SU R V I V A L OF RODENT MALARIA MEROZOITES I N T H E LYMPHATIC N E T W O R K :
POTENTIAL ROLE I N CHRONICITY OF T H E INFECTION
LANDAU I.*, CHABAUD A.G.*, MORA-SILVERA E.*, COQUELIN F.*, BOULARD Y.*, RÉNIA L.** & SNOUNOU G.***
Summary:
Experiments performed during the last few years, lead us to hypothesise the existence of latent asexual forms of murine Plasmodium. In the present report we examined the organs of infected animals and describe novel structures, which we call merophores, containing merozoites which have resisted lysis seen with other asexual stage parasites. We propose that these merozoites represent a latent form of the parasite. Merophores were also found in the lymphatic circulation, and were demonstrated by subinoculation to have retained their viability.
Depending on the parasite species two types of merophores were observed. For P. yoelii nigeriensis merophore sacks, with the latent merozoites found inside vesicles, were usually observed.
Merophore leucocytes, where latent merozoites dispersed in the cytoplasm of macrophages or neutrophils, were solely seen with P. vinckei petteri. Both structures were seen in P. chabaudi chabaudi infections. Merophores were found in lymph nodes of rodents after the asexual parasitaemia had apparently subsided.
They were formed soon after schizogony, principally in the spleen, either by pitting or by macrophage phagocytosis. Merophore numbers appeared to be proportional to the number of maturing schizonts. We propose that merophore formation and their circulation in the lymphatics play an important role in the pattern of recrudescences and chronicity of rodent malaria infections. It is further suggested that the lymphatic network, a privileged pathway for many parasites, might play a similar role in human malaria infections.
KEY WORDS : chronicity, latency, lymphatic, merophore, merozoite,
Plasmodium chabaudi, Plasmodium vinckei, Plasmodium yoelii, recrudescence.
Résumé :
SURVIE, DANS LE RÉSEAU LYMPHATIQUE, DES MÉROZOÏTES DES PLASMODIUMS MURINS. RÔLE PROBABLE DANS LA CHRONICITÉ DE L'INFECTION Les travaux poursuivis au cours des récentes années sur le paludisme des rongeurs nous ont conduit à supposer l'existence de mérozoïtes latents. Dans les appositions ou les coupes de différents organes des rongeurs infectés, il existe des structures, non encore décrites, que nous nommons mérophores. Ces structures contiennent des mérozoïtes qui ont résisté à la lyse observée avec les autres formes parasitaires.Dans nos hypothèses ces mérophores constituent des formes de latence du parasite. Ils se trouvent dans le réseau lymphatique et, comme l'indiquent les subinoculations ils conservent leur infectivité.
Selon l'espèce plasmodiale étudiée deux types morphologiques sont observés : "Sacs mérophores" presque toujours pour P. yoelii nigeriensis, avec les mérozoïtes latents rassemblés dans une vésicule, ou
"leucocytes mérophores" presque toujours pour P. vinckei petteri, avec les mérozoïtes latents dispersés dans le cytoplasme d'un neutrophile ou d'un macrophage, les deux types de formations coexistent pour P. chabaudi chabaudi. Des mérophores ont été observés dans les ganglions lymphatiques de rongeurs après la disparition des stades asexués du sang. Ils se forment peu après la schizogonie,
essentiellement dans la rate, soit par "pitting" soit par phagocytose des macrophages, le nombre de mérophores semble proportionnel à l'intensité de la schizogonie. Nous pensons que la formation et la circulation des mérophores dans le réseau lymphatique joue un rôle essentiel dans la chronicité et les rythmes de recrudescences particuliers à chaque espèce de Plasmodium murin. Il est suggéré que le réseau lymphatique, refuge privilégié de nombreux parasites, joue un rôle comparable dans les paludismes humains.
MOTS CLES : chronicité, latence, lymphatique, mérophore, mérozoïte, Plasmodium chabaudi, Plasmodium vinckei, Plasmodium yoelii, recrudescence.
INTRODUCTION
I
n the natural host Thamnomys rutilans, the course o f malaria infections is characteristically chronic and life-long. For instance all the Thamnomys older* Laboratoire de Biologie Parasitaire et Laboratoire de Protozoologie et Parasitologic Comparée (EPHE), Muséum National d'Histoire Naturelle, 61, rue Buffon, 75231 Paris Cedex 05, France.
** U 445 INSERM, Institut Cochin de Génétique Moléculaire, Hôpital Cochin, Bâtiment Gustave Roussy, 27, rue du Faubourg Saint- Jacques, 75014 Paris, France.
*** Department of Infection and Tropical Medicine, Imperial College School of Medicine, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, United Kingdom.
Correspondence: Irène Landau.
Tel.: (+33) (1) 40 79 3500 - Fax: (+33) (1) 40 79 3499.
e-mail: [email protected]
than two months, captured during March-April 1 9 6 5 and 1 9 6 6 at the experimental field station o f La M a b o k e near M'Baiki (Central African Republic), w e r e found to b e infected with o n e o r m o r e Plasmodium species, with the parasitaemia mostly < 0.01 % and never m o r e than 3 %. W h e n maintained in captivity, these wild caught Thamnomys maintained low-grade infections w h i c h persisted all their life ( u p to two years) (Landau
& Chabaud, 1 9 9 4 ® . In a later study the course o f infec- tion o f two T. rutilans captured in the Central African Republic and kept in captivity in Paris, was followed for 17 month by carrying out subinoculations every 15 days (Landau & Gautret, 1 9 9 8 ) . T h e three parasite species, w h i c h naturally co-exist in this host, e a c h pre- s e n t e d a characteristic c o u r s e o f infection. P. cha- baudi chabaudi w a s almost always found ( 9 / 1 0 subi- n o c u l a t i o n s ) , while P. vinckei petteri w a s d e t e c t e d
Parasite, 1999, 6, 311-322 311
Mémoire
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999064311
nearly every other inoculation. P. yoelii nigeriensis on the other hand remained undetectable for five months and then could only be observed in only one of the two Thamnomys but only in one of four subinocula- tions (Landau & Gautret, 1998).
