Preface (2003)

The Primate Malarias was published in 1971 and summarized knowledge

on different species of Plasmodium that develop in non-human primates. It has served as
a foundation for subsequent investigations on these parasites, their primate hosts, and
their mosquito vectors. The Division of Parasitic Diseases of the Centers for Disease
Control and Prevention has made this electronic version available to the scientific
community in hopes that it will stimulate and support continued interest in these parasites
and their contribution to the understanding and control of malaria.

This electronic version was produced by James J. Sullivan, Gregory Noland, and
Leanne Ward, Biology and Diagnostics Branch of the Division of Parasitic Diseases.

William E. Collins
Division of Parasitic Diseases
Atlanta, Ga.
March 2003

Suggested citation:

CD-ROM. The primate malarias [original book published 1971]. Coatney GR, Collins
WE, Warren M, Contacos PG. Division of Parasitic Disease, producers. Version 1.0.
Atlanta, GA: CDC; 2003.
THE
PRIMATE
MALARIAS

G. ROBERT COATNEY, Ph. D., D. Sc. in Public Health (hon.), Sc.D. (hon.) Formerly:
Chief, Laboratory Parasite Chemotherapy, National Institute of Allergy and Infectious
Diseases, NIH, Bethesda, Md.; Professor of Pharmacology, Louisiana State University
Medical School. Presently: Visiting Professor of Community Health Practice, Howard
University Medical School; Visiting Lecturer in Tropical Public Health, Harvard
University School of Public Health; Visiting Professor of Pharmacology, Tulane
University School of Medicine; Member, Expert Panel on Malaria, World Health
Organization; Member, Commission on Malaria, Armed Forces Epidemiological Board,
Department of Defense; Consultant on Malaria, Center for Disease Control, Atlanta, Ga.
WILLIAM E. COLLINS, Ph.D. Research Biologist, Section on Primate Malaria,
Laboratory Parasitic Diseases, NIH, Chamblee, Ga.
McWILSON WARREN, Ph.D. Scientist Director, Center for Disease Control, Central
America Malaria Research Station, San Salvador, El Salvador, C.A.
PETER G. CONTACOS, Ph.D., M.D. Head, Section on Primate Malaria, Laboratory of
Parasitic Diseases, NIH, Chamblee, Ga.; Member, Expert Panel on Malaria, World
Health Organization; Associate Member, Commission on Malaria, Armed Forces
Epidemiological Board, Department of Defense.

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE

National Institutes of Health
National Institute of Allergy and Infectious Diseases
Bethesda, Maryland 20014
 1971

Library of Congress Number 71-610655

For sale by the Superintendent of Documents

U.S. Government Printing Office
Washington, D.C. 20402
Price $7
Stock Number 1744-0005

ii
 Dedicated to

DR. DON EDGAR EYLES

4 September 1915 to 4 October 1963

and

to the inmates at the United States Penitentiary,

Atlanta, Georgia, who volunteered to accept
infection with human and simian malarias.

iii
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 Preface

THIS book is about the malaria parasites of Primates. It deals

with the parasite as seen in the vertebrate host and in the mosquito with comments on
each of these hosts as their life habits or geographical location may contribute to the
biology of the plasmodium. The knowledge that primates have malaria is not new, but
the fact that lower primates, especially the monkeys and apes, harbor malarias infective
to man, and, which produce disease in him, is a relatively new concept and one of
special significance in the light of worldwide programs of malaria eradication and
control.
Because of the dramatic suddenness with which this concept burst upon the world
community of malariologists, we have included a detailed account of the initial
happenings and their ramifications during the next ten years.
The first section deals with the evolution of the Haemosporidia, historical review,
ecology of the hosts, and life-cycle including relapse. In subsequent sections each
species is dealt with separately, beginning with its discovery and taxonomy, the cycle
in the peripheral blood, the sporogonic cycle, the cycle in the tissues, the course of the
infection, host specificity, and immunity and antigenic relationships. Pathology,
chemotherapy, and the clinical aspects of the disease process are considered outside the
scope of this work except as they may have a direct bearing on the specific malaria
under discussion.
In dealing with individual species, it is recognized that, at present, the key to their
identification rests with the cycle in the blood. It is not unlikely, however, that in the
hands of the zoologist, knowledge of the sporogonic cycle will contribute greatly to
species identification. In support of this concept, we have included comparison studies,
done under controlled conditions, which, in some situations confirm, and in others pose
some doubt, as to taxonomic identities and species relationships. The cycle in the
tissue, except in a broad sense, can contribute little in this regard because these stages
fail to show different, distinct, and constant morphological characteristics which permit
species identification. Blood smear preparations must be used in the routine
identification of species in man and lower animals but the specialist may find it
advantageous to turn to the cycle in the mosquito or, to examine the serologic
relationships for absolute identification in closely related species.
The course of the infection in the normal vertebrate host is described and
illustrated, followed by its manifestations in other hosts, man or lower primates, or vice
versa, if data are available.
The portion dealing with host specificity treats that item in both the vertebrate and
invertebrate hosts in order to cast light on distribution and host specificity. Likewise,
the discussion of immunity and antigenic relationships is treated in the light of
speciation.
The synonomy of the malaria species is discussed in the introductory portion of a
given chapter but for the human malarias it is given in tabular form, too, directly
preceding the text. In presenting the presently accepted zoological name of the
vertebrate host, we have relied on the Handbook of Living Primates by Napier and

v
Napier, 1967. In cases where the synonomy seemed clouded, we accepted the name
given in the Checklist of Palearctic and Indian Mammals by Ellerman and Morrison-
Scott, 1951. For the correct synonomy of the mosquitoes we accepted A Synoptic
Catalog of the Mosquitoes of the World by Stone, Knight, and Starcke, 1959.
The literature on human malaria and some of the simian malarias, too, is so vast
that we elected to base this work on selected references considered to be the most
interesting and relevant through 1969; in a few instances, because of the importance of
the work, we have included work published in 1970. Most references have been seen
by one of us and where that was not possible, the listed reference is followed by (NS) =
not seen.
The colored plates are an important part of this work and in planning for them we
decided on certain requirements which would have to be met in order for a plate to be
included: (1) the material for the plate must be our own, (2) the smear preparations
would be stained by one of us and under exactly the same conditions as to stain, time,
and pH, and (3) all of the illustrations would be done by a single artist working under
the direct supervision of either McWW or GRC.
Our greatest difficulty was in fulfilling the first requirement. It took us something
over two years to obtain an infection of Plasmodium reichenowi and one of us
(McWW) made a special trip to Borneo in order to get material for the P. pitheci plate.
We were never able to obtain an animal infected with P. rodhaini or parasitized blood
for infecting our own animal; as a consequence, there is no plate depicting this parasite.
We had to relax our requirements in the case of the malarias of lemurs. The material for
that plate is copied from the work of the original describers.
This work was started in early 1966 by the senior author and Dr. McWilson
Warren while colleagues in the Laboratory of Parasite Chemotherapy at the National
Institutes of Health in Bethesda, Maryland, and continued, with interruptions, during
the three years the senior author was Professor of Pharmacology at the Louisiana State
University Medical School, New Orleans, Louisiana and finished, with the
collaboration of Drs. William E. Collins and Peter G. Contacos, at the Center for
Disease Control, Atlanta, Georgia.

ATLANTA, GA. G.R.C.

December, 1970 W.E.C.
McW.W.
P.G.C.

vi
 Acknowledgements

SPECIFIC acknowledgement is made for support, facilities,

and special services received from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health; the Department of Pharmacology, Louisiana
State University Medical School, New Orleans, La.; and the Center for Disease
Control, Health Services and Mental Health Administration, Atlanta, Ga.
We wish to thank the Director of the Bureau of Prisons, Department of Justice,
and his staff and the inmate volunteers for allowing us to carry on transmission studies
at the U.S. Penitentiary, Atlanta, Ga. We are indebted for numerous contributions from
Dr. G. M. Jeffery, who was Chief, Laboratory of Parasite Chemotherapy, NIAID, NIH,
during 1966-1969. We also extend thanks to the Institute for Medical Research, Kuala
Lumpur, West Malaysia, the South Carolina State Hospital, Columbia, South Carolina,
and to the Delta Regional Primate Research Center, Covington, Louisiana for their
interest and cooperation in many of the studies reported here.
Particular acknowledgement is made of important contributions to this monograph
by Mrs. Gertrude Nicholson, Medical Illustrator, NIH, who painted the colored plates;
by Mr. J. C. Skinner, Biologist, who prepared the charts and graphs; by Mrs. Nancy
Herbon, formerly of the Louisiana State University staff, for valuable aid and,
especially, for her diligence in locating many hard-to-find references; and to Mr.
Clinton S. Smith, Biologist, for the unique photographs of the sporogonic and EE
stages.

vii
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 Contents
Page

Preface …………………………………………………………………………………………….v
Acknowledgements ……………………………………………………………………………...vii

