CHRONOBIOLOGY OF PLASMODIUM CHABAUDI CHABAUDI:
ANALYSIS OF HOURLY RECORDED TOTAL AND DIFFERENTIAL PARASITAEMIA DURING A SCHIZOGONIC CYCLE
CHIMANUKA B.*, ***, FRANÇOIS G.**, TIMPERMAN G.**, VANDEN DRIESSCHE T.*** & PLAIZIER-VERCAMMEN J.*
S u m m a r y :
Detailed total and differential parasitaemia curves of asexual Plasmodium chabaudi chabaudi erythrocytic stages were recorded and analysed. Female, inbred, CBA/Ca mice were infected with the virulent IP-PC 1 strain after in vitro synchronization of the parasites. Thin blood smears were made on an hourly basis, and the total and differential parasitaemia of ring forms, trophozoites and schizonts were counted after Giemsa staining. These curves reveal information that remains hidden when less detailed curves are examined: the duration and periodicity of the schizogonic cycle, the existence of a plateau, indications of a schizont withdrawal from the peripheral blood, the timing of the rise of the parasitaemia at each schizogony, and the invasion rate of the merozoites. In the perspective of developing a rational and efficient strategy for chronotherapy of malaria, such information should be taken into account.
KEY WORDS : malaria, Plasmodium chabaudi chabaudi, chronobiology, parasitaemia curves, schizogonic cycle.
R é s u m é : CHRONOBIOLOGIE DE PLASMODIUM CHABAUDI CHABAUDI : ANALYSE DES COURBES HORAIRES DES PARASITÉMIES TOTALES ET DIFFERENTIELLES PENDANT UN CYCLE SCHIZOGONIQUE
Des courbes détaillées de parasitémie totale et différentielle des stades érythrocytaires asexués de Plasmodium chabaudi chabaudi ont été établies et analysées. Des souris femelles CBA/Ca ont été infectées avec la souche virulente IP-PC 1 après une
synchronisation in vitro. Des frottis sanguins ont été faits chaque heure. Les parasitémies totales et différentielles ont été évaluées après coloration au Giemsa et comptage des formes en anneaux, des trophozoites et des schizontes. Les courbes obtenues ont révélé des détails qui seraient restés cachés si l'intervalle entre les échantillonages avait été plus long, notamment la durée et la périodicité du cycle schizogonique, la présence d'un plateau, des indications sur une disparition des schizontes du sang
périphérique, le moment de la montée de la parasitémie après la schizogonie et le taux d'invasion des mérozoïtes. En vue du développement d'une stratégie rationelle et efficace pour la chronothérapie de la malaria, les informations révélées por ce type de courbes devraient être prises en considération.
MOTS CLÉS : malaria, Plasmodium chabaudi chabaudi, chronobiologie, courbes de parasitémie, cycle schizogonique.
INTRODUCTION
I
n general, the course o f a malaria infection is m o n i t o r e d b y e x a m i n i n g daily m a d e G i e m s a stained smears. However, the s h a p e o f the cor- responding curve, displaying the total parasitaemia as a function o f time, d o e s not reflect the detailed e v o - lution o f the schizogonic cycle. This implies that impor- tant relevant data remain hidden if only daily o b s e r - vations are m a d e .Against this background w e present the analysis o f total and differential parasitaemia curves o f P. chabaudi
* Faculteit Geneeskunde en Farmacie, Vrije Universiteit Brussel, Laar- beeklaan 103, B-1090 Jette, Belgium.
** Prins Leopold Instituut voor Tropische Geneeskunde, Nationales- traat 155, B-2000 Antwerpen, Belgium.
*** Département de Biologie Moléculaire, Université Libre de Bruxelles, rue des Chevaux, 67, B-1640 Rhôde Saint-Genèse.
**** Laboratoire d'Entomologie et Parasitologie Médicale, Centre de Recherche en Sciences Naturelles, Lwiro, D.S. Bukavu, Kivu, Congo.
Correspondence : В. Chimanuka.
Phone : (32) 2 477 45 98 - Fax : (32) 2 477 41 13.
chabaudi in their rodent host b a s e d u p o n hourly m a d e thin b l o o d smears. P. chabaudi chabaudi is an interesting experimental model (Landau et al., 1 9 9 3 ) and shares a n u m b e r o f biological features with P. fal
ciparum (Jarra & B r o w n , 1985; Gilks et al., 1 9 9 0 ) . B e s i d e s , P. chabaudi chabaudi infections are syn
c h r o n o u s , with a period o f 2 4 hours, and d e p e n d o n the circadian rhythm o f the host ( C a m b i e et al., 1990;
Landau et al., 1990, 1 9 9 1 ) .