When laboratory born T. rutilans were experimentally infected with either one of the above three parasite species, and the course of infection followed for 12 to 58 weeks by subinoculation, the infections presented a similar course to that observed in wild caught ani
mals. The parasitaemias reached a peak of 3 % or less, and the chronicity of the infection and the pattern of recrudescences were also found to be similar to those observed with natural infections. The fact that com
parable results were obtained when the animals were inoculated with sporozoites or with fresh infected blood, strongly suggested that the different patterns of recrudescences were due to erythrocytic parasites, and could be accounted for by postulating the existence of latent parasites, i.e. parasites which survive for extended periods before initiating further cycles of replication.
We had previously demonstrated the existence of latent merozoites in murine malaria parasites through a number of biological observations (summarised by Landau & Chabaud, 1994<3). The selective resistance of merozoites over other asexual blood stages to cryopreservation (Montalvo et al., 1 9 8 8 ) , was exploited to confirm that red blood cell (RBC) inva
sion can be delayed by 12 to 18 hours following ino
culation of P. y. nigeriensis or P. c. chabaudi mero
zoites (Landau & Chabaud, 1980). The presence of such forms in the blood could not however be demonstrated with ease during an ongoing infection.
The observation of Plasmodium parasites in the lym
phatic circulation (Landau et al., 1995) suggested a mechanism for the localisation and survival of such latent merozoites.
In the present work detailed investigation of various organs and of their lymphatic network resulted in the detection of latent merozoites in animals infected with each of three different rodent Plasmodium species.
Depending on the parasite species, these latent forms occurred either as "merophore leucocytes", where the merozoites were dispersed separately in the cytoplasm of a macrophage or neutrophil, or as "merophore sacks", where the occasionally numerous merozoites were gathered inside a vesicle. Viability of these latent merozoites was confirmed by subinoculation of lym
phatic fluid.
Confirmation that malaria parasites can survive as latent forms possibly in the lymphatic circulation sug
gests a mechanism for recrudescences and for the chronicity of infections, and has implications to the interpretation of drug resistance studies.
MATERIALS AND METHODS
BIOLOGICAL MATERIAL
W hite male mice IOPS OF1 (Swiss IFFA- Credo, France) weighing between 20 and 25 grams were used. Some mice, infected by P. y. nigeriensis, were splenectomised at D5 (post- inoculation). Infections were also analysed in Wistar outbred rats (Charles Rivers).
Three species of murine malaria parasites were used
Plasmodium chabaudi chabaudi, strain 864 VD, Plasmodium vinckei petteri, strain 106 HW and Plasmo
dium yoelii nigeriensis.
O B S E R V A T I O N M E T H O D S
Optical microscopy was used for the observation of merophores which are only easily detectable in impres
sion smears from various organs: liver, spleen, bone marrow, kidney, brain, and with the renal, mediastinal, lumbar and mesenteric lymph nodes. These smears were fixed by Bouin's fluid and stained by the Giemsa- colophonium method. Some sections of lymphoid organs fixed with Carnoy's fluid were stained with the Giemsa-colophonium method. The ultrastructural observations were performed according to Seureau et
al. (1980).PARASITE ENUMERATION
As indicated below, the merozoites are rarely free, and are mostly to be found either dispersed in the cyto
plasm of a leucocyte (merophore leucocyte), or gathered inside a transparent generally spherical sack (merophore sack). Estimation of the number of mero
phores in the spleen and the lymph nodes, is difficult because the impression smears are not homogenous and only an approximation of their numbers can be given. The smears and the sections were examined with the help of an ocular reticule. Fields of equiva
lent thickness were selected, i.e. with roughly the same numbers of cells. The following approximations were made, a) Merophore sacks: for 100 grids ins
pected, an average of 17,000 nuclei were counted for the impression smears of the spleen. A comparable number of nuclei was counted in sections, 5 um thick, with 50 grids. The volume corresponding to 50 grids of sections is 100 x 100 x 5 x 50 = 2.5 10
6um
3. A spleen of 1 g, approximately 1 cm
3or 1,012 um
3, thus contains 10
1 2/2.5 x 10
6or 400,000 cells. Thus when one merophore sack per 100 grids is found, a mouse spleen weighing 0.2 g, was estimated to contains 80,000 sacks, and b) merophore leucocytes: in spleen impression smears the percentage of neutrophil and macrophage latent merozoite carriers was calculated in relation to the total number of neutrophils.
312 Mémoire Parasite, 1999, 6, 311-322
SAMPLING OF LYMPH
T h e lymph was s a m p l e d from the intestinal lymph canal (ILC) w h i c h drains the lymph from the liver, the mesentery, part o f the intestine, several lymph n o d e s and the spleen, a particularly important organ. It is necessary to handle the vessel without provoking the slightest h a e m o r r h a g e , and this was verified by e x a mination o f the s m e a r e d and Bouin-fixed G i e m s a - stained cellular pellet o b t a i n e d after centrifugation o f the samples in micro-haematocrit tubes. Catheterisation o f the intestinal lymph canal was performed using the surgical t e c h n i q u e described by Waynforth ( 1 9 8 0 ) . In order to best visualise the lymph vessel the rat was given 0.8 ml o f olive oil o n e hour before the opera
tion.