SECTION 1—GENERAL
1. Evolution of the Primate Malarias …………………………………………………..1
2. Historical Review …………………………………………………………………. 11
3. Ecology of the Hosts in Relation to the Transmission of Malaria …………………19
4. Life Cycle and the Phenomenon of Relapse ……………………………………….29

SECTION 2—VIVAX-TYPE PARASITES

5. Plasmodium vivax ………………………………………………………………….43
6. Plasmodium cynomolgi ………………………………………………………….…69
7. Plasmodium eylesi …………………………………………………………………99
8. Plasmodium gonderi ……………………………………………………………...107
9. Plasmodium hylobati……………………………………………………………...117
10. Plasmodium jefferyi ………………………………………………………………125
11. Plasmodium pitheci……………………………………………………………….137
12. Plasmodium schwetzi ……………………………………………………………..141
13. Plasmodium simium ………………………………………………………………153
14. Plasmodium youngi ……………………………………………………………….163

SECTION 3—OVALE-TYPE PARASITES

15. Plasmodium ovale…………………………………………………………………171
16. Plasmodium fieldi …………………………………………………………………185
17. Plasmodium simiovale..............................................................................................197

SECTION 4—MALARIAE-TYPE PARASITES

18. Plasmodium malariae ..............................................................................................209
19. Plasmodium brasilianum ..........................................................................................231
20. Plasmodium inui .......................................................................................................245
21. Plasmodium rodhaini ...............................................................................................259

SECTION 5—FALCIPARUM-TYPE PARASITES

22. Plasmodium falciparum ...........................................................................................263
23. Plasmodium coatneyi ...............................................................................................289
24. Plasmodium fragile ………………………………………………………………..301
25. Plasmodium reichenowi …………………………………………………………...309

ix
 CONTENTS (Continued)
SECTION 6—OTHER TYPE PARASITES
26. Plasmodium knowlesi …………………………………………………………….317
27. Plasmodium girardi ………………………………………………………………335
28. Plasmodium lemuris ……………………………………………………………...339

SECTION 7—INDEXES
Author Index …………………………………………………………………………...343
Subject Index …………………………………………………………………………..353

x
SECTION 1
GENERAL
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 1
Evolution of the Primate Malarias
for the insect in contrast to the situation in many
THE evolution of the of the vertebrate infections. Traditionally, high
Haemosporidia has occupied the attention of levels of pathogenicity in a host-parasite system
many investigators and detailed discussions of are considered to be indicative of a recent
the subject have been presented by Baker association. There is undoubtedly an increased
(1965), Ball (1943, 1960), Bray (1957, 1963), potential for survival in any parasite that creates
Bruce-Chwatt (1965), Christophers (1934), as little discomfort as possible for its host and
Garnham (1955, 1963, 1966), Manwell (1955), obviously there is no real lasting advantage to a
and others. The subject is extremely complex parasite which destroys its host. Ultimately, this
since not only must the evolution of the logic can be extended and symbiosis becomes
protozoan be accounted for but the concomitant the goal of all parasites. If this tenant is
evolutionary development of both the vertebrate accepted, it is reasonable to assume that less
and invertebrate hosts as well. Conclusive proof pathogenic associations are older than those in
has not been presented to substantiate a which the whole gamut of the host's resistance
particular evolutionary scheme for these mechanisms are forced into play. It is thus
parasites and their hosts and it seems unlikely reasoned, by many workers, that the malaria
that incontrovertible evidence to support any parasite has been in association with the insect
particular theory will be forthcoming. However, for a longer period than it has with the vertebrate
the subject does provide exciting grist for the host and, therefore, the coccidian ancestor of the
evolutionary biologist to the extent that now it is Haemosporidia was originally an insect parasite.
more than an academic exercise. There are some The first of these two arguments is
very practical considerations to the evolutionary plausible and difficult to refute. Christophers
trends in the primate malarias and these will be (1934) suggested that the association between
discussed in more detail later. dipterans and plasmodia was established because
Most workers agree that the malaria of the tree dwelling habits of mosquitoes, birds,
parasites descended from a coccidian ancestor. rats, and monkeys. This argument does not
However, there are two schools of thought as to appear to be very strong and is based on the
whether this ancestor was parasitic in an insect assumption that since the initial vertebrate
host or in the intestine of a vertebrate. The infections were possibly in reptiles, these
strongest evidence for the theory of an insect animals were tree dwellers also. Such an
origin of the malaria parasites has been put forth explanation does not help to understand the
by Huff (1938, 1945) and concentrates on two failure of the protozoans to develop in fleas,
fundamental observations: 1) The parasites are ticks, lice, and mites since certainly these
found in a very diverse group of vertebrates ectoparasites intimately share an environment
(reptiles, birds, and mammals) but the with their hosts which probably antedated the
invertebrate hosts are always dipterans. dipterans in the development of a blood-sucking
Development patterns in the insect are similar in activity. Thus Christopher's argument is an
all the species of malaria parasites and the interesting one but seems much less convincing
invertebrate hosts form a much closer group than than the evidence presented by Huff to support
do the vertebrate hosts of the same parasites. 2) the probable insect origin of the coccidian
The Haemosporidia are relatively apathogenic ancestor of the contemporary members of the

1
 PRIMATE MALARIAS 2

genus Plasmodium. (The same idea was put developmental stages of malaria parasites found
forth by Fantham (1936) but without in the vertebrate host seem to be reversed if the
elaboration). parasite ancestor had been an insect coccidian.
It is interesting to note how a different In other words schizogony and gametogony
emphasis can influence the interpretation of the should be in the mosquito host and sporogony in
same information. Huff considers the dipteran the vertebrate host.
hosts of malaria parasites to be closely related The first of these arguments is interesting
when compared to the vertebrate hosts of the but the parasite could well have evolved after
same parasites. At the same time, Bruce-Chwatt the blood sucking dipterans were on the scene.
(1965) refers to the same group of insects as The second argument is important in that it
"diverse". Thus the same information has been reveals the potential of the coccidian intestinal
used to support the insect origin of the malaria parasites to invade tissues, but such a potential
parasites (Huff) and the antithesis of this point did not have to be realized.
of view (Bruce-Chwatt). The last two arguments of Bray and
The second body of evidence is, in our Garnham are possibly the most convincing in
view, somewhat less convincing. The absolute defense of a vertebrate coccidian as the ancestor
associations between pathogenicity and the age of the malaria parasites in that they seem to offer
of a particular host-parasite system is possibly the greatest challenge when one is searching for
too simple. One cannot argue with the biological alternative explanations.
success of symbiosis over parasitism. However, It is our conclusion that the immediate
we have to agree with Ball (1960) in that ancestor of the plasmodia is still not clear.
although long host-parasite associations However, the greatest weight of available
frequently result in lowered pathogenicity there evidence seems to support the position that a
is no reason to assume that this is always the tissue invading parasite of the vertebrate
case. In addition, it should not be assumed that a intestine, in a reptile, found its way into the
new host-parasite association will be pathogenic. blood of the animal and eventually into a
If we accept the proposition that the blood-sucking dipteran. The peculiar association
developmental sequence was from free living of mammalian malarias and culicines probably
forms, to commensals, to parasites, then reflects an early development of rather
apathogenicity actually preceded pathogenicity. circumscribed feeding habits on the part of the
Support of a coccidian parasite of the mosquitoes. The perplexing absence of
vertebrate intestine as the ancestor of the involvement in the life cycles of these parasites
Plasmodiidae comes primarily from Manwell by other blood sucking arthropods, namely fleas,
(1955), Bray (1957, 1963), Baker (1965), ticks, mites, and/or lice remains one of the most
Bruce-Chwatt (1965), and Garnham (1966). difficult aspects of this problem to understand if
Manwell (1955) notes that malaria parasites the parasites did arise in the vertebrate and
occur in birds and reptiles which probably therefore were presumably provided with many
preceded the blood sucking diptera in their opportunities to adapt to some, if not all, of this
evolution. Baker emphasizes the tendency of latter group of the ectoparasites.
coccidians of the vertebrate intestine to become The long period between the initial efforts
tissue parasites as further evidence to support the of the primitive tissue invading coccidian to
intestinal coccidian as the most likely ancestor adapt to the blood stream of a vertebrate and the
of the malaria parasites. Garnham (1966) points digestive tract of a blood sucking dipteran, and
out the difficulty in visualizing the existence of the development of our contemporary genera
the plasmodia as dipteran parasites without and species of the malaria-like parasite remains
intervention of a vertebrate if the life cycle in obscure. Undoubtedly there were many failures
these insects was similar to that seen in as new vertebrate and invertebrate hosts were
contemporary species of Plasmodium. Bray tried. However, these efforts were successful to
presents a well constructed discussion of this a large degree since the genus Plasmodium alone
subject in which he points out that the is now found in all major groups of terrestrial
3 EVOLUTION OF THE PRIMATE MALARIAS

