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British Medical Bulletin (1982) Vol. 38, No. 2, pp.

117-122

LIFE-CYCLE OF PRIMATE MALARIA PARASITES R S Bray & P C C Garnham


al. 1981), and the other in which the sporozoite evades the
Kupffer cell and enters a hepatocyte directly (Shortt, 1948).
Finally there is the possibility of active invasion of a hepatocyte
via an endothelial lining cell. By 40—48 hours tiny malaria
THE LIFE-CYCLE OF PRIMATE parasites 3 urn in diameter, consisting of a single nucleus and a
MALARIA PARASITES little cytoplasm, can be found in hepatocytes (Shortt et al. 1954;
Krotoskiera/. 1980, 1981, 1982).
Three days after infection these intrahepatocytic forms may
R S BRAY PhD DSc or may not have differentiated into schizonts or hypnozoites
P C C GARNHAM CMG MD DSc FRS (latent forms of tissue stages), depending on whether the species
of malaria parasite causes true relapses or not. Hypnozoite
Department of Pure and Applied Biology development will be considered in section Id.
Imperial College of Science and Technology The exoerythrocytic schizonts' growth is accompanied by
London nuclear division and an increase in cytoplasmic volume. Mature
schizonts may measure 30-70 urn in diameter and grossly
1 General features of the life-cycle of primate malaria enlarge their host cell, pushing the hepatocyte nucleus to one
parasites side and frequently flattening it. No reaction is caused in the
a Introduction of sporozoites and exoerythrocytic
schizogony liver until the schizont bursts. At maturity a piece of cytoplasm
b Cycle in the blood joins with each nucleus to form daughter parasites—the
c Development in anopheline mosquitoes merozoite—measuring about 1 um in diameter. The schizont

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d Relapses and host cell then rupture, releasing merozoites into the blood
2 Special features of important species stream (Garnham, 1966).
a Plasmodium (Plasmodium) vivax
b Plasmodium (Plasmodium) ovale b Cycle in the Blood
c Plasmodium (Plasmodium) malariae
d Plasmodium (Laverania)falciparum The merozoites from the liver are probably short-lived and
e Plasmodium (Plasmodium) cynomolgi and subspecies must find and enter an erythrocyte within minutes. Merozoites
/ Plasmodium (Plasmodium) knowlesi contain an apical complex including organelles which, after
References orientation, come into contact with a site on the erythrocyte
surface, which is probably a specific receptor for that species of
malarial merozoite. The merozoite organelles induce in-
1 General Features of the Life-Cycle of Primate Malaria vagination of the erythrocyte surface and the merozoite enters
Parasites the erythrocyte via the invagination possibly by capping; that is
We wish to emphasize two factors which are of primary the merozoite maintains the original points of contact between
importance in the study of primate malaria: (0 the alternation of itself and the erythrocyte membrane and moves these points of
two hosts—vertebrate and invertebrate—in the life-history of contact back along its own surface membrane and slips into the
the parasite in which three steps occur, two intracellular in the erythrocyte. The merozoite has an envelope consisting of an
vertebrate and the third extracellular in the anopheline mosquito. outer plasma membrane and two inner membranes and acquires
Both sexual and asexual growth and division occur; (ii) a surface coat while in the plasma. On entering the erythrocyte
chronobiology. The timetable of events in all stages is highly it loses the two inner membranes and sheds the surface coat.
significant, for clinical diagnosis, for taxonomic differentiation to After entry into the erythrocyte, the merozoite develops a
subspecies and as a survival mechanism for the parasite. vacuole and becomes a ring form, shaped like a signet ring, with
the nucleus as the seal. The ring grows by increasing the
cytoplasm until the vacuole disappears; the cytoplasm may
a Introduction of Sporozoites and Exoerythrocytic Schizogony remain compact or become amoeboid, and pigment—a break-
When a female anopheline mosquito infected in the salivary down product from haemoglobin—appears in the cytoplasm.
glands with a primate malaria parasite (Plasmodium spp.) bites, The parasite feeds on the erythrocyte by encircling and taking in
the sporozoites in the saliva are injected by the proboscis as it small amounts of erythrocytic cytoplasm which is largely
probes in the skin. It is not known whether the mosquito feeds haemoglobin. Digestion occurs within food vacuoles, with the
directly from a capillary or from a pool formed by the probing production of the typical yellow brown to black malarial
proboscis or, if from a capillary, whether the proboscis is pigment particles as an end-product (see Table I). At this stage
introduced upstream or downstream in the capillary. In any the parasite is known as the trophozoite. The next stage, the
case there is no doubt that the sporozoites reach the blood schizont, is heralded by the division of the nucleus, which it is
stream quickly and efficiently and few are carried into the assumed has increased its DNA content and become X-ploid,
lymphatic system (see Bray, 1981). where X is the eventual number of daughter cells to be
The sporozoites circulate in the blood stream for an hour at produced. The nucleus divides three to five times to form 8—32
the most but probably for much less (Fairley et al. 1947; Sinden nuclei (see Table I). At this stage the parasite occupies much or
& Smith, 1982). Their pathway from blood to liver hepatocyte, all of the erythrocyte and the pigment tends to concentrate into
where they can be found 40 hours later in the case of P. a single mass. Possibly the feeding process may cause small
cynomologi bastianellii of macaque monkeys (Krotoski et al. pieces of the parasite to be broken off and left at the site of
1982), is a matter of conjecture. Two pathways have been feeding near the plasmalemma of the erythrocyte, and it may be
suggested, one in which the sporozoite is taken up by a Kupffer these pieces that stain with Romanovsky stains to give the
cell and spends some time there before actively leaving that cell characteristic stippling to the host erythrocyte caused by many
and invading a hepatocyte (Verhave et al. 1980; Smith et primate malaria parasites (see Table I).