T h e lymph c o l l e c t e d was diluted in saline and a given volume was inoculated intra-peritoneally to mice. Daily b l o o d smears w e r e performed and the time to reach a parasitaemia o f 5 % was r e c o r d e d in order to eva
luate the numbers o f infective stages present in the ino
culum. T h e calculation w a s b a s e d o n the results o f Beaute-Lafitte et al. ( 1 9 9 3 ) w h e r e the n u m b e r o f hours necessary for P. y. nigeriensis to reach a parasitaemia o f 5 % w a s determined for inocula o f increasing dilu
tions o f freeze-thawed infected b l o o d . According to t h e s e authors a 5 % parasitaemia is r e a c h e d only
160 hours after a single merozoite is inoculated.
A ligature o f the ILC was performed in o n e rat and five m i c e to study its c o n t e n t s histologically. T h e rodents w e r e given olive oil, o n e hour before the o p e ration, anaesthetised and the ILC e x p o s e d . T w o liga
tures w e r e performed approximately 1 c m apart and the m o u s e was sacrificed b y C 02 inhalation. It was then immersed into Carnoy's fixative during o n e hour before the ILC was removed, transferred into Carnoy's fixative for a further twelve hours, and p r o c e s s e d for histology.
RESULTS
MEROPHORES
M
e r o p h o r e s w e r e mainly o b s e r v e d at the e n d o f schizogony w h e n the leucocytes scavenge in the o r g a n s w h e r e parasite maturation takes p l a c e , that is in all the viscera. T h e highest concentration o f m e r o p h o r e s w a s s e e n in the spleen.Latent merozoites a p p e a r e d in different forms. S o m e o c c u r r e d as a few dispersed units inside a l e u c o c y t e w h i c h m a y b e a m a c r o p h a g e o r a polymorph, and s o m e w e r e e n c l o s e d in m e r o p h o r e sacks. T h e n u m b e r o f m e r o z o i t e s and pigment masses c o n t a i n e d in m e r o p h o r e s a c k s indicate that s o m e c o n t a i n e d m o r e than o n e schizont. T h e s e m e r o p h o r e sacks a p p e a r mostly
e x t r a - c e l l u l a r and w e r e very likely f o r m e d inside m a c r o p h a g e s w h i c h have lysed.
M e r o p h o r e sacks
O p t i c a l m i c r o s c o p y s h o w e d t h e m e r o p h o r e s a c k (Figs 1A-E, 2A, 2C, 2E, 3A, 4 A - B ) as a clear rounded structure, w h i c h c o n t a i n e d o n e o r several mature and ruptured schizonts. It was found inside a m a c r o p h a g e or was frequently extra-cellular. T h e merozoites w e r e dispersed in the interior o f the cavity with o n e to three pigment masses. Up to 4 0 merozoites may b e s e e n inside the sack. T h e pigment masses w e r e identical to those o b s e r v e d in the mature schizonts o f the blood.
T h e r e w a s n o sign o f degeneration or digestion. Other p h a g o c y t o s e d e l e m e n t s in t h e p r o c e s s o f b e i n g digested, w e r e s o m e t i m e s present e l s e w h e r e in the host cell.
T h e m e r o p h o r e sacks differed from schizonts s e e n digested in m a c r o p h a g e s by their s m o o t h rounded contour, by the regular dispersion o f merozoites inside a transparent cavity, and b y their healthy a p p e a r a n c e and normal staining affinities.
W h e n the m e r o p h o r e s a c k s w e r e extra-cellular, either b e c a u s e t h e m a c r o p h a g e w a s d e a d o r b e c a u s e it mechanically burst w h e n the smear was performed, their m o r p h o l o g y was distinct from that o f schizonts inside red b l o o d cells: the vesicles w e r e apparently empty apart from the merozoites and a few pigment granules. T h e n u m b e r o f m e r o z o i t e s and pigment masses was an indication o f the n u m b e r o f mature schizonts a s s e m b l e d in the sack. T h e host cell m e m brane was stained by the antibody Mac-2 (Mora-Silvera et al., 1 9 9 7 ) , thus confirming that the host cell b e l o n g s to the m o n o c y t e - m a c r o p h a g e line.
In the red pulp o f the s p l e e n o f mice infected with P. y. nigeriensis, electron m i c r o s c o p y revealed the fol
lowing, a) an extra-cellular immature m e r o p h o r e sack w h i c h was a round structure, 6-7 um diameter, contai
ning several merozoites and o n e o r two pigment gra
nules. T h e parasitophorous vacuole, which was pre
sent and surrounded by remains o f the erythrocytic cytosol, w a s very v a c u o l a t e d and slightly e l e c t r o n d e n s e . T h e w h o l e was surrounded by the red b l o o d cell external m e m b r a n e (Fig. 5A). T h e merozoites were o f average electron density, possessed typical structures ( r h o p t r i e s a n d d e n s e m i c r o n e m e s , h o m o g e n o u s nucleus, dense cytoplasm), and w e r e healthy in appea
rance, b) M e r o p h o r e s a c k s in w h i c h the m e m b r a n e o f the parasitophorous v a c u o l e had aiptured, releasing the merozoites into the ghost o f the red cell. T h e cytosol o f the erythrocyte had disappeared and the only limiting m e m b r a n e was the e n v e l o p e o f the red b l o o d cell (Fig. 5 B ) . c ) W e have also o b s e r v e d a macrophage containing at least seven merozoites inside a phagocytic v a c u o l e limited by a single m e m b r a n e .