vertebrates and is represented by a large number the ancestor of the New World monkeys was
of distinct species. As the malaria parasites isolated from the main Old World line at a very
evolved, they maintained certain basic features pivotal point when a variety of new
of morphology (particularly pigment production morphological and physiological features were
in the erythrocytic stages), life cycle (all being tested for their ability to survive. Thus, the
presumably have schizogonic development in non-human primates of the Western Hemisphere
both fixed tissue cells and circulating blood cells are monkey-like, reflecting a close ancestral
of the vertebrate host) and host range (all relationship to the African and Asian forms.
undergo a sexual period of development with the However, differences between the two great
subsequent production of sporozoites in the groups are in many ways quite marked,
alimentary tract and haemocoele of a dipteran). revealing an extensive period of isolated
To discuss the evolution of all the species of development.
Plasmodium is beyond the scope of this work; The Old World primate stock undoubtedly
so, we will direct our attention to a more produced not only the common monkeys of Asia
detailed examination of those forms found in and Africa but all of the anthropoids as well.
primates. There is a considerable amount of controversy
The differentiation of the primate malarias concerning the precise line through which man
is relatively recent and the trends that produced eventually evolved. Three major theories on the
the currently known aspects of speciation and branching points between pongids and hominids
distribution are, in our opinion, part of a were reviewed by Mayr (1963). Briefly, these
dynamic process which is still not completed. three theories are: 1) that the hominid line
Many theories have been advanced in an effort branched from the common stem before the
to correlate the distribution of contemporary living anthropoids split into three different lines;
primate plasmodia with various known aspects 2) that the hominid line branched off after the
of their vertebrate hosts; i.e., physiology, gibbon line but before the pongids split into
distribution, and ecology. The evolutionary lines that eventually gave rise to Pan and Pongo;
relationships of most parasites must necessarily and 3) that the hominid line branched off from
be speculative since direct knowledge is the line of the African apes at a comparatively
confined to very recent history. This is recent date and long after the pongid line had
particularly true of the malaria parasites since split into an Asiatic (Pongo) and an African
they were discovered less than 100 years ago. (Pan and Homo) branch. Fossil records
Prior to this century, we have only vague supporting either of these theories are limited
historical references to clinical entities which we but hemoglobin and serum protein data make the
struggle to attribute to currently known disease third alternative more likely. Supportive
agents. Therefore, an understanding of the evidence for the closeness of the hominid and
evolution and interrelationships of the African lines of evolution has been reviewed and
contemporary species of primate plasmodia and expanded by Dunn (1966), with particular
associated aspects of host and geographic reference to their parasites.
distribution will depend, for the most part, on Efforts to orient the evolution of the
our knowledge of primate evolution. malaria parasite to the general trends of primate
Different approaches to the evolution of evolution have been successful only to a limited
primates vary in detail but there are some extent. It would be convenient to pick up the
concepts on which there is considerable story with a primitive hepatocystis-plasmodium
agreement. There seems little doubt that there like ancestor in one of the lemuroid progenitors
was a lemuroid-tarsieroid ancestor for all of the of contemporary primates and follow the
contemporary primate species and that the development of both the parasite and the
evolutionary changes resulting in today's primates through successive evolutionary stages.
simians and anthropoids began in the middle or Unfortunately, such an approach runs into
late Eocene some 35-40 million years ago. In difficulties when the current distribution of the
addition, there is little conflict over the idea that primates and their plasmodia is considered.
 PRIMATE MALARIAS 4

There are true plasmodia in the lemurs of comparatively recent offshoot from the African
Madagascar but the biological isolation of this ape line if we assume that the malaria parasites
island presents many problems in evolutionary were evolving at the same time and through the
origins. Also, considerable difficulty is found in same lines as the primates. However, only one
accounting for the distribution of malaria species of plasmodia (P. gonderi) has been
parasites among the living species of monkeys. identified in African monkeys although
The proliferation of species in Asia presents a Hepatocystis is quite common in these animals.
probable picture of long association of a It is difficult to understand the evolutionary
host-parasite system and indicates a considerable sequence that would yield six species of
amount of success on the part of the parasite. If Plasmodium representing three basic types in the
we confine our examination of parasite-primate apes and man in Africa and in the process see
evolution to southern Asia, then we encounter the development of only one species of one type
relatively little difficulty in arriving at a in monkeys in the same area. It is possible that
sequence of events which lead directly to current ancestors of living African monkeys had malaria
associations of monkeys, apes, and man and parasites at one time which have been lost
their respective malarias. subsequently. However, these factors seem to
We can readily postulate a probable indicate that the malaria parasites in apes and
aperiodic hepatocystis-like parasite in a man in this area were introduced relatively
cercopithecoid ancestor of the Old World recently rather than the end product of a long
monkeys. After an ecological separation of evolutionary process involving both the host
Asian and African monkey groups, the parasite animals and their parasites.
in the Asian monkeys began to undergo a series Problems are also encountered in the
of changes which gradually produced malaria Western Hemisphere when the primates and
parasites with biological and morphological their malaria parasites are considered. The
characteristics with which we are familiar today. evolutionary line of the New World primates
It is probable that a quartan branch from this was isolated from the Old World line quite early.
main line gave rise to contemporary tertian and There are no hepatocystis-like parasites in New
quartan species in various primates as well as World monkeys. The success of the primates in
the successful but geographically limited the Western Hemisphere is well demonstrated
quotidian experiment, P. knowlesi. Meanwhile, by the large variety of genera and species of
Hepatocystis resulted from a more conservative these animals found in Central and South
and less innovative series of changes from the America. The Haemosporidia have not been as
primitive tissue coccidian than was involved in prolific; there are only two species described
the evolution of the plasmodia. The stimuli from the many potential host animals.
responsible for the development of the malaria Plasmodium simium seems to be limited in both
parasites were either not associated with African host and geographic distribution being found
primates or, malaria-like parasites developed but only in howler (Alouatta sp.) and woolly-spider
were not initially successful. Thus, in Asia a (Brachyteles arachnoides) monkeys in the
reasonable hypothesis concerning the forests of southern Brazil (Deane et al, 1969).
simultaneous development of primates and their Plasmodium brasilianum has a much wider
malaria parasites can be proposed. However, range, but there are large areas, with suitable
since there are both primates and primate host animals, where it is not found, and, where
malarias in Africa and in the Western natural infections in the same genera of
Hemisphere, any hypothesis concerning the monkeys, vary considerably even over relatively
origins must encompass these forms, too. short parts of its range. These factors would
In Africa, the human and ape malaria seem to indicate that these host-parasite
parasites are remarkably similar and at least two associations are much more recent than the 35
species, P. malariae and P. schwetzi, may be million years since the apparent isolation of the
shared. This situation fits well with the primitive ancestral primates of the New World
hypothesis that the hominid line was a by continental drift. Finally, the two species of
5 EVOLUTION OF THE PRIMATE MALARIAS

plasmodia from this area are virtual duplicates of numerous occasions, his malaria parasites into
the human P. vivax and human and chimpanzee the apes and monkeys and his fellow hominids
P. malariae, a phenomenon that severely of West and Central Africa. It was in this
challenges even the most extravagant views of African environment that the biological
convergent evolution. It is almost as though experimentation on the part of the parasites
these parasites arose full grown like the which eventually produced Plasmodium
mythological appearance of Athena from Zeus' schwetzi, P. ovale, and the P. falciparum-P.
head. reichenowi complex took place. It seems
The evolution of the Haemosporidia has probable that the ancestors of P. malariae and
been reviewed by Baker (1965), Bruce-Chwatt possibly even P. vivax came out of Southeast
(1965), and Garnham (1966) where efforts were Asia pretty much as we know them now. The
made to correlate the evolution of the primates older quartan parasite found an environment in
and their malaria parasites. Some of the which it could readily develop and thus the
problems associated with such an effort have quartan malaria species of man and the African
been pointed out in the preceding paragraphs. apes are considered generally to be conspecific.
We believe that the most uncomplicated and, The situation was less sympathetic for the
therefore, possibly, the most likely explanation tertian parasites. The indigenous negroid
for the peculiar features of host and geographic hominids in Africa were even then probably not
distribution, as well as species proliferation of very receptive to this latter group and
the primate malarias, has not been given a full adaptations were necessary. From the vivax-like
hearing. Our suggestion is, that the nidus of the stem developed a morphologically similar
primate plasmodia universe lies somewhere in species, P. ovale, that was capable of surviving
the jungles of Southeast or South Central Asia in the indigenous hominds. At the same time, a
and that there, there has been a simultaneous similar type of adaptation was taking place in
development of non-human primates and their the anthropoid cousins of the hominids in the
malaria parasites. Possibly during the early area with the development of an additional
Pleistocene, some one to two million years ago, species from the same stem, P. schwetzi.
there appeared the first of many incursions into Coatney (1967) proposed that P. schwetzi was in
this reasonably stable millieu by a new and more all likelihood a chimpanzee equivalent of P.
highly evolved primate, from the west and north, ovale. Such a relationship would not be
in the form of an ancient hominid ancestor. This surprising in the light of the hypothesis being
invader, the product of a long evolutionary presented here. However, it would be difficult, if
sequence in Central Africa, was already not impossible, to say whether P. ovale and P.
demonstrating that peripatetic feature so schwetzi arose simultaneously from the
characteristic of his descendants. The invader introduced P. vivax stem, or, whether the
was still a hunter and forest dweller and, development was sequential in man and then the
therefore, shared the forest environment, chimpanzees, or, the reverse. Probably the most
including anopheline mosquitoes, with the more interesting and most recent manifestation of
primitive native primates of the area and their attempts by the tertian parasite stem to adapt to
malaria parasites; the latter, probably already African primates is seen in the development of
possessed characteristics of morphology and the P. falciparum-P. reichenowi complex. Three
periodicity similar to those we recognize today. features of P. falciparum seem to set it apart.
The situation was thus ideal for the introduction First, the presence of crescent-shaped
of the parasites into this new primate. Such a gametocytes; second, the tendency toward deep
potential still exists, when man and non-human circulation schizogony; and third, the apparent
primates intimately share the same jungle absence of a mechanism, either relapse or long
environment. term recrudescence, by which the parasite can
The peripatetic nature of the restless more safely assure its survival. It is interesting
primate increased and over the next 500,000 to that each of these characteristics can be seen to a
one million years introduced, probably on greater or lesser extent in parasites of
 PRIMATE MALARIAS 6