MATERIALS AND METHODS
MICE
i x - w e e k - o l d , f e m a l e , i n b r e d , p a t h o g e n - f r e e , CBA/Ca mice (Kingman, England) w e r e kept in plastic translucent c a g e s at 21 °C and 5 0 % rela
tive air humidity. T h e i r p h o t o p e r i o d i c rhythm was s e t b y a r e g i m e n o f 1 2 h o u r s l i g h t ( 1 3 h 0 0 - 01 h 0 0 ) / 1 2 hours dark ( 0 1 h 0 0 - 1 3 h 0 0 ) . T h e mice w e r e handled conform with national and international legislation and guidelines.
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1997044319
CHIMANUKA В.. FRANÇOIS G., TIMPERMAN G , VANDEN DRIESSCHE T., PLAIZIER-VERCAMMEN J .
PARASITE AND INFECTION
T h e virulent IP-PC1 strain o f P. chabaudi chabaudi was inoculated into a СВА/Са m o u s e . W h e n a p a r a s i t e m i a o f 3 0 - 5 0 % was r e a c h e d and late schizonts predomi
nantly occurred, 1 ml o f b l o o d w a s s u s p e n d e d in RPMI 1640 ( G i b c o B R L ) s u p p l e m e n t e d with 10 % foetal calf serum (FCS; GibcoBRL), and centrifuged. T h e recovered erythrocytes containing mature schizonts w e r e resus- p e n d e d in the same medium and incubated at 37 °C for 3 0 minutes to liberate the merozoites. After centri- fugation, the supernatant containing the merozoites w a s centrifuged again, and the brownish pellet w a s r e s u s p e n d e d in 2 5 0 μl o f P B S (pH 7 . 3 ) and immedia
tely i.v. injected into another m o u s e at 10 h 0 0 . W h e n its parasitaemia had reached a level o f about 10 % (ring forms), 1 06 infected red b l o o d cells in sterile Alsever medium (pH 6 . 2 ) were i.v. injected into 4 receptor m i c e at 11 h 0 0 . T h e e x p e r i m e n t was started w h e n the para
sitaemia o f these m i c e r e a c h e d a level o f 5-10 % and about 3 0 %, respectively.
TOTAL AND DIFFERENTIAL PARASITAEMIA
After G i e m s a staining o f thin b l o o d smears, the total p a r a s i t a e m i a ( n u m b e r o f i n f e c t e d red b l o o d c e l l s e x p r e s s e d as a p e r c e n t a g e o f the total n u m b e r o f red b l o o d cells) and the differential parasitaemia ( n u m b e r s o f ring forms, trophozoites, and schizonts, e x p r e s s e d as p e r c e n t a g e s o f the total n u m b e r o f infected red b l o o d cells) w e r e determined. Ring forms were defined as the stages d e v e l o p i n g b e t w e e n the attachment o f merozoites to the erythrocytes and the d i s a p p e a r a n c e o f the ring specific vacuole, while trophozoites did not display such a v a c u o l e and their cytoplasm w a s larger than the o n e o f the ring s h a p e d parasites. Schizonts o c c u p i e d most o f the host erythrocyte cytoplasm, with a very d e n s e a n d / o r dividing nucleus. F o r e a c h s m e a r a m i n i m u m o f 1 0 0 i n f e c t e d r e d b l o o d c e l l s w a s c o u n t e d .
Fig. 1. — Time course of total parasitemia of Plasmodium chabaudi chabaudi (IP-PC1) infected CBA/Ca mice ( · and o), based upon the evaluation of hourly made thin blood smears. Photoperiodic rhythm of the mice set by a cycle of 12 h dark (01 h 00-13 h 00V12 h light (13 h 00-01 h 00). a) Initial parasitemia 5-10 %; b) Initial parasi- taemia ca. 30 %.
T h e s e observations c o r r e s p o n d well with t h o s e m a d e o n the infected m i c e displaying a higher initial para
sitaemia (ca. 3 0 % ) (Fig. LB). Furthermore, these results are in agreement with those obtained in a series o f ana
l o g o u s e x p e r i m e n t s carried out with infected female o u t b r e d O F 1 m i c e ( B . Chimanuka, unpubl. o b s . ) . No a n a e m i a w a s noticed after the s c h i z o g o n y at 3 0 %. T h e parasitaemia r e a c h e d a plateau o f 6 0 - 7 0 % at the e n d o f w h i c h the crisis o c c u r r e d .