Parasite. 1999, 6, 311-322
Mémoire 313
Fig. 1. - Merophore sacks of Plasmodium yoelii nigeriensis. Impression smears (except A: section) Giemsa-colophonium (x 1,750). PI = post
inoculation.
A) Splenectomised mouse. Day 5 (PI). Parasitemia 33 %. Merophore sack inside a leucocyte, in the cortical sinus of a renal lymph node.
B) Splenectomised mouse. Day 5 (PI). Parasitaemia 33 % Merophore sack containing approximately 40 merozoites in a renal lymph node.
C) Mouse day 25 (PI). Parasitaemia 0.07 %. Merophore sack in a renal lymph node.
D) Mouse day 6 (PI). Parasitaemia 26 %. Merophore sack in the spleen.
E) Mouse day 4 (PI). Parasitaemia 53 % Merophore sack in a mediastinal lymph node.
T h e s e merozoites l o o k e d healthy by contrast with the degenerated aspect o f the leucocyte which s h o w e d the beginning o f cytoplasmic vacuolisation, rupture o f the nuclear e n v e l o p e , and a h o m o g e n o u s and granular nucleus (Fig. 5C, the l e u c o c y t e is in the process o f b e i n g phagocytised by a neighbouring m a c r o p h a g e ) . M e r o p h o r e l e u c o c y t e s
B y optical microscopy s o m e merozoites were observed inside m a c r o p h a g e s and neutrophils o f the spleen, the l y m p h n o d e s a n d e v e n , s o m e t i m e s o f t h e b l o o d
(Figs 2 B , 2D, 2F, 3 B - D ) . O n e to ten o f these merozoites w e r e dispersed in the cytoplasm o f the host cell w h i c h s o m e t i m e s also c o n t a i n e d o n e or t w o pigment gra
nules. T h e host cell and the merozoites w e r e appa
rently intact.
M e r o p h o r e s isolated inside l e u c o c y t e s w e r e o b s e r v e d b y electron m i c r o s c o p y during an ultrastructural study o f the spleen o f a m o u s e infected b y P. v. petteri (Fig. 5 D ) . T h e y appeared as round phagocytic vacuoles, e a c h containing a single merozoite and limited by a single m e m b r a n e m o r e or less applied against the
314 Mémoire Parasite, 1999, 6, 311-322
Fig. 2. - Merophores of P. c. chabaudi in the spleen. Impression smears. Giemsa-colophonium (x 1,750).
A) Mouse day 13 (PI). Parasitaemia 16 %. Merophore sack inside a macrophage in the spleen.
B) Mouse day 14 (PI). Post-crisis. Parasitaemia 20 %. Left, merophore leucocyte (macrophage); right, merophore leucocyte (polymorph).
C) Same mouse as B. Merophore sack inside a macrophage.
D) Mouse day 12 (post-inoculation). Parasitaemia 26 %. Merophore leucocyte (polymorph).
E) Mouse day 14 (PI). Parasitaemia 20 %. Merophore sack inside a macrophage in the spleen.
F) Mouse day 11 (PI). Parasitaemia 8 %. Merophore leucocyte (polymorph).
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Mémoire315
L A N D A U I , C H A B A U D A . G . , M O R A - S I L V E R A E . , C O Q U E L L N F . , B O U L A R D Y . , R E N I A L. & S N O U N O U G .
Fig. 3. - M e r o p h o r e s o f P. vinckeipetteri in t h e s p l e e n . I m p r e s s i o n s m e a r s . G i e m s a - c o l o p h o n i u m ( x 1 , 7 5 0 ) . A ) M o u s e d a y 7 ( P I ) . P a r a s i t a e m i a 2 1 %. M e r o p h o r e s a c k i n s i d e a m a c r o p h a g e in t h e s p l e e n .
B ) M o u s e d a y 8 ( P I ) . P a r a s i t a e m i a 1 6 %. M e r o p h o r e l e u c o c y t e ( p o l y m o r p h ) . C ) M o u s e d a y 8 ( P I ) . P a r a s i t a e m i a 4 5 %. M e r o p h o r e l e u c o c y t e ( m a c r o p h a g e ) . D ) M o u s e d a y 8 ( P I ) . P a r a s i t a e m i a 4 5 %. M e r o p h o r e l e u c o c y t e ( p o l y m o r p h ) .
Fig. 4. - Section of merophores of P. y. nigetiensis inside the intestinal lymph canal of mice fixed in situ after ligature. Sections. Giemsa- colophonium (x 1,750).
A) P. y. nigetiensis left, red blood cell parasitized by a trophozoite and merophore sack with three merozoites. Insert: same merophore.
in the adjacent section with three other merozoites.
B) P. y. nigetiensis. Merophore sack.
316 Parasite. 1999, 6, 311-322
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SURVIVAL OF MALARIA PARASITES IN THE LYMPHATICS
Fig. 5. - Transmission electron microscopy of merophores.
A) Merophore formation of P. y. nigeriensis. Six merozoites (M) (one of them still attached to the pigment granule) are enclosed in a common parasitophorous vacuole (PV) inside a red blood cell (RBC) with a vacuolated cytosol (x 140,000).
B) Merophore sack of P. y. nigeriensis with four sections of scattered merozoites. Of the red blood cell (E) only the plasmic membrane remains (x 15,000).
C) Macrophage with eight merozoites of P. y. nigeriensis inside a phagosome. The host-cell (N nucleus, V vacuoles) is in the process of being destroyed by another macrophage; note the pseudopodia (arrow) of the other macrophage.