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LIFE-CYCLE OF PRIMATE MALARIA PARASITES R S Bray & P C C Garnham

TABLE I. Some comparative and diagnostic characters of the four human malaria parasites
P. faldparum P vfvax P. ovah P malarias

Duration of primary exoerythrocytlc cycle (days) 51 8 9 14-15


Prase nee of hypnozofltea + + —
Number of exoerythrocytlc meroioltes 30000 10000 15000 15000
Enlargement of nucleua of hepatocyte _ _ + +
Average diameter of mature exoerythrocytlc schlzont (pun) 60 46 60 65
Duration of erythrocytic cycte (nouns) 48 48 50 72
Nature of the host erythrocyte Young Retlculocytes and Retlculocytes and Normocytes
young normocytes. young normocytes
Duffy group positive
Enlargement of hoot erythrocyte — ++ + —
Stippling of host erythrocyte Maurer's clefts Schuff ner's dots Schuffner's dots Zlemann's stippling
Pigment colour Black Yellow—brown Dark brown Dark brown to black
Number of erythrocytic merozoltei 8-32 8-24 4-8, 8
occasionally 16
Shape of gametocytes Crescent Round Round Round
Duration of mosquito cycle at 27 °C (days) 10 8-9 12-14 14-16
Pigment pattern in oocysts Chains and rows Prince of Wales feathers Crossed lines Clustered late on
periphery
Diameter of mature oocyst (nm) 55 50 45 40

The maturation of the schizont and the production of chemotaxic gradient, it penetrates the macrogamete and the two