non-human primates in Asia. The potential of the monkeys, in certain areas, of Central and
the Haemosporidia for producing South America. The older and, apparently, the
crescent-shaped gametocytes has been well more stable quartan P. malariae has become
demonstrated by the bird parasites. In the well adapted as P. brasilianum to a variety of
primates, P. coatneyi and P. fragile are most monkeys in many parts of Central and South
closely related to P. falciparum-P. reichenowi. America. The tertian parasites have adapted less
In the latter parasites, there is a definite tendency readily in the New World monkeys with only
toward the production of oval gametocytes, limited success as P. simium in the howler and
especially in the younger stages (Fig. 27, Plate wooly-spider monkeys of Brazil. The P.
XLII). Deep circulation schizogony is present in falciparum line has not, in so far as is presently
P. coatneyi and P. fragile of Asian monkeys. known, found a suitable host among the
The possession of a true relapse mechanism nonhuman primates of the Western Hemisphere.
appears to be limited among the primate The hypothesis that primate malarias arrived in
malarias. This phenomenon has been seen in P. the New World with Europeans and their West
vivax, P. cynomolgi, P. ovale, P. fieldi, and P. African slaves in the 16th century is not new.
simiovale, all of which are tertian parasites in Recent authors who have defended this point of
stippled cells. Relapse is not known to occur in view have included Boyd (1949), Jarcho (1964),
P. fragile or in P. coatneyi, and is apparently and Dunn (1965). However, there is a
absent in all the quartan species, including P. considerable body of literature that supports the
malariae. Thus P. falciparum and P. reichenowi existence of human and other primate malarias
are not far removed from the main stem of in pre-Columbian America. Bruce-Chwatt
evolution in the primate malarias when we (1965) has presented an extremely interesting
consider that there are other similar and well written article on this subject in which
developments from the tertian ancestor among he summarizes the three kinds of evidence
Asian non-human primates. which are used to support the extreme antiquity
The dynamics of this relationship between of malaria in America; i.e., linguistic, botanical,
the malaria parasites and the primates probably and historical.
remained fluid for thousands of years. As the The linguistic evidence seems to be
migratory habits of the hominid increased he primarily oriented around the appearance in the
carried his parasites to all areas of Asia, Africa, Indian dialects, from both Mexico and Peru, of
and Europe and, if anopheline mosquitoes were words which were roughly translated to mean
present, a focus of infection was established. chills and fever. The earliest translations were
Eventually, in the 16th century, man introduced made by Spanish soldiers and priests who were
his malaria parasites into the last remaining familiar with chills and fever (probably from
fertile area for their development. With the malaria) and, therefore, their interpretation of
arrival of the southern European conquerors and Indian words referring to general classes of
their West African slaves into the Carribean illness may not have been without bias. In
area, the last large malaria free primate addition, "chills and fever" is not a syndrome
population, in an appropriate environment, had unique to malaria.
been drawn into a process that had begun The botanical evidence is probably even
millions of years previously in the monkeys of more fraught with uncertainty than the linguistic
Southeast and/or South Central Asia. This latest evidence. The romanticism that has developed
experiment is, of course, still underway in the around the discovery of the effectiveness of an
jungles of Central and South America. At the extract of cinchona bark against malaria has left
present time, the parasites have had unqualified us with a number of intriguing stories but little
success in human populations of the area, both concrete evidence (see Haggis, 1941).
indigenous and recently introduced. The Considerable importance is given to the belief
situation in the non-human primates has been that the use of cinchona was widespread among
less successful but at least two of the human the New World Indians for the treatment of
forms seem to have established themselves in fevers. Since cinchona extract is known to be a
7 EVOLUTION OF THE PRIMATE MALARIAS

febrifuge there is no reason to assume that the is little, if any, support.) The advocators for the
fever being treated was malaria. Undoubtedly existence of malaria in pre-Columbian America
there were many febrile agents in the area. usually postulate that the parasites were
Finally, there is a growing body of evidence as introduced with man. Most anthropologists
reviewed by Jarcho (1964) that cinchona was agree that man arrived in America via a land
not widely used in Indian medicine. bridge from northeastern Asia between 15,000
The historical evidence is, in our opinion, and 25,000 (possibly as much as 40,000) years
not only controversial as Bruce-Chwatt points ago. More recent movements of small numbers
out but, also, unconvincing. There is no doubt of people from islands in the Central and Eastern
that the early Spanish explorers in the Caribbean Pacific to South America would also seem
and along the Atlantic coasts of Central and possible since the epic voyage of the Kon-Tiki.
Northern South America suffered terribly from Finally, there is now no doubt that the Vikings
outbreaks of disease. However, the evidence for reached the shores of North America 500 years
these early problems being due to malaria is before Columbus. Thus, there were at least three
vague in the extreme. The earliest slaves opportunities for man to have introduced malaria
probably arrived in Cuba in the first decade of into the Western Hemisphere prior to the advent
the 16th century. They undoubtedly brought of the Conquistadores, with their malarious
malaria with them, but the Europeans slaves, into the tropical area of the Caribbean.
themselves were also subject to malaria and What are the chances that any or all of the
undoubtedly assisted in establishing the migrants were infected with malaria when they
parasites in the New World. Bruce-Chwatt arrived? The land bridge from Asia connected
(1965) notes that since the number of slaves was two areas which have been, during historical
initially small, it is doubtful if large scale times at least, incompatible with the
epidemics could have started with such a small transmission of malaria, and the evidence
source of parasites. There are two factors to be available indicates that the climate at the time of
emphasized here; first, the evidence that the the migrations was at least as severe as today
early disease outbreaks in the Spanish colonies (Hopkins, 1959). Additionally, there is no
were due to malaria is, to say the least, tenuous. evidence that there was any malaria-like illness
Second, in the settlement areas, the Indians, as among North American Indians prior to
non-immunes, constituted an immediately historical times.
available and readily infectable population. Assuming that Homo sapiens did cross the
There is no doubt that human malaria became Pacific to reach the shores of South America, it
well established early in the Spanish conquest of is probable that such a movement was made in
Central and South America. Actually, this is not stages using the islands of the Central and
surprising considering the hardiness of the Eastern part of this area. These islands have
parasite and the fact that it was introduced into been, and are now, free of anophelines and
an area with a surfeit of both vertebrate and hence malaria in historical times, a biological
invertebrate hosts and an environment for condition that did not occur suddenly and
transmission that has proved to be most therefore probably reaches back some distance
efficient. into prehistory.
Possibly the most convincing evidence for Finally, during the time the Vikings were
the post-Columbian introduction of malaria into journeying to Iceland, Greenland, and North
the New World is the difficulty in finding any America, their home area was free of malaria.
other means by which it could have arrived. (We Therefore, it would seem difficult, if not
assume that there is no question that primate impossible, for malaria to have accompanied any
malarias were introduced into the New World. of the pre-Columbian human invasions into the
The alternative would be that these parasites Western Hemisphere.
evolved in the Western Hemisphere and became There are, undoubtedly, many flaws in the
established only recently in the Old World hypothesis we have presented. However, it does
primates; a proposal for which we believed there account for most of the known aspects of the
 PRIMATE MALARIAS 8

