepte le 15 juin 1994
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75,449-456.
CAlIlARD V., BEAUTE-1.AFrrrE A., CHAfiAUD A.G., GINSBURG H. :
and lANOAV L Stage sensitivity of Plasmodium vinckei
petteri to Quinine, Mefloquine and Pyrimethamine.
journal ofParasitology, (in press).
CAMBIE G., 1.ANDAV L et CHABALU A. G. : Niches horaires des
trois especes de Plasmodies coexistant chez un Rongeur
de Centrafrique. Comptes Rendus de l'Academie des
Sciences de Paris, 1990,310, 183-188.
CAMBIE G., CAILLARO V., BEACTE-LAFllTE A., GINSBVRG H.,
CHABAlJU A.G. and I.ANOAC L : Chronotherapy of malaria:
Identification of drug-sensitive stage of parasite and
timing of drug delivel)' for improved therapy. Anna/es de
Parasitologie Humaine et Comparee, 1991, 66, 14-21.
DEHARO E., GAlITRET P., GINSBURG H., CHABAUD A. and 1.ANDAV
L : Synchronization of P. yoelii nigeriensis and P. yoelii
killicki infection in the mouse by means of Percoll-glu-
cose gradient stage fractionation. Determination of the
duration of the schizogonic cycle. Parasitology Research,
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GRVLET 0., 1.ANDAV L et BACCAM D. : Les Isospora du moi-
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(
T he present study demonstrates that very clo-sely related parasites like the subspecies of P.vinckei may show important chronobiological
differences. In all the subspecies studied, the length
of the schizogonic cycle and the duration of each
stage appeared to be identical. The main differences
concern the relationships of the parasites with the cir-
cadian rhythms of the host. It has been shown
(Montalvo et al., 1988) that, in ordinary laboratory
conditions, the timing of the schizogony of P. v. pet-
teri was independent of the hosts' rhythm. The pre-
sent study indicates that P. v. vinckei shows the same
behaviour. In both cases merozoites from the frozen
thawed inoculum penetrate immediately into red
blood cells and establish the time of schizogony
which occurs 24 hours post-inoculation. On the
contrary the cycle of the closely related P. v. fentum is
under the control of both, the time of inoculation,
like P. v. petteri and P. v. vinckei, and the hosts
rhythm, like P. cbabaudi (Cambie et al., 1990) which
is unusual, parasite cycles nonnally being governed
either by one or the other. Whatever the time of ino-
culation, merozoites of P. v. /entum appear to wait for
the favourable time to penetrate and the subsequent
cycle is set with schizogony occuring either at 06:00
or at 18:00. We have no deHnite explanation for this
duality of timing. It bears perhaps a relationship with
the biology of the vectors biting twice during the
night, at dawn and dusk. Finally, P. y. yoelii differs
little from the other yoelii subspecies killicki and
nigeriensis (Deharo et al., 1994), the cycle of which
also last 18 hours. The existence of a "timing niche" is
very common in polyparasitic infections by sporo-
zoans. The 12 species of Isospora described by Gmlet
et al., 1982 in the domestic sparrow occupy a diffe-
rent localisation in the gut but also their oocysts are
fonned and excreted at a definite time of the day, dif-
ferent from one species to the other. The analysis of
available data on the chronobiology of the rodent
malarias lead to the conclusion that differences exist
not only between parasites coexisting in the same
host but also between the subspecies of a same spe-
cies occuring in different localities. It would be very
surprising if the biology of the human malarias diffe-
red very much from that of the other animal malarias.
In fact workers are increasingly aware of the biodiver-
sity of the human parasites in the Held; the duration
of the schizogonic cycle, the timing of the different
stages, the degree of synchronicity are variable para-
meters of which it is important to be aware, for che-
motherapeutic or epidemiological studies.
W e are very much indebted to R.for his revision of the manuscript.
REFERENCES
S. Bray
~P~aras~i~te~,':199~4.:..,!.cl,~2~3~5:':2~39~ •••••••••••L 2'\9


Plasmodium vinckei vinckei, P. v. lentum and P. yoelii yoelli : chronobiology of the asexual cycle in the blood

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