D) Merophore macrophage of P. v. petteri (M). Note the unaltered appearance of the parasite organelles and the absence of lysosomes around the merozoites and inside the cytoplasm of the macrophage (x 3,000).
Parasite, 1999, 6, 311-322
317Mémoire
m e r o z o i t e . T h e i n t e r m e m b r a n e s p a c e w a s e l e c t r o n clear, containing little or n o material. Up to five o f these v a c u o l e s c o u l d b e found per l e u c o c y t e section.
Each c o n t a i n e d o n e unaltered merozoite with easily r e c o g n i s a b l e organelles.
T h e s e structures differed from the merozoites in the c o u r s e o f elimination. T h e latter w e r e electron d e n s e with altered organelles. T h e y w e r e inside a p h a g o s o m e , in c l o s e contact with primary l y s o s o m e s w h i c h discharged an electron d e n s e material filling the pha- g o l y s o s o m a l cavity thus formed.
SPECIFIC LOCALISATION AND ABUNDANCE OF THE MEROPHORES
T h e localisation and a b u n d a n c e o f m e r o p h o r e s varied from o n e s p e c i e s to the other. Results are presented in the following tables. T a b l e I: P.y. nigeriensis in the spleen, T a b l e II: P. y. nigeriensis extrasplenic, T a b l e III:
P. c. chabaudi, and T a b l e IV: P. v. petteri.
P. y. nigeriensis (Fig. 1) gave rise to n u m e r o u s m e r o - phore sacks, with large, round merozoites, often extra
cellular. M e r o p h o r e l e u c o c y t e s w e r e e x c e p t i o n a l .
R o d e n t D a y % P MS
1 1 0 0
2 2 0 . 1 3 0
3 5 0 . 4 0 0
4 3 14 3
5 3 21 3 5
(. 3 2 6 3 0
7 3 2 7 17
8 3 3 D 3
9 4 5 1 5
id i 3 0 0
11 4 5 3 18
12 5 19 4 0
1 3 6 s 0
14 6 2 6 6 1
15 7 9 10
1 6 7 r 0
17 7 m 9
18 8 5 i 2 5
1') 11 52 I
2 0 12 7 5 6
2 1 12 1 0 3 4
22 1 8 4 0 10
2 3 2 5 0 . 0 7 1
2 4 8 19 6
2 5 10 2 8 3
2 6 17 2 8 ( p c ) 5
2 7 17 4 4 ( p c ) 6
2 8 2 0 18 ( p c ) 4
Day: day of autopsy (post-inoculation). % P: parasitemia, (pc): post- crisis. MS: number of merophore sacks counted on 100 reticular grids of impression smears.
Table I. - Abundance of merophore sacks in the spleen of mice infected with P. yoelii nigeriensis.
R o d e n t D a y % P O r g a n
2 9 3 1 5 m e d i a s t . 1 . n.
3 0 4 51 r e n a l 1. n.
3 1 4 51 r e n a l 1. n.
31 4 5 1 m e s e n t . 1 n .
31 4 5 1 l u m b a r 1. n.
11 4 5 3 m e d i a s t . 1. n.
1J •i 5 3 b o n e m a r r o w
3 2 5 3 3 r e n a l 1. n.
3 3 5 4 4 m y o c a r d i u m
3-4 6 6 7 b o n e m a r r o w
15 _ 9 b r a i n
2 3 25 0 . 0 7 r e n a l 1. n.
3 5 2 7 0 b l o o d
3 1 8 P a r a s i t e , 1 9 9 9 , 6, 3 1 1 - 3 2 2
M o u s e D a y % P M S M L
1 11 8 5 2 0
2 12 2 6 2 3 2 0
3 12 > 5 0 0 4
4 13 2 8 0 3
5 13 4 1 8 2
6 13 7 5 3 10
7 13 7 6 5 2
8 L4 1 0 0
9 14 2 7 6 2
10 14 51 1 0 1
11 14 2 0 ( p c ) 3 1
12 15 17 ( p c ) 3 1
M o u s e D a y % P MI.
1 6 1 11!
2 4 5(1
3 S 8 50
4 8 9 50
5 8 16 4 0
6 8 17 4 0
7 8 4 ^ 4 0
8 9 32 10
9 9 6 6 8 0
10 9 > 8 0 2 0
1 1 10 3 1 2 0
12 11 4 8
13 11 12 3 0
14 11 15 8 0
15 12 2 0
16 12 11 0
17 13 3 3 20
Day: day of autopsy (post-inoculation). % P: parasitaemia. 1. n. : lymph node, mediast.: mediastinal, mesent.: mesenteric.
Table II. - Extrasplenic merophore sacks detected in mice infected with P. yoelii nigeriensis.
Day: day of autopsy (post-inoculation). % P: parasitaemia. (pc): post- crisis. MS: number of merophore sacks counted on 100 reticular grids of impression smears of the spleen. ML: number of merophore leu
cocytes per 1,000 leucocytes.
Table III. - Abundance of merophore sacks and merophore leuco
cytes in the spleen of mice infected with P. chabandi chabaudi.
Day: day of autopsy (post-inoculation). % P: parasitaemia. ML:
number of merophore leucocytes per 1,000 leucocytes.
Table IV. - Abundance of merophore leucocytes in the spleen of mice infected with P. vinckei petteri.