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merozoites occupy an interval of time characteristic of each nuclei fuse, probably within minutes, to form a diploid zygote.
species of malaria parasite and lasting 24, 48 or 72 hours, thus The zygotes, still containing the pigment of the macro-
giving rise to the typical quotidian, tertian and quartan gametocyte, elongate over the next 12 hours and move through
periodicity (see Table I). The merozoites containing a single the blood clot to the gut epithelium of the mosquito. Between 24
nucleus are budded off from the schizont, leaving a residual and 48 hours after fertilization the zygotes, known as ookinetes,
body containing the pigment. At this time the host erythrocyte penetrate the gut epithelial cells and come to rest between the
disintegrates, releasing merozoites and residual body into the gut epithelial cell layer and the basal lamina that covers the gut
plasma. The residual body is phagocytosed mostly by fixed- on the coelomic cavity side. About two to three days after the
tissue macrophages and the merozoites in the non-immune host infective blood meal the ookinetes have secreted a thin cyst wall
invade new erythrocytes. and probably undergone meiotic division to become haploid
After one or more cycles in the blood the sexual phase of the oocysts. The nucleus must now organize its DNA in order to
parasite begins. The exact stimulus is unknown. The nucleus undergo the number of schizogonic-like divisions necessary to
becomes diploid in the macrogametocyte (female) and octoploid form up to 10000 daughter cells. Such a process resembles in
in the microgametocyte (male). The gametocytes are usually some ways the exoerythrocytic schizogony in the next stage of
round (crescentic in one case), contain a single nucleus and the cycle. The oocyst will undergo repeated nuclear divisions at
scattered pigment; when mature they fill the host erythrocyte. a rate dependent upon the species of malaria parasite and the
They also give rise to the stippling of the host erythrocyte temperature (see Table I). The pigment, which can be seen in
typical of the species. fresh young oocysts before repeated division obscures it, lies in
Hawking et al. (1968) showed that, in synchronous infections patterns typical of the species (see Table I). During nuclear
of several species of Plasmodium, gametocytes matured during division of the oocyst the cytoplasm is thrown into an
the night and suggested that this represented a device whereby anastomizing sponge-work and eventually the nuclei are
their presence in the blood at this time coincided with the biting arranged along the surface of this network. Instead of a
habits of the nocturnal anopheline mosquitoes. sponge-work, the contents of the oocyst are in some species
condensed into spheres 4 or 5 urn in diameter, and resemble the
c Development in Anopheline Mosquitoes sporoblasts described by the earlier workers. From the surface
The sexual cycle is completed when a suitable species of of the network or of the sporoblasts, small protrusions form into
mosquito bites the individuals in whose blood mature which a nucleus and other organelles pass; in this way the fine
gametocytes are circulating. The macrogametocyte escapes sporozoites are developed. The latter are slightly curved and
from the erythrocyte and becomes a free macrogamete in the about 11-14 urn long; they eventually become free and emerge
lumen of the mid-gut of the mosquito; the nucleus undergoes into the haemocoelomic cavity, through rents in the old oocyst
reduction division. The microgametocyte undergoes internal wall.
organization, including spindle formation, and then emerges These free sporozoites are carried all over the mosquito by the
from the erythrocyte, and its nucleus undergoes a series of haemocoelomic fluid but concentrate, probably under chemotaxic
extraordinarily rapid divisions (Sinden, 1981) to form eight new control, in the salivary glands of the mosquito. Here by a totally
nuclei. From the cytoplasm eight filaments are extruded, each unknown process they invade the acinal cells of the salivary
equipped with a flagellum, and into these filaments the nuclei glands and mature into infective forms. They then penetrate
migrate completing the formation of the microgametes. This further into the salivary glands and appear in the ramifications
process takes about 10-20 minutes after blood is ingested by the of the salivary duct where they are able to infect the next
mosquito and is induced by various factors, including tempera- primate that the mosquito bites, by flowing with the saliva into
ture and CO 2 partial pressure. The microgamete, once formed, the wound.
breaks off from the now shrunken body of the former
microgametocyte and moves off driven by the flagellum in d Relapses
search of the macrogamete. When the haploid microgamete The malaria parasites of primates that cause true relapses are
finds a haploid macrogamete, perhaps by moving along a P. vtvax and P. ovale of man and a number of malaria parasites

118
LIFE-CYCLE OF PRIMATE MALARIA PARASITES R S Bray & P C C Garnham

FIG. 1. Exoarythrocytic development of various primate malaria parasites

Rfaldpanim

Rovala

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RvWax NIcoiMvi Moscow strain (R v. hibarnans)

Rvtvax North Korean strain


-M-

• « / » • - • Wood rhmtr, (R)

Rylvax St.EIzab*th,Madagascar,North M a n itraint

Rv1va« Chasaon atrafn (and Rcynomotgl, P.fWdl, Rslmlovata)