biology of the primate malarias especially with zoonotic potential in human malaria. At the
reference to morphology, host, and geographic present time, the potential role of the simian and
distribution. The intimate relationships between anthropoid malarias in human infections is still
the plasmodia of man and non-human primates primarily a matter of speculation.
is possibly as recent as one million years, and The genus Plasmodium has been very
there were probably fairly consistent successful in monkeys, apes, and man in Asia
interchanges throughout the long history of since it infects naturally every genus of
man's development. Obviously, such an contemporary higher primate in the area. All of
exchange was related not only to the biology of the primate malarias have their origin in Asia
the parasites in question but to the specific except P. falciparum and P. ovale which,
ecological niches which were occupied by man according to the hypothesis presented here,
and other primates. As man evolved from a probably originated in Africa. The tertian
hunter and forest dweller into a planter and species are found in a variety of host animals
builder of houses, he became more and more which may be a reflection of the much less
separated from the vectors of non-human specific vector requirements in these parasites
primate plasmodia and the biology of the now than in the quartan species, thereby providing
more or less separate groups of parasites began more opportunities for their introduction into
to diverge. The trend toward the separation of new hosts. Plasmodium knowlesi is the only
the malaria parasites of man and non-human quotidian malaria of primates. It has particular
primates has probably been progressing for vector relationships which, combined with the
thousands of years. However, the potential for lethal impact on monkeys other than the natural
exchange remains. This was demonstrated by the host, have served to keep it confined to the
arrival of malarious Europeans and Africans in jungles of Malaysia, the Philippines, and,
the New World early in the 16th Century and, possibly, some islands in Indonesia.
through intimate sharing of anophelines with In Africa, there has been a great reduction
native primates, malaria was introduced to them. over that seen in Asia not only in the number of
Such exchanges still occur, as shown by two species present but in the host and geographic
recent reports of natural infections in man with range in which they have been successful. It is
malaria parasites of monkeys. Chin, et al (1965) proposed that all of these species developed
documented a human infection with P. knowlesi from malariae-vivax stock introduced from Asia
from West Malaysia and in the same year Deane by a hominid ancestor. In Africa, the tertian
et al reported a human infection with the parasites have had considerable success, while
vivax-like P. simium which was believed to have P. malariae is the only quartan species found
been contracted in a forest area outside São and the quotidian stem was too fragile to survive
Paulo. In each of these situations, man had in this new and apparently hostile environment.
introduced himself into the forest environment In American primates, the trend toward a
where transmission of malaria among the reduction in species is more pronounced. The
nonhuman primates was constant. success of the human forms in indigenous
When such exchanges of parasites occur primates in the New World is limited to only
today and man is on the receiving end, we have two species. It may be, that in the near future, P.
a zoonosis. The situation with regard to the brasilianum and P. simium will be made
monkey parasites of the New World can be conspecific with P. malariae and P. vivax,
described as an anthroponosis or a reverse respectively. Such a situation presents the
zoonosis if the point of view is particularly problem of how extensive a period of isolation
anthropocentric. Thus, the study of the in a new host animal, associated with what level
evolutionary history of the primate malarias of physiologic and/or morphologic changes, is
becomes something more than an academic required for a new species of parasite to be
exercise, since such information may provide an recognized. We support the position that the
insight into future trends in the relationships human P. vivax and P. malariae are the
among these parasites and help to assess the immediate and recent ancestors of P. simium and
9 EVOLUTION OF THE PRIMATE MALARIAS

P. brasilianum, but believe that these latter isolation of Madagascar from the mainstreams of
parasites are nevertheless valid species, at least either Asian or African animal evolution.
for the present, in spite of the close morphologic Plasmodium girardi and P. lemuris are poorly
similarities between the monkey and human understood parasites which may be examples of
forms. Therefore, it would seem that the only convergent evolution arising from the same
really startling innovation in the development of hepatocytis-like ancestor as the other plasmodia
the primate plasmodia outside of Asia has been of which P. foleyi (also from the lemur and
the rise of the falciparum-like parasites of considered by Garnham (1966) to be more
Africa. The remainder of the species in both probably Hepatocystis than Plasmodium) is the
Africa and the New World are closely related to more direct, contemporary descendant.
the stock Asian forms. Undoubtedly, there are alternative
The evaluation of the role of simian and explanations for the evolutionary processes
anthropoid malaria in human disease would not which gave rise to the current distribution of the
be complete without a word on possible new primates and their malaria parasites. The
simian malarias. Recent successes in cultivating hypothesis proposed here seems logical in the
human malaria parasites in owl monkeys from light of our present knowledge, suggesting that
South America and splenectomized gibbons the origin of at least the primate malarias was in
from Asia have served to remind us again that Asia and that man in his wanderings has been
Homo sapiens was a possible source of the responsible for the introduction of these
simian and anthropoid parasites of Africa and parasites into African and New World primates.
the New World and, that the evolutionary For thousands of years man and non-human
processes that brought about this development primates occupied the same environment with
are dynamic and contemporary. New species of what was probably a fairly free interchange of
simian malaria may still be adapting in the malaria parasites. However, man's development
jungles of Africa or South America. as an agricultural animal tended to separate him
The current status of our knowledge makes ecologically from his simian and anthropoid
it difficult, if not impossible, to relate the relatives and at the same time from their malarial
evolution of primate malarias to the earlier parasites. This trend has continued until it is
offshoots of the same ancestral line in reptiles, obvious that today the relationships between the
birds, rodents, bats, and a few ungulates. Rodent human and non-human primate malarias are very
and bat plasmodia are found only in Africa. Two tenuous and, in the overall aspects of human
of the three ungulate species are African and one health, of limited importance. However, it is
is Asian. No non-primate mammalian malarias probable that occasional cases of human malaria
are found in the New World. The most puzzling of simian origin will be detected in deep jungle
aspect of the primate plasmodia is the presence areas of Asia, Africa, and South America once
of two species in lemurs on the island of malaria eradication has advanced to a point
Madagascar. These are malaria parasites in a where such cases can be individually evaluated.
true but primitive African primate. Probably this
enigma reflects the long period of biological
REFERENCES
BAKER, J. R., 1965. The evolution of parasitic protozoa (pages BRUCE-CHWATT, L. J., 1965. Paleogenesis and
1-27). In: Third Symposium of the British Society for paleoepidemiology of primate malaria. Bull. Wld. Hlth.
Parasitology, Blackwell Scientific Publications, Oxford. Org. 32 : 363-387.
BALL, G. H., 1943. Parasitism and evolution. Am. Nat. 77 : CHIN, W., CONTACOS, P. G., COATNEY, G. R., and
345-364. KIMBALL, H. R., 1965. Naturally acquired quotidian
BALL, G. H., 1960. Some considerations regarding the sporozoa. type malaria in man transferable to monkeys. Science
J. Protozool. 7 : 1-6. 149 : 865.
BOYD, M. F., 1949. Malariology, 2 Vol. W. B. Saunders, CHRISTOPHERS, R., 1934. Malaria from a zoological point of
Philadelphia, Pa. view. Proc. Royal Soc. Med. 27 : 99 1 -1000.
BRAY, R. S., 1957. Studies on the exo-crythrocytic cycle in the COATNEY, G. R., 1967. Simian malarias in man: facts,
genus Plasmodium. London School of Hyg. & Trop. implications, and predictions. Am. J. Trop. Med. &
Med. Memoir No 12. pp. 192. Hyg. 17 :147-155.
BRAY, R. S., 1963. The exo-erythrocytic phase of malaria DEANE, L. M., DEANE, M. P., and NETO, J. F., 1965. Studies on
parasites. Int. Rev. Trop. Med. 2 : 41-75. transmission of simian malaria and on a natural
 PRIMATE MALARIAS 10