RESULTS
TOTAL PARASITAEMIA CURVES
The detailed time c o u r s e o f the total parasi- taemia o f m i c e with an initial parasitaemia o f 5-10 % is given in figure la. B y the e n d o f the dark period a plateau level with a constant parasitaemia o f ca. 12.5 % w a s r e a c h e d , w h i c h lasted the full length o f the light period plus 2-3 hours o f the dark period.
At the e n d o f the plateau the parasitaemia s h o w e d a distinct d e c r e a s e with a minimum level roughly cor
responding with the middle o f the s e c o n d dark period.
T h e n it rose sharply and r e a c h e d a n e w plateau level with a parasitaemia o f ca. 3 7 . 5 %.
INVASION RATES
Invasion rates w e r e o b t a i n e d by dividing the values o f t w o c o n s e c u t i v e plateau levels. Figure la yields a rate o f about 3, while a value o f about 2 is d e d u c e d from figure lb. In previous e x p e r i m e n t s ( B . Chimanuka, unpubl. o b s . ) w e o b t a i n e d higher invasion rates o f 10 and 8, w h e n initial parasitaemias w e r e 0.1 and 1 %, respectively. T h e correlation b e t w e e n initial parasi
taemia and invasion rate is clearly negative (r = - 0 . 8 7 ) . DIFFERENTIAL PARASITAEMIA CURVES
T h e d e v e l o p m e n t o f ring forms, t r o p h o z o i t e s , and schizonts, c o r r e s p o n d i n g with the total parasitaemia curves o f figure la, is s h o w n in figure 2. G a m e t o c y t e s
3 2 0 Parasite, 1997, 4, 319-323
Mémoire
Local time (h)
Local time (h)
CHRONOBIOLOGY OF THE MALARIA PARASITE PLASMODIUM CHABAUDI
were not observed throughout this experiment. T h e duration o f the d e v e l o p m e n t a l stages was estimated at 12 hours for the rings, 8 hours for the trophozoites, and 4 h for the schizonts.
T h e proportion o f ring forms rose sharply at the begin
ning o f the e x p e r i m e n t and remained almost constant at a level o f about 9 5 % until the e n d o f the first light period. T h e n it d e c r e a s e d to reach a minimum level o f 10 % in the middle o f the s e c o n d dark period. T h e proportion o f trophozoites o n the other hand was o n the d e c r e a s e w h e n the observations b e g u n and stayed at a very low level ( < 5 % ) during the light period.
T h e n it increased to reach a p e a k level o f about 7 5 % o f the total n u m b e r o f parasites b y the middle o f the s e c o n d dark period. T h e schizont curve s h o w e d p e a k s at 2 5 - 3 0 % and 4 0 - 4 5 %, e a c h o f them occurring 7-8 hours after the onset o f the dark periods, i.e. 2-3 hours after the a p p e a r a n c e o f the trophozoite p e a k s . In between, the proportion o f schizonts did not e x c e e d 5 % o f the total n u m b e r o f infected erythrocytes. T h e differential distribution curves s h o w e d a cyclic pattern, as illustrated by the observations m a d e after the first 24 hours o f the experiment.
DISCUSSION
A
nalysis o f the total parasitaemia curves (Fig. 1) reveals the duration and the periodicity o f the s c h i z o g o n i c cycle, the e x i s t e n c e o f a plateau, indications o f schizont withdrawal from the peripheral blood, the timing o f the rise o f the parasitaemia, and the invasion rate o f the merozoites. T h e initial increase o f the total parasitaemia (Fig. 1a) during the s e c o n d half o f the dark period c o r r e s p o n d s with the appearance o f ring forms (Fig. 2 ) after the release o f m e r o zoites by mature schizonts. W h e n the total parasi
taemia r e a c h e s a plateau, ring forms are predominant and a c c o u n t for at least 9 5 % o f the infected erythro
cytes (Fig. 2 ) . Other authors ( H o m m e l et al, 1 9 8 2 ; B o y l e et al., 1 9 8 3 : C o x et al., 1 9 8 7 ; Gilks et al., 1 9 9 0 ) m e n t i o n e d only part o f these possibilities or used the curves for different purposes. T h e position o f the para
sitaemia drop at the e n d o f e a c h plateau phase per
fectly c o i n c i d e s with the rising slope o f the schizont peak. Similar drops w e r e o b s e r v e d with P. chabandi adami by Gautret ( 1 9 9 3 ) . This p h e n o m e n o n has b e e n e x p l a i n e d b y the disappearance o f schizonts from the peripheral b l o o d and their sequestration in the inner organs, mainly the liver and the spleen ( C o x et al., 1 9 8 7 ; Gilks et al., 1 9 9 0 ) . This peripheral withdrawal was estimated at 8-10 % o f the preceeding plateau level.