0 U 24 tim !• 4«

The thickness of the horizontal lines is Intended to indicate the proportion of the sporozoites that differentiate into immediately growing
schizonts or into the various populations of hypnozoites destined to cause relapses. All timings, other than the immediately growing
schizogony cycle, are necessarily approximate or even notional
(R): relapse if a primary attack has occurred
(P): primary attack if an early primary attack has not occurred

119
LIFE-CYCLE OF PRIMATE MALARIA PARASITES R S Bray & P C C Garnham

of apes and monkeys, which share with P. vivax and P. ovale the population of hypnozoites destined to bring about the
the characteristics of a 48-hour blood cycle and Schuffner's dots relapse commences to grow and undergoes exoerythrocytic
on the infected erythrocyte surface (Garnham, 1977). We schizogony, forming merozoites that invade the blood. "Reac-
define a true relapse as renewed parasitaemia following a period tivated" or "relapse" schizonts were first described by Shortt &
in which the blood contains no detectable parasites. Malaria Garnham (1948) in P. cynomolgi infections and have since been
parasites for which no evidence exists for relapses and whose seen by other observers. In recent experiments, Krotoski et al.
sporozoites are thought not to differentiate into hypnozoites in (1982) have described "relapse" schizonts varying from 16 urn
the liver include P. falciparum and P. malariae of man, P. to 38 urn in diameter at 50 and 105 days after sporozoite
reichenowi of apes, and the quotidian and quartan (72-hour infection.
blood cycle) malaria parasites of monkeys. In these parasites Additional populations of hypnozoites, in our belief, will
we believe all sporozoites develop directly into hepatic schizonts. cause further relapses, with the blood becoming clear of
The sporozoites of the relapsing malaria parasites differen- parasites between relapses. The number of populations of
tiate into either hypnozoites or developing schizonts in varying hypnozoites and the number of hypnozoites per population will
proportions, depending upon the species and strain (see section vary with the strain and species of parasite involved and its
2a and fig. 1). The hypnozoites remain dormant as single- characteristic relapse pattern (see fig. 1).
nucleated intrahepatocytic round bodies of about 4—5 urn in The characteristics of the human malaria parasites are
diameter. We believe that at the predetermined time for a tabulated in Table I. Figure 2 shows the life-cycle of a primate
relapse (e.g. 8—10 months in temperate-zone P. vivax infections) malaria parasite in diagrammatic form.

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FIG. 2. The life-cycle of a primate malaria parasite

1: sporozoites Injected into skin by the mosquito; 2: 2-day-old exoerythrocytic form in a hepatocyte; 3, 4, 5: growing exoerythrocytic
schizonts; H: hypnozoites in hepatocytes; 6: mature exoerythrocytic schizonts bursting, releasing merozoites into the blood; 7: erythrocyte;
8, 9: growing trophozoites; 10, 1 1 : growing schizonts; 12: mature schizont releasing merozoites; 13, 14, 15, 16, 17: erythrocytic cycle
repeated; 18, 19: growth of the microgametocyte; 20, 2 1 : growth of the macrogametocyte; 22: mosquito has taken gametocytes up into
its mid-gut; 23: exflagellation of the microgametocyte; 24: macrogametocyte escapes from erythrocyte to become a macrogamete; 25:
microgamete; 26: macrogamete about to be fertilized; 27: zygote or ookinete; 28, 29, 30: oocyst growth on the mid-gut surface; 3 1 :
odcyst bursting, releasing sporozoites; 32: sporozoites in the mosquito salivary glands