REFERENCES—Continued
infection of man with Plasmodium simium in Brazil. GARNHAM, P. C. C., 1966. Malaria parasites and other
Bull. Wld. Hlth. Org. 35 : 805-808. haemosporidia. Blackwell Scientific Publications,
DEANE, L. M., FERREIRA NETO, J. A., OKUMURA M., and Oxford. pp. 1114.
FERREIRA, M. 0., 1969. Malaria parasites of Brazilian HAGGIS, A. W., 1941. Fundamental errors in the early history of
monkeys. Rev. Inst. Med. Trop. São Paulo 11 : 71-86. cinchona. Bull. Hist. Med. 10 : 417-459; 568-592.
DUNN, F. L., 1965. On the antiquity of malaria in the western HOPKINS, M., 1959. Cenzoic history of the Bering land bridge.
hemisphere. Human Biology 37 : 385-393. Science 129 : 1519-1528.
DUNN, F. L., 1966. Patterns of parasitism in primates: HUFF, C. G., 1938. Studies on the evolution of some disease
phylogenetic and ecological interpretations with producing organisms. Quart. Rev. Biol. 13 : 196-206.
particular reference to the Hominoidea. Folia Primatol. HUFF, C. G., 1945. A consideration of the problems of evolution
4 : 329-345. of malarial parasites. Rev. Inst. Salub. enferm. trop.,
FANTHAM, H. B., 1936. The evolution of parasitism among the Mex. 6 : 253-258.
protozoa. Scientia. Milano 49 : 316-324. JARCHO, S., 1964. Some observations on disease in prehistoric
GARNHAM, P. C. C., 1955. The comparative pathogenicity of North America. Bull. Hist. Med. 38 : 1-18.
protozoa in their vertebrate and invertebrate hosts. MANWELL, R. D., 1955. Some evolutionary possibilities in the
Symposium No. 5 of Soc. Gen. Microbiol.: 191-206. history of malaria parasites. Ind. J. Malariol. 9 :
GARNHAM, P. C. C., 1963. Distribution of simian malaria 247-253.
parasites in various hosts. J. Parasit. 49 : 905-911. MAYR, E., 1963. Animal species in evolution. Harvard University
Press, Cambridge, Mass.
 2
Historical Review
infected people. For approximately the next
THE malaria parasite is a 1500 years, man did little about malaria but
well organized, highly adapted end-product of suffer and die from it. Then, in the middle of the
thousands of years of biological evolution which 17th century, the bitter extract from the bark of a
would mark it as successful, regardless of the New World tree was found to have remarkable
criteria. Species of the genus Plasmodium are powers against these intermittent fevers so
currently found in every group of strictly common in the Mediterranean area. The
terrestrial vertebrates and adaptive capacities romance surrounding the discovery of quinine
have been particularly well demonstrated in has served to obscure the real story, and today,
birds and primates where the level of speciation there are a number of versions from which to
is high. When one considers the complexity of choose. It now seems doubtful that the Countess
the life cycle, including the necessity for of Chinchon was ever treated for malaria with
adjustment to two decidedly different host Peruvian bark, but her name was used as the
environments, the precise combination of genus name for the Cinchona tree, by way of a
temperature and humidity required, plus vector spelling error, by Linnaeus. Cinchona bark first
and vertebrate host habits necessary to assure appeared in European medical literature in
continued transmission of the parasite, its Heyden's Discours et Aris sur les flus de ventre
survival in nature alone, is amazing. Recent Doloureux published in Antwerp in 1643. The
history has confirmed the hardiness of these efficacy of the extract was known by that time
parasites. Man's concentrated attacks on the and it is probable that it had been used in Lima
parasites and their vectors have met with only and possibly Spain, earlier. Its value was soon
limited success and a great deal of frustration. recognized and thus began a long period of
Even areas freed of human malaria remain in a intrigue and frustration associated with efforts to
precarious position because the parasites keep secure sufficient quantities of the extract to treat
up an unrelenting pressure on their borders, the increasing number of malaria infections in
maintaining a constant threat of reinvasion with the world.
the production of epidemics such as occurred in The 18th century saw three advances which
Ceylon in 1968. can, in the light of our present knowledge, be
The chronicle of human malaria begins in recognized as important. Lancisi noted the
pre-history and continues for thousands of years presence of black pigment in human brains and
through a tortuous path of sickness and death. spleens, but did not associate these changes with
The full antiquity of the man-malaria parasite malaria, and in 1717 suggested a relationship
association remains obscure, but Hippocrates, between marsh insects and the occurrence of
"The Father of Medicine" and the first malaria. In 1775, Torti recognized that quinine
malariologist, described the various malaria was not therapeutic for all fevers, thus
fevers of man 400 years before the birth of effectively separating the malarias from human
Christ. Progress in understanding the disease febrile illnesses of other etiologies. It can be
was slow, but about 30 A.D., Celsus described seen that the status of knowledge of malaria at
two types of tertian fevers; 150 years later, the beginning of the 19th century was at a very
Galen recognized the appearance of these fevers low level. Meanwhile, the problem was
with the summer season and a jaundice in becoming more acute due to European

11
 PRIMATE MALARIAS 12

explorations and settlements in the tropics of Baltimore, in P. falciparum in man. In 1891,

Asia. In addition, the malarias introduced into Romanowsky developed a stain which allowed
the New World in the 16th and 17th centuries for differential identification of blood parasites,
had found fertile ground, and, by the early including malaria.
1800's, severely challenged man's ability to The animal nature of the malaria parasite
survive in parts of the American tropics and had fairly wide acceptance by the last decade of
subtropics. the 19th century. It is during this period that
The 19th century proved to be a some of the most dramatic and far-reaching
momentous one in the field of malaria. Initially, work in the field of malaria was to take place.
information continued to be fragmented and The possible association of insects and the
uncoordinated. In 1820, two French chemists, transmission of malaria had been discussed in
Pelletier and Caventou, isolated the active various parts of the world for a long time. As
anti-malaria component from quinine powder. early as 1848, Nott published a paper in the
Boyle (1831) while working in Africa gave U.S.A. which contrasted yellow fever and
credence to the swamp theory of malarial remittent fever and proposed that mosquito
etiology. The probable relationship between involvement was the best explanation for the
malaria and swamps was so firmly established occurrence and distribution of both kinds of
that it had given the two most frequently used fever. In 1856, Burton noted that some African
names to the disease mal’aria, later shortened to tribes believed that mosquitoes were responsible
one word malaria, and paludisme. The black for certain fevers. The development of filarial
pigment, first noted by Lancisi, was specifically larvae in mosquitoes was demonstrated by
associated with malaria by Schutz (1848) when Manson in 1883, and in 1893, Smith and
he observed it in the internal organs of patients Kilborne reported the transmission of Texas
who had died of malaria. Meckel in 1847, cattle fever by ticks.
without recognizing its true importance, Information on the probable association of
probably saw malaria parasites for the first time insects and certain disease syndromes had no
when he described black pigment in doubt begun to accumulate on both sides of the
protoplasmic masses in the blood of a patient Atlantic before Ronald Ross's epoch making
with fever; it was not until 1879, however, that work on mosquitoes and malaria in India;
Afanasiev proposed that these bodies might be communications were poor, however, and there
the agents of the disease. was little opportunity to correlate the various
The scene was now set for the dramatic observations arising from such divergent points
events which crowded into the last 20 years of as Russia (Danilewsky) and the U.S.A. (Nott).
the 19th century. In 1880, Laveran, working in Patrick Manson, who encouraged, stimulated,
North Africa, described exflagellation of the and advised Ross, was committed to the idea
Plasmodium falciparum male gametocyte in the that insects were involved in disease
blood of a malarious patient. The reception of transmission.
Laveran's discovery was less than enthusiastic. Ross's early results, reported by Manson in
Most of the scientific world was convinced of 1896, included the observation of exflagellation
the bacterial etiology of malaria. Meanwhile, by P. falciparum gametocytes in the stomach of
according to Garnham (1966), Danilewsky a mosquito. Manson (loc. cit.) therefore
(1884) was able to observe parasites of malaria concluded that the mosquito's stomach provided
in the blood of wild birds, and, in the same year, an appropriate medium for the development of
Laveran's influential countryman, Pasteur, the malaria parasite outside the vertebrate host.
became convinced of the soundness of the In the same year, according to Russell (1955),
former's observations. The true nature of the Theobald Smith came to the conclusion that
organisms seen by Laveran was finally made mosquitoes were vectors of malaria. The
clear by MacCallum in 1897 who, while in following year, Ross reported the presence of
Canada, observed fertilization in bird malaria pigmented bodies in spotted winged mosquitoes
and later, at the Johns Hopkins Hospital in after the insects had fed on patients with malaria.
13 HISTORICAL REVIEW