T h e apparent discrepancy observed b e t w e e n the dura
tion o f the p r e s e n c e o f ring forms in this paper ( 1 2 h ) and in other reports ( 6 h) (Cambie et al., 1990; Gaillard et al, 1 9 9 2 ; Gautret et al., 1 9 9 5 ) is due to the fact that our ring forms c o i n c i d e with the c o m b i n e d ring and young trophozoite forms described by the m e n t i o n e d authors.
Schizonts o f other species such as P. falciparum and P. b e r g h e i are sequestered in the cerebral capillaries and are not frequently s e e n in the peripheral b l o o d (Kwiat- kowski & G r e e n w o o d , 1 9 8 9 ; Lambert & Grau, 1989).
This explains w h y severe P. falciparum malaria attacks can even o c c u r with little or n o evidence o f parasites in the e x a m i n e d b l o o d films (Carme et al, 1989). It means that a more p r o n o u n c e d drop in the total para
sitaemia curve can b e e x p e c t e d in the c a s e o f P. fal
ciparum. In principle, this p h e n o m e n o n could b e deve
loped as a diagnostic parameter for the onset o f cerebral P. falciparum malaria, as sequestration is the key ini
tiating event for this pathology (Warrell, 1987).
Fig. 2 . — Time course of differential para
sitaemia of Plasmodium chabaudi chabaudi ( I P - P C 1 ) infected CBA/Ca mice, based upon the evaluation of hourly made thin blood smears. Data corresponding with the total parasitaemia of figure LA. Proportions of ring forms ( · ) , trophozoites ( Δ ) , and schi
zonts ( π ) expressed as percentages of the total number of infected erythrocytes. Pho
toperiodic rhythm of the mice set by a cycle of 1 2 h dark ( 0 1 h 0 0 - 1 3 h 0 0 V 1 2 h light
( 1 3 h 0 0 - 0 1 h 0 0 ) .
Local time (h)
C H I M A N U K A В . , F R A N Ç O I S G . , T I M P E R M A N G . , V A N D E N D R I E S S C H E T., P L A I Z I E R - V E R C A M M E N J .
The invasion rate with a value o f 2, d e d u c e d from figure ìb), c o r r e s p o n d s with the o n e given b y others ( B o y l e et al., 1983)· H o w e v e r , invasion rates o f about 10 and 8 obtained in other experiments ( B . Chimanuka, unpubl. o b s . ) c o r r e s p o n d with the m e a n n u m b e r o f merozoites p r o d u c e d p e r mature schizont (Landau, 1 9 6 5 ) . T h e negative correlation b e t w e e n invasion rate and initial parasitaemia could b e e x p l a i n e d b y the fact that at high parasitaemia levels less erythrocytes are available for invasion b e c a u s e many o f them are lysed at e a c h s c h i z o g o n y , i m m u n e reactions are m o r e pro- n o u n c e d (Jayawardena, 1 9 8 1 ) , multiple invasion o f red b l o o d cells o c c u r s , a n d free m e r o z o i t e s c o u l d aggregate both within themselves and with pigment and m e m b r a n e s , as has b e e n s h o w n for P. falciparum (Vernot & W a s s e r m a n , 1 9 9 0 ) .
The timing o f the administration o f antimalarial drugs to t h e target parasite stage has b e e n p r o p o s e d b y many authors ( Y a y o n et al., 1983 ; C a m b i e et al., 1991 ; Caillard et al., 1 9 9 2 ; Landau et al., 1 9 9 2 ) . In such a stra- tegy, the parasitaemia curves presented in this report could b e c o m e an important tool.
ACKNOWLEDGEMENTS
W
e appreciate t h e participation o f D. Hen- drix, P. Desai, T. Scorza, E. С. Chimf- w e m b e , T. S t e e n a c k e r s , a n d J . Lisgarten in e x p e r i m e n t s a n d discussions, and thank R. Wynants and M.-Th. D e l o b e l for taking care o f t h e m i c e . W e thank Prof. R. Hamers for initiating this w o r k and Prof. I. Landau for her critical comments. Financial support from the U N D P / W o r l d B a n k / W H O Special Pro
gramme for Research and Training in Tropical Diseases (TDR) is gratefully a c k n o w l e d g e d . B.C. thanks t h e Belgian Cooperation Administration ( A B O S / A G C D ) for his research grant.
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R e ç u le 21 avril 1 9 9 7 A c c e p t é le 2 4 juillet 1 9 9 7
CHRONOBIOLOGY OF THE MAL.RU PARASITE PLASMODIUM СНABAUDI