Mémoire
SURVIVAL OF MALARIA PARASITES IN THE LYMPHATICS
P. c. chabaudi (Fig. 2 ) s h o w e d fewer m e r o p h o r e sacks, with round and small merozoites, often disposed per
ipherally. T h e y w e r e found intracellularly m o r e often than those o f P. y. nigeriensis. T h e m e r o p h o r e m a c r o p h a g e s w e r e small and rounded and usually contained a n a k e d schizont with a pigment grain in its centre.
P. v. petteri (Fig. 3 ) hardly e v e r s h o w e d m e r o p h o r e sacks. T h e m e r o p h o r e s w e r e mostly in neutrophils and s e l d o m in m a c r o p h a g e s .
LYMPHATIC CIRCULATION
Catheterisation and lymph inoculation
O n e m o u s e infected with P. y. nigeriensis and ten rats infected b y either P. y. nigeriensis (eight rats), or P. berghei ( t w o rats) w e r e successfully o p e r a t e d on.
Half o f the lymph drawn was inoculated into m i c e and the inoculum proved to b e infective nine times out o f eleven. Using the tables established by Beaute-Lafitte et al. ( 1 9 9 3 ) b a s e d o n the length o f the prepatent period, the quantity o f merozoites inoculated w a s esti
mated at b e t w e e n 5 0 and 2 , 5 0 0 merozoites per ml o f lymph ( T a b l e V ) .
R o d e n t S t r a i n o/oP D a y N b . M / m l
Rat 1 P. y. nig. 2 J 3 5 0 - 1 0 0
Rat 2 P. y. nig. 0 . 1 0 .13 1 2 5
Rat 3 P. y. nig. 2 .17 2 , 0 0 0
Rat 4 P. y. nig. 3 J4 0
Rat 5 P. y. nig. 2 0 J 7 1 2 5 - 1 , 2 5 0 Rat 6 P. y. nig. 4 J 7 7 1 5 / 1 8 5 - 1 , 8 5 0
Rat 7 P. y. nig. 2 J 8 2 , 5 0 0
Rat 8 P. y. nig. 0 . 2 0 .136 8 5 - 8 0 0
Rat 9 P. b. N K 6 5 0 . 1 0 .16 0
Rat 1 0 P. b. A N K A 2 6 J 8 5 5
M o u s e 1 P. y. nig. 3 8 J 5 1 0 0 - 1 , 0 0 0
Day: day of autopsy (post-inoculation). % P: parasitaemia. Nb.M./ml lymph: evaluation of the number of merozoites, per ml of lymph.
Table V. - Estimation of the number of infective stages in the lymph of the intestinal canal of rodents infected with P. yoelii nigeriensis and P. berghei.
T h e n u m b e r o f merozoites p e a k e d shortly before the c r i s i s , w h e n p a r a s i t e n u m b e r s w e r e m a x i m u m . H o w e v e r , the n u m b e r s o f latent merozoites s e e m e d to cumulate with the duration o f infection. T h e lymph o f a rat with a l o w parasitaemia ( 0 . 1 5 % ) , 36 days post inoculation w a s s h o w n to b e infective.
Ligatures
Histological observations made on the intestinal lymph canal (ILC) o f three m i c e and o n e rat infected with P.
y. nigeriensis (Table VI) s h o w e d that it contained a few intra-erythrocytic parasites o f all stages, a few free merozoites, and s o m e uninfected RBCs (Fig. 4 A ) . T h e m o r p h o l o g y o f the parasitised red b l o o d cell w a s
R o d e n t % P N
Rat 1 4 0 . 4 - 0 . 5
M o u s e 1 2 4 1.7
M o u s e 2 32 2 . 3
M o u s e 3 5 0 9
% P: parasitaemia. N: Number of parasites per 100 leucocytes.
Table VI - Abundance of parasites, at day 6 (post-inoculation), in histological sections of the intestinal lymph canal, of rodents infected
with P. yoelii nigeriensis.
altered and differed from that o f infected cells inside neighbouring b l o o d vessels, in that they w e r e m u c h paler and only a thin surrounding pellicle remained visible.
S o m e schizonts in the lymph a p p e a r e d fully mature with well defined merozoites, and a few free m e r o zoites w e r e s e e n inside the ILC o f a m o u s e . In addi
tion to erythrocytic parasitic stages, rare m e r o p h o r e sacks (Fig. 4 B ) w e r e identified in sections. However, it is possible that s o m e parasites identified as mature schizonts w e r e in fact free m e r o p h o r e sacks.
In sections o f the ILC, parts o f the mesenteric and c o e - liac lymph n o d e w e r e o b s e r v e d . Parasitised RBCs and structures w h i c h l o o k e d like free or intracellular m e r o p h o r e sacs w e r e s o m e t i m e s s e e n in the cortical and cortico-medullar sinuses and in the hylus and the beginning o f the efferent lymph vessel.
Ligatures o f the ILC o f two mice heavily infected ( 4 9 % and 3 8 % ) with P. c. chabaudi w e r e performed at mid
night (time o f s c h i z o g o n y ) . It a p p e a r e d that similarly to P. y. nigeriensis, the ILC transports erythrocytic stages o f P. c. chabaudi and altered parasitised ery
throcytes. A m e r o p h o r e m a c r o p h a g e with a merozoite inside the cytoplasm was identified in the efferent vessel o f the c o e l i a c lymph n o d e .