At that point, Ross was forced to interrupt his malariae were made by Grassi and Feletti in
investigations on human malaria, but was soon 1890. However, the situation was not as
(1898) able to observe the sporogonic cycle of a clear-cut as would be indicated by this short
bird malaria (P. relictum) in the mosquito, and, statement. Chaos was the order of the day in the
to transmit the parasite to healthy sparrows. taxonomy of the malaria parasites for many
It is at this time that the famous, years. Numerous generic names were proposed,
acrimonious and long-lived conflict began including Haemamoeba, Oscillaria, Laverania,
between the British and Italian malariologists and Haemomonas. Confusion continued well
over priority in the transmission of malaria by into the 20th century over whether all of the
mosquitoes. Bignami, in 1898, successfully parasites belonged to one species or to several.
infected a volunteer with P. falciparum by the In the confusion, it is difficult to know exactly
bites of mosquitoes collected from a malarious which parasite was under observation by some
area. There is no serious challenge to the of the early authors. There is little doubt that
validity of either Ross's or Bignami's work. The Laveran saw and described parasites of what
acrimony arose over who was the first to was to become P. malariae in 1881, and that he
demonstrate the role of the mosquito in malaria. first used the species name "malariae." It is also
It would seem that Bignami's was the first report clear that Laveran first saw the gametocytes of
but both Ross and Manson insist that the ideas P. falciparum, but he firmly believed that all of
for the Italian investigations came from the parasites belonged to one species. In 1890,
unpublished reports of Ross's work. Grassi and Feletti described and illustrated two
Confirmation of the mosquito role in the parasites that were to become P. vivax and P.
transmission of malaria came in 1900 when malariae. Seven years later, Welch (1897)
Manson arranged for three people from the proposed the name Haematozoon falciparum for
London School of Tropical Medicine to spend the parasite with the crescent-shaped
the summer near Ostia in the Roman Campagna. gametocytes which Laveran had seen some 17
Their days were spent in various excursions in years earlier. The end result of this taxonomic
the vicinity, but each night was passed in a chaos is now well known. The genus name
screened hut. The three did not come down with Plasmodium of Marchiafava and Celli was
the disease, although transmission of malaria maintained for all species. The species name of
continued at its usual high rate in the the parasite described by Grassi and Feletti as
surrounding area. Final proof came when Haemamoeba vivax was eventually accepted to
mosquitoes previously fed on a malaria patient designate the benign tertian parasite of man,
in Rome were allowed to bite healthy volunteers Plasmodium vivax. Welch's specific name
in London. The London participants in these gained wide acceptance for the malignant tertian
experiments, including Manson's son, came parasite and Plasmodium falciparum became a
down with typical vivax malaria two weeks after permanent part of the malaria literature. With P.
exposure. The complete cycle of P. falciparum malariae, the situation is even more confused. In
was observed by Grassi, Bignami, and 1890, Grassi and Feletti gave malariae as the
Bastianelli in 1899 and in the same year, specific name for the quartan parasite and on the
Bastianelli and Bignami accomplished the same basis of priority, that name and date is valid.
feat with P. vivax. The Italian studies on the The 20th century part of the malaria story
sporogonic cycle of malaria were summarized in began in what might be termed organized
what was to become a classical monograph by confusion. The last two decades of the 19th
Grassi in 1900. century had produced more information than
Meanwhile, the same group of Italian could readily be assimilated, but a firm basis for
workers had been busy with other facets of the the epidemiologic investigations, which were to
rapidly developing fund of knowledge about dominate work in the early part of the 20th
malaria. Credit for the genus name Plasmodium century, had been established. Three different
goes to Marchiafava and Celli (1885) and the human parasites were recognized (though not
initial differential descriptions of P. vivax and P. universally accepted) both clinically and
 PRIMATE MALARIAS 14

morphologically, and the role of the anopheline difficult to overestimate the contribution that
mosquito in the transmission of the parasite had thousands of patients, in many such institutions,
been established. have made to our understanding of malaria.
The stage was now set for the development There is no doubt that the evolution of
of new concepts of control based on biological concepts of control and eradication have been
aspects of mosquito transmission and the the predominant feature story of malaria during
erection of some type of barrier between man the 20th century. However, significant strides
and the insect vector of the disease. In 1900, have been made in its biology, parasitology, and
Gorgas began his momentous work on the therapy as well. One of the most important new
control of malaria and yellow fever in Havana developments was the discovery of the
and following his success there, he transferred exoerythrocytic stages of the primate malarias.
his activities, in 1907, to the Panama Canal Zone This story is reviewed in detail in Chapter 6.
where he achieved equal, if not greater, results Actually, Grassi suggested as early as 1900 the
in reducing the incidence of two deadly possibility that the sporozoite did not develop
diseases-malaria and yellow fever. There is little directly into blood parasites. However, it was
doubt that Gorgas, LePrince, and Carter must, in not until 1948 that Shortt and Garnham finally
a large measure, be given credit for the ultimate demonstrated the pre-erythrocytic stages of a
completion of the Panama Canal. During the primate malaria.
same period, Watson began his classical work of No discussion of the history of man's
draining the salt marshes, which made parts of conflict with malaria would be complete without
the west coast of Malaya habitable. During the consideration of the plasmodia of other animals.
next two decades evolved the well known The genus is extremely widespread in reptiles,
concepts of ditching and draining which birds, and mammals, and, in the light of this
achieved such notable results in malaria control monograph, a brief review of the development
in the southern United States and in Italy. In of our knowledge of the malarias of non-human
1939, the Malaria Service of Northeast Brazil primates is in order. The evolution of an
was organized to combat the populations of understanding of the plasmodia of non-human
Anopheles gambiae which had been introduced primates parallels, in many ways, and
from Africa. This effort at species eradication complements, the story of human malaria. The
was enormously successful and the mosquito is first plasmodium-like parasite (P. kochi)
still absent from the area. The control of malaria described from non-human primates was not a
took a giant step forward in the late 1930's and true malaria but a member of the genus
early 1940's with the development of residual Hepatocystis which was subsequently reported
insecticides and synthetic antimalarials. from a number of species of African monkeys
However, the history of these developments is (Kossel, 1899; Laveran, 1899). Most of the early
outside the scope of this work. observations on the blood protozoa of
The capacity of a malaria fever to non-human primates were made on animals
frequently improve the condition of people imported into Europe from Africa, Asia, and
suffering from general paralysis of the insane by America. Laveran (1905) saw the parasite, later
Wagner-Jauregg (1922) may be one of the most described by Halberstaedter and Prowazek
important discoveries in the history of malaria in (1907) as Plasmodium pitheci, in the blood of
that it allowed for the systematic study of the Javan orangutans housed in Berlin. In the same
disease under controlled conditions. It was in year, Halberstaedter and Prowazek examined
such mental hospitals as those in Milledgeville, blood of Macaca irus and M. nemestrina
Georgia and Columbia, South Carolina in the imported from Indonesia and Borneo and found
U.S.A.; Horton Hospital in England; and in another malaria parasite which they described as
Bucharest and Socola, Roumania that studies on P. inui. In the same year, while Halberstaedter
induced malarias revealed much of our and Prowazek were working in Berlin, Mayer,
information on pathology, biology, drug working with Asian monkeys at the Hamburg
responses, and relapse mechanisms. It would be Institute, described a parasite from the blood of a
15 HISTORICAL REVIEW

Macaca irus, from Java, which he named P. The malarias of apes created a major
cynomolgi. The following year, a cacajao problem in taxonomy. Much of the confusion
(Brachyurus calvus) from Brazil was arose because, for some time, their parasites
encountered in a circus in Hamburg and Gonder were considered to be the same as their three
and von Berenberg-Gossler found a parasite in morphological counterparts in man. Plasmodium
its blood which they named P. brasilianum. In reichenowi was given specific rank in 1922 by
1932 and 1933, Sinton and Mulligan reviewed Sluiter, Swellengrebel, and Ihle. In 1939,
the confused situation with regard to the malaria Brumpt gave the P. vivax and P. malariae
parasites of monkeys, and, in a well reasoned counterparts the specific names of P. schwetzi
two part paper, managed to bring some order out and P. rodhaini, respectively. Previous efforts
of the chaos. The African parasites fell into two had failed to establish these parasites in man, but
obvious categories-one without schizogony in Rodhain (1940) working with P. malariae
the blood which remained as Plasmodium (later transferred it, by blood inoculation, to the
to become Hepatocystis) kochi. The second chimpanzee, and, in 1948, by the same route,
parasite did have multiplication in the peripheral transferred P. rodhaini from the chimpanzee to
blood and the authors considered it to be more man. As a result of this work, some investigators
closely related to the P. inui of Asia; they have synonymized P. rodhaini with P. malariae.
therefore established P. inui var. gonderi, The It is of interest in this connection that, as early as
Asian parasites also received careful attention. A 1920, Mesnil and Roubaud had transferred P.
parasite described but not named by Knowles vivax to the chimpanzee by blood inoculation
and Das Gupta (1932) from a M. irus but had not attached any significance to it.
(=fascicularis) from Singapore was considered Recently, Contacos et al (1970) infected human
by Sinton and Mulligan (loc. cit.) and was given volunteers with P. schwetzi, via mosquito bites,
specific rank as P. knowlesi, on the basis of and pointed out the close similarity between the
morphological characters and on the presence of blood forms of P. schwetzi and of P. ovale.
a 24- rather than a 48-hour schizogonic cycle. Coatney (1968) suggested that P. schwetzi might
The parasite of Mayer, P. cynomolgi, was actually be P. ovale. The true relationship of
believed to be a separate species also, but the these ape- and man-forms is still to be
investigators (Sinton and Mulligan, loc. cit.) determined.
considered the information available at that time The taxonomic status of the malaria
to be too meager; they therefore established P. parasites of Asian apes is much less confused.
inui var. cynomolgi. Only one species, P. pitheci, has been found in
So well had Sinton and Mulligan done their orangutans. The situation with the gibbons is
work that the only change made later in their interesting in that taxonomic difficulties do not
taxonomic arrangements was elevation of the arise with the four distinct species known from
two "varieties" of P. inui to specific rank. these animals, but the systematic position of the
Rodhain and van den Berghe (1936) found the apes themselves is so confused that it is
African parasite to be tertian in periodicity rather sometimes difficult to clearly establish the type
than quartan, as in P. inui, and gave P. gonderi host for a given parasite.
specific rank. Mulligan (1935) established P. The parallel histories of the malarias of
cynomolgi as a species separate from P. inui on human and non-human primates first became
the basis of morphology and its tertian mixed when it was believed that the malaria
periodicity. parasites of the great apes of Africa were
The work of Sinton and Mulligan (loc. cit.) identical with those of man. Then in 1932
became the base on which the future taxonomy Knowles and Das Gupta reported the infection
of the monkey malarias was to be developed. of a human volunteer, by the inoculation of
Workers in Malaya and India in the 1960's faced blood stages of a parasite from the Malaysian
a much less complicated problem, and with monkey, M. irus. This parasite was later
reasonable confidence, described five new described as P. knowlesi by Sinton and Mulligan
species of malaria in monkeys from these areas. (1932). In 1934, Ionesco-Mihaiesti et al
 PRIMATE MALARIAS 16