120
LIFE-CYCLE OF PRIMATE MALARIA PARASITES R S Bray & P C C Garnham
2 Special Features of Important Species past two or three centuries into the New World by immigrants
from Europe and West Africa; the strain then became adapted
Some of the characteristic features of the life-cycle of two to the local monkey and gradually spread through the forest
groups of parasites are discussed below: the characteristic The life-cycle and morphology of the two parasites appear to be
human species P. vivax, P. ovale, P. malariae and P.falciparum identical, and man and South American monkeys are suscep-
and the simian species that occur rarely as human zoonoses and tible to both (Coatney et al. 1971). The anopheline vector of .P.
that have also been used extensively as models of human rodhaini (= P. malariae) is unknown; it seems that in
malaria in research (P. cynomolgi and P. knowlesi). present-day conditions man would not be bitten by sylvatic
a Plasmodium (Plasmodium) vivax mosquitoes and thus a zoonosis does not occur. Under
laboratory conditions P. brasilianum (= P. malariae) is easily
This species is a relapsing parasite causing benign tertian transmitted to man by the bite of infected mosquitoes.
malaria of man; it has a largely subtropical distribution though The quartan periodicity of this parasite in the blood is
it occurs frequently in South-East Asia and New Guinea. Its reflected by the long duration of sporogony in the mosquito and
sporozoites differentiate in the liver into schizonts or hyp- exoerythrocytic schizogony in the liver (see Table I). The
nozoites. The schizonts grow for 8 days and then produce persistence of the parasite for many years, perhaps even for a
merozoites, which produce a primary clinical malaria attack lifetime, was thought to denote a long succession of relapse
usually about 15 days after the introduction of sporozoites. cycles in the liver. The relapse cycle in other forms of malaria is
Some northern strains of P. vivax, such as the Russian strains of now known to be solely initiated by sporozoites. No late
Nicolaev (1949) and Tiburskaja et al. (1968) (so-called P. vivax "relapse" exoerythrocytic stages have been detected in livers
hibernans), and the Hainan (China) strains (so-called P. vivax infected with quartan parasites. The infection is as persistent
multinucleatum) may have few, or no, sporozoites which form

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after inoculation with infected blood as with sporozoites. Both
schizonts immediately so that early primary attacks are the original attack and the relapses are easily cured by
unknown (see fig. 1). Tropical strains tend to show a number of schizontocidal drugs such as chloroquine. Individuals with no
randomly distributed relapses following an early primary attack. symptoms frequently infect recipients when acting as blood
Intermediate subtropical strains may or may not show an early donors. All these factors confirm that persistent hypnozoites in
primary attack and will usually have a late primary attack or a the liver do not occur in P. malariae infections or infections with
relapse at about 9 months after infection. Obviously these other quartan parasites.
patterns fit the seasonal activity of Anopheles of the area.
Figure 1 shows how we presently think the various strains of P. None of the quartan parasites enlarge their host erythrocytes
vivax sporozoites develop. and all cause Ziemann's stippling of the erythrocyte, a
dust-like blush, on heavy Romanovsky staining. The exo-
During the erythrocytic development of P. vivax all forms erythrocytic schizont causes enlargement of the host hepato-
can be found in the peripheral blood. Merozoites of P. vivax cyte nucleus.
have a receptor that attaches to a site upon young human
erythrocytes, which is (or is associated with) the Fy Fy (Duffy
group) antigen, so only Duffy-group-positive individuals suffer d Plasmodium (Laverania) falciparum
P. vivax malaria (Miller et al. 1976). This is the basis for the This parasite belongs to a different subgenus by virtue of the
innate resistance of subjects of pure Negro lineage who are crescentic shape and lengthy development of its gametocytes. It
uniformly Fy Fy — ve. P vivax greatly enlarges its host erythro- does not cause relapses, and renewed activity in P. falciparum
cyte and causes it to become regularly stippled when stained. It infections springs from persisting if undetected erythrocytic
forms golden-brown pigment and its gametocytes are round. infections and is called a "recrudescence". The parasite causes
malignant tertian malaria, and, as the name implies, may kill. It
b Plasmodium (Plasmodium) ovale is a largely tropical parasite and is the great scourge of Africa
This species is another relapsing parasite of man which and South-East Asia.
resembles P. vivax. It is largely confined to Africa and its All the sporozoites of P. falciparum develop directly into
relapse patterns are not well known. It differs from P. vivax in schizonts, and hypnozoites do not occur (see Table I and fig. 1).
having a nine-day liver schizogony where it enlarges the host The liver schizogony occupies 5$ days and schizonts reach
hepatocyte nucleus (see Table I) and in causing an oval 50 um in diameter. The erythrocytic schizogony occurs in the
distortion of its host erythrocyte during the making of thin blood blood space of inner organs. The ring forms appear in the
smears, which can be preserved and used as a diagnostic tool if peripheral blood after merozoite invasion of young erythrocytes
the blood smear is dried quickly. It also possesses slightly larger and persist there until just before the black pigment appears. At
and more irregular nuclei, produces eight nuclei as a rule in the this stage a stippling known as Maurer's clefts appears, which
primary attack and displays dark brown pigment in the when stained shows as streaks, commas or large dots. The para-
erythrocytic phase. In the oocyst stage it shows two lines of sitized erythrocyte then disappears from the peripheral blood
pigment usually crossed. and will reappear only when containing a new ring form or a
fully mature crescentic gametocyte. Further development occurs
c Plasmodium (Plasmodium) malariae in the capillaries of the spleen, liver, lung, heart, brain and other
This species, causing quartan malaria of man, used to be of organs and notably in the intervillous spaces of the placenta in
cosmopolitan distribution, though it tended to be common in the pregnant woman who is more susceptible to infection with
certain parts of the world, e.g. tropical Africa, Guyana and parts this parasite. The cause of this sequestration may be an
of India. It is a natural parasite of the chimpanzee in which it adherence between distortions on the infected erythrocyte
was originally named P. rodhaini, now regarded as a synonym surface and endothelial lining cells (Luse & Miller, 1971;
of P. malariae. The quartan parasite of South American Aikawa, 1977), though this cannot be the case in the placenta
monkeys, P. brasilianum, is thought by many workers to (Bray & Sinden, 1979). The means of retention of the
represent the result of the introduction of P. malariae in the developing gametocyte is unknown. The gametocyte of P.