DISCUSSION
CHRONIC INFECTIONS
I
n their vertebrate hosts malaria parasites survive for e x t e n d e d periods in the face o f active i m m u n e defences. In the a b s e n c e o f treatment parasites, w h o s e n u m b e r s reach a high level during the primary p e a k w h i c h follows inoculation o f n o n - i m m u n e hosts, are usually cleared from the circulation by predominantly s p e c i e s and strain-specific immune m e c h a n i s m s (Talliaferro, 1 9 4 9 ; S n o u n o u et al., 1 9 8 9 ) . T h e subse
quent course o f the infection is characterised b y the distinct e p i s o d e s o f r e c r u d e s c e n c e w h e r e the parasites reappear in the circulation for short periods, usually at l o w e r levels than in the primary parasitaemia. In a n u m b e r o f natural hosts the infection is thought to b e
Parasite, 1999, 6, 311-322
Mémoire 319
life-long, and different Plasmodium species and strains often display a characteristic pattern o f recrudescent episodes. Dormant liver stages k n o w n as hypnozoites and w h i c h a c c o u n t for s o m e o f these recurrent epi
sodes, are only found in a few o f the primate malaria parasites n a m e l y P. cynomolgi, P. vivax and P. ovale, but c o u l d not b e d e m o n s t r a t e d for P. falciparum, P. malariae ( w h e r e infections o f m o r e than 3 0 years duration have b e e n r e c o r d e d ) , n o r for any o f the murine Plasmodium s p e c i e s T h e m e c h a n i s m s w h i c h w o u l d explain the p h e n o m e n o n o f protracted recru
d e s c e n c e s remain to b e elucidated.
T h r e e n o n - m u t u a l l y e x c l u s i v e p o s s i b i l i t i e s c o u l d explain the a b s e n c e o f circulating parasites b e t w e e n r e c r u d e s c e n c e s : 1) the n u m b e r o f infected red b l o o d cells is maintained at such l o w levels, presumably b y i m m u n e m e c h a n i s m s , as to m a k e t h e m undetectable b y m i c r o s c o p y o r e v e n subinoculation, 2 ) parasites which survive c l e a r a n c e o f e a c h parasitaemic wave are s e q u e s t e r e d away from the peripheral circulation or 3 ) the parasite p r o d u c e s latent forms w h i c h a l o n e sur
vive clearance, and probably remain sequestered away from the peripheral circulation.
Previous observations o f malaria infections in animals m o d e l s lent support to the c o n t i n u e d p r e s e n c e o f viable parasites in certain organs following c l e a r a n c e o f infected red b l o o d cells from the peripheral circu
lation (Jerusalem, 1 9 6 8 ; Suzuki, 1 9 7 4 ) . T h e e x i s t e n c e o f latent forms o f the asexual erythrocytic parasite has b e e n generally c o n s i d e r e d unlikely. In the a b s e n c e o f drug or i m m u n e pressure, parasites in "arrested deve
l o p m e n t " within the red b l o o d cell are yet to b e demonstrated. T h e m e r o z o i t e w a s d i s c o u n t e d as a candidate since in vitro observations have resulted in the conviction that merozoites w o u l d not survive as free cells unless they penetrate erythrocytes within a few minutes o f their release from schizonts ( J o h n s o n et al., 1 9 8 0 ) . H o w e v e r , in vivo observations in murine models indicated that invasion b y s o m e merozoites can b e delayed up to 18 hours, and it w a s postulated that these merozoites w o u l d s e q u e s t e r outside the b l o o d circulation (Landau & Chabaud, 1 9 9 4 « ) . Free parasites including merozoites w e r e subsequently demonstrated in the thoracic duct and s p l e e n lymph vessels (Landau et al., 1995), and in the cortical sinus o f the renal lymph n o d e ( B o u l a r d et al., 1 9 9 6 ) o f infected mice.
MEROPHORE FORMATION
In this article w e demonstrate that m e r o p h o r e s , novel structures containing only merozoites, are formed in s o m e organs but mainly in the spleen, and can b e found in the lymphatic circulation w h e r e they retain their viability. M e r o p h o r e s w e r e o b s e r v e d with three s p e c i e s o f murine Plasmodium.
T h e formation and quantity o f m e r o p h o r e s a c k s a n d m e r o p h o r e l e u c o c y t e s w a s directly r e l a t e d to t h e n u m b e r o f mature schizonts in the spleen. T h e e x a mination o f spleen impression smears shortly after the time o f s c h i z o g o n y s h o w e d intense p h a g o c y t o s i s and the p r e s e n c e o f many m e r o p h o r e s . Several m e c h a n i s m can b e suggested. Schnitzer et al. ( 1 9 7 2 , 1 9 7 3 ) analysed the various m e c h a n i s m s o f filtration and destruction o f parasitised cells in the s p l e e n b y e l e c t r o n m i c r o s c o p y . At the e n d o f schizogony, m a c r o p h a g e s and neutro
phils destroy the altered and infected RBC's, extra-cel
lular parasites and pigment. H o w e v e r , inspection o f impression smears o f organs and ultrastructural o b s e r vations s h o w e d that many merozoites remained unda
m a g e d and form the specialised structures n a m e l y the m e r o p h o r e s . Crosby ( 1 9 5 7 and 1 9 5 9 ) designated the
"pitting function o f the spleen" as the capacity o f this organ to extract inclusions from the RBCs. In order to reach the v e n o u s sinuses and the b l o o d circulation ery
throcytes trapped inside the splenic cords must pro
c e e d through the inter-endothelial slits o f the sinus wall. T h e s e slits are at the most 0.5 p m wide, and only allow the passage o f fully deformable erythrocytes. T h e portion o f an infected RBC w h i c h contains the para
site may remain outside the sinus while the remainder o f the cell g o e s through the slit. A scission b e t w e e n these t w o parts o c c u r s and results in the "pitting" o f the parasite (Schnitzer et al., 1912, 1 9 7 3 ) . M e r o p h o r e sacks w h i c h w e r e frequently s e e n in an extra-extra
cellular position, m a y derive from such pitting.