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LIFE-CYCLE OF PRIMATE MALARIA PARASITES R S Bray & P C C Garnham

falciparum takes about 10-11 days to develop, as opposed to f Plasmodium (Plasmodium) knowlesi
about two days for P. vivax. When mature it appears in the This common parasite of South-East Asian kra monkeys is
peripheral circulation and is available for uptake by a female unique among the primate species in possessing a quotidian
mosquito. (24-hour) periodicity in the blood. For this and other reasons (h
is sequestered to some degree in inner organs) its pathogenicity
to unnatural hosts such as the rhesus monkey is extremely high;
e Plasmodium (Plasmodium) cynomolgi and Subspecies the rapid erythrocytic schizogony in the blood causes the
P. cynomolgi of Asian macaque monkeys closely resembles P. infection to build up so quickly that effective immunity has no
vivax in all respects, and the type and its subspecies P. c. time to develop and the rhesus monkey dies from a fulminating
bastianellii form ideal models for certain strains at least of the parasitaemia.
human parasite. So much so, that laboratory transmission from The duration of the exoerythrocytic schizogony is also
monkey via mosquitoes to man took place first accidentally then shorter (5$ days) than that of similar stages in other primate
deliberately in both the USA and England. The parasite is so species of the subgenus Plasmodium. P. knowlesi is not a
infectious to man that it seems zoonotic transmission must be relapsing parasite as hypnozoites do not occur. Sporogony is
common in the enzootic areas in South-East Asia, though also short (8-9 days). The blood stages do not enlarge the host
infection has not been reported. The human disease is mild and erythrocyte but do erratically cause some irregular stippling of
might well remain unrecognized. the erythrocyte.
The blood stages of P. cynomolgi are slightly less amoeboid P. knowlesi is a proved zoonosis (Chin et al. 1965); an
and smaller than those of P. vivax. Exoerythrocytic schizogony American soldier became infected in the Malayan jungle and
is slightly faster. developed malaria about 10 days later. This species has in the

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The discovery by Krotoski et al. (1980, 1982) of the past been used for malaria therapy and after about 170 blood
hypnozoite and its significance relative to relapses (discussed in passages became highly virulent for man, although in earlier
section Id) was made using P. c. bastianellii. As P. cynomolgi, transfers the pathogenicity for man was low.
like P. vivax, is unable to infect Negroes it can be assumed that
its merozoites like those of P. vivax and P. knowlesi invade
ACKNOWLEDGEMENTS
erythrocytes via a receptor which is, or is associated with, the
Duffy group antigen site. We would like to thank Jill Hammond who drew the figures.

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