SPECIFIC CHARACTERISTICS OF THE MEROPHORES T h e formation o f m e r o p h o r e s in the organs o f infected animals and their p r e s e n c e in the lymphatic circulation strongly suggests that they represent a latent form o f the parasite. W e p r o p o s e that m e r o p h o r e s are adap
tive structures w h i c h ensure the longevity o f Plasmo
dium infections, and explain the r e c r u d e s c e n c e s which characterise the c h r o n i c c o u r s e o f these infections.
T h e d i f f e r e n t r e c r u d e s c e n c e p a t t e r n s w h i c h a r e o b s e r v e d with the three murine Plasmodium s p e c i e s studied a p p e a r to correlate with the type o f m e r o phores, s a c k or l e u c o c y t e , found and the red b l o o d cell preference o f these three parasites. P. yoelii nige- riensis has n u m e r o u s m e r o p h o r e sacks and practically n o m e r o p h o r e leucocytes. This parasite preferentially invades reticulocytes. It m a y b e hypothesised that reti
culocytes, w h i c h are less motile and m o r e sticky than n o r m o c y t e s (Key, 1 9 2 1 ) , are readily p h a g o c y t o s e d b y m a c r o p h a g e s w h e n they contain schizonts o f P. yoelii, and thus form the s o u r c e o f the characteristic m e r o p h o r e sacks. Most m e r o p h o r e sacks a p p e a r to reach the b l o o d via the lymph canals, but a small n u m b e r remained inside the lymphatic c h a n n e l s e v e n after the peripheral parasitaemia has cleared. T h e s e persisting
320 Parasite, 1999, 6, 311-322
Mémoire
forms might explain t h e late r e c r u d e s c e n c e s at the lengthy intervals s e e n in t h e natural host. B y contrast, P. vinckeipetteri exclusively invades mature cells, a n d leucocytes w o u l d mainly ingest merozoites released from mature schizonts. I n d e e d this parasites forms practically n o m e r o p h o r e s a c k s a n d latent m e r o z o i t e s are usually s e e n dispersed mostly inside neutrophils and s o m e t i m e s inside m a c r o p h a g e s . T h e s e m e r o p h o r e leucocytes may explain the frequent early, and the rela
tively less c o m m o n late, r e c r u d e s c e n c e s . P. c. cha- haudi-mkcted m i c e p r o d u c e equally abundant m e r o p h o r e s a c k s a n d m e r o p h o r e leucocytes, a n d exhibit frequent r e c r u d e s c e n c e s . It is possible that, as t h e result o f t h e smaller size o f P. chabaudi mature schi
zonts (average o f six m e r o z o i t e s ) a n d merozoites, t h e fraction o f infected RBC's w h i c h g o o n to form m e r o - phores is higher in this s p e c i e s . It is interesting to note that in m a n y instances w h e r e highly chronic infections are observed, the size o f the schizonts is small, for e.g.
P. malariae in m a n , P. atheruri in t h e p o r c u p i n e (Landau et al, 1 9 8 3 ) , a n d P. coulangesi in t h e Mada
gascar lemurs (Landau et al, 1 9 8 9 ) .
CONCLUSION
T h e duration o f h u m a n malaria infections, inclu- ding P. falciparum c a n e x t e n d to m o r e than o n e year, often sub-clinically a n d despite an effective i m m u n e r e s p o n s e (Eyles & Y o u n g , 1 9 5 1 ; Arez et al, 1 9 9 9 ) . It h a s also b e e n n o t e d that sensi
tive parasites s o m e t i m e s reappear despite adequate antimalarial treatment (White, 1998). Metabolically inac
tive drug-resistant merozoites in the m e r o p h o r e w o u l d provide an e s c a p e m e c h a n i s m in the face o f inhibitory drug levels. Parasites originating from t h e s e latent forms could then initiate further waves o f parasitaemia.
W h e n these r e c r u d e s c e n c e s o c c u r at sub-optimal drug concentrations, the selection o f drug resistant parasites w o u l d b e p r o m o t e d .
W e predict that m e r o p h o r e structures similar to those s e e n in rodent malaria will b e found in Plasmodium s p e c i e s infecting h u m a n s a n d other primates. Histolo
gical observations o f h u m a n infections are for t h e most part confined to post-mortem samples o f perni
cious malaria. Observation o f infected RBC's in white b l o o d cells w o u l d b e ascribed to destructive p h a g o cytosis, as d e v e l o p m e n t o f parasites in these cells is currently excluded. T h e formation o f latent erythrocytic malaria parasites a n d their trafficking via t h e lymphatic network h a v e important implications for t h e interpre
tation o f epidemiological, immunological a n d clinical observations. Murine malaria infections offer a most suitable system for further studies o f these latent forms.
SURVIVAL OF MALARIA PARASITES IN THE LYMPHATICS
It is interesting to note that mature schizonts o f human parasites in w h i t e b l o o d cells w e r e d e s c r i b e d b y Bignami in 1 8 9 0 a n d Golgi in 1 8 9 3 ( q u o t e d b y Thayer, 1 9 0 1 ) , w h i c h prompted them to suggest that late recru
d e s c e n c e s might originate from parasites preserved as resistant "spores" in h u m a n phagocytes.
ACKNOWLEDGEMENT
W
e are particularly grateful to R.J.M. Wilson (NIMR, L o n d o n ) for reviewing t h e manuscript critically a n d making e x c e l l e n t impro
vements.
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Reçu le 17 juin 1999 Accepté le 24 septembre 1999
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