successfully infected mental patients with a transmission studies, and pathogenesis. At the
parasite which they believed to be P. inui but same time, unfortunately, Eyles's discovery (loc.
which was later identified as P. knowlesi cit.) raised the unwelcome specter of an animal
(Garnham, 1966). Earlier, Clark and Dunn reservoir for malarias infective to man. This
(1931) failed to infect man with the blood stages situation underwent intensive study in Southeast
of P. brasilianum of the New World. The Asia from 1961 to 1965 and the results form the
relationship between human and non-human substance of this monograph. The investigators
primate malarias remained primarily of concluded that the simian malarias of Asia might
academic interest even though Mesnil and produce an occasional infection in man, but
Roubaud (1920) had produced infection in the were not considered to be a serious public health
chimpanzee with P. vivax and Rodhain (1948) problem. It was recognized, however, that such
was able to induce infection in man with the P. cases might threaten the eradication of malaria
malariae-like parasite of chimpanzees. All of in the tropics where natural infections occur in
these infections were blood induced and seemed monkeys and apes.
to be of little natural significance. Then, in 1960, The 1970's find man's struggle against the
Eyles et al reported a natural infection of man malaria parasite still unfinished. The dream of
with P. cynomolgi. True, it had occurred in the eradication is no longer considered attainable
laboratory, but the transmission was within the foreseeable future in parts of Central
accomplished by a mosquito. The fact that America, South America, Africa, and Asia. The
simian malaria was a true zoonosis was finally XXIInd World Health Assembly, meeting in
established by Chin et al (1965) with the report Boston in 1969, called for a reevaluation of the
of a human infection with P. knowlesi acquired concept of eradication with a reversion to
in the jungles of peninsular Malaysia. More classical approaches to control where necessary.
recently, various workers have been successful Giant strides have been made against the
in infecting owl monkeys (Aotus trivirgatus) disease, especially through the eradication
with human malarias (Young et al, 1966; Porter programs, but we have probably reached the
and Young, 1966; Geiman and Meagher, 1967; limit imposed by the techniques available and
Geiman and Siddiqui, 1969). the last quarter of this century may need to be as
With the primate malarias, malariologists innovative as was the first quarter, if we are to
were provided with a tool for the controlled make real progress against the age-old
study of various aspects of malaria including scourge-malaria.
drug responses, relapse mechanisms, immunity,

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CHIN, W., CONTACOS, P. G., COATNEY, G. R., and GARNHAM, P. C. C., 1966. Malaria parasites and other
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GEIMAN, Q. M. and SIDDIQUI, W. A., 1969. Susceptibility of a MULLIGAN, H. W., 1935. Descriptions of two species of monkey
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18 PRIMATE MALARIAS

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(NS) = Not seen.

 3
Ecology of the Hosts
in Relation to the Transmission
of Malaria
ASIA
BECAUSE the ecology of
human malaria has been discussed by many Parts of Southeast Asia are the only areas
authors, we will focus our main attention on the for which reasonably complete information is
transmission biology of the malarias of non- available, and proven natural vectors of non-
human primates; man and his malarias will be human primate malaria have been identified
included, but only in those situations where the only from West Malaysia (Wharton and Eyles,
two groups of primates and their parasites 1961; Wharton et al, 1962; Eyles et al, 1963;
become part of the same transmission system. and Cheong et al, 1965). In 1960, the U.S.
The level of speciation in the plasmodia of Public Health Service (NIH, LPC) established a
non-human primates is high; 17 species have cooperative project with the Institute for
been described from Asia, 3 from Africa (not Medical Research in Kuala Lumpur, Malaysia,
including 2 from Madagascan lemurs), and 2 to investigate the potential for the natural
from the New World. The fundamental ecology transmission of malaria parasites of monkeys to
of the malarias of apes and monkeys is similar to man. The baseline information on the ecology of
that of the human species. Susceptible malaria in the area was essentially complete
mosquitoes must live for a sufficient period of because the personnel of the Institute for
time to allow for completion of sporogony and Medical Research had maintained a high degree
they must have a predilection for returning to the of interest in the bionomics of anophelines of the
same group of vertebrates for a second blood Malay Peninsula for many years.
meal. In addition, it is essential that the parasite The NIH-IMR team approached the
be able to maintain an infection in the vertebrate problem on an ecological basis, studying
host long enough for the vector to become separately, but by the use of essentially the same
infected. Some degree of chronicity is required, techniques, the several distinct types of
too, since either the rapid destruction of the environments found on the Malay Peninsula
parasite by the vertebrate host or the rapid which supported monkeys and their malarias
destruction of the vertebrate host by the parasite (Fig. 1). The basic techniques used in these
would be detrimental, if not fatal, to the studies were described in detail by Wharton et al
maintenance of the transmission system. Our (1963). Briefly, they consisted of monkey-baited
approach to the discussion of the ecology of the net traps erected on platforms in the forest
malarias of apes and monkeys has for canopy and run in conjunction with human-
convenience and continuity been organized on a baited net traps, of similar design, on the ground
geographical basis. nearby. Mosquito collections were made from
these traps from sunset to sunrise, and all

19
20 PRIMATE MALARIAS

FIGURE 1.—Vectors of human and animal malarias in various ecological zones in West Malaysia (modified from Wharton
et al, 1964).

anophelines taken in the traps were dissected nemestrina. The anopheline fauna in this area was
and examined for malaria parasites. Sporozoite- dominated by members of the Anopheles umbrosus
positive salivary glands were inoculated group. Natural malaria infections were known to
intravenously into malaria-free rhesus monkeys. occur frequently in this species complex in
The 3 basic types of environments in the area Malaysia and Hodgkin (1956) suspected that some
are: fresh water swamp forests, primary rain of these infections might be of monkey origin.
forests, and mangrove forests. As was expected, most of the anophelines
Fresh Water Swamp Forests. This type caught in human- and monkey-baited net traps
of forest covers a large portion of lowland belonged to the A. umbrosus group, however, A.
Malaysia either as valleys, in which water letifer was the only species which invaded the
collects during the rainy season, or extensive flat monkey-baited net traps in the canopy in significant
areas, which maintain some level of water numbers. Sporozoites from a large number of
through much of the year. These areas support naturally infected mosquitoes (including A.
considerable populations of monkeys, including umbrosus and A. roperi as well as A. letifer) were
both macaques (Macaca sp.) and langurs inoculated into malaria-free monkeys, but no
(Presbytis sp.). Investigations were conducted in infections resulted. During the same period,
a swamp forest north of Kuala Lumpur, experimental infections were being studied in the
Malaysia where malaria infections had been laboratory, in Kuala Lumpur, to establish the level
found in both Macaca fascicularis and M. of susceptibility of Malaysian anophelines to
 ECOLOGY OF THE HOSTS IN RELATION TO THE TRANSMISSION OF MALARIA 21

monkey malaria. Plasmodium cynomolgi (B A. leucosphyrus group of mosquitoes, are never far
strain), which was originally isolated from M. from any location in West Malaysia. Finally, it
irus (= fascicularis) from the east coast of should be noted that 20 A. pujutensis were trapped
Malaysia near Kuantan (Garnham, 1959), was in the swamp forest environment but none was
used as the laboratory parasite model for these found infected. This mosquito is of more than
studies. Anopheles letifer from the swamp forest passing interest because it is a member of the A.
study area, located on the opposite side of the leucosphyrus group in which 3 species are proven
main Malaysian mountain range from the B vectors of simian malaria in Malaysia.
strain type locality of P. cynomolgi, were Primary Rain Forests. The rain forests of
exposed to experimental infections. Oocyst Southeast Asia occupy hill areas over parts of
development was poor and no sporozoite Indochina, Thailand, Burma, Malaysia, Indonesia,
differentiation was seen. These results seemed to and the Philippines, and all of these areas have
preclude the involvement of A. letifer in the certain biological features in common. All support
transmission of simian malaria even though this populations of non-human primates, and, wherever
anopheline would invade the forest canopy to investigations have been carried out, non-human
feed on monkeys. Eventually, the malaria primate malarias have been found. A typical high
infections in the A. umbrosus group of canopied rain forest, in a mountainous area
mosquitoes in this environment were attributed approximately 20 miles north and east of Kuala
to P. traguli of the Malaysian mouse deer, Lumpur, was selected as a site for the investigation
Tragulus javanicus, (Wharton et al, 1963). The of the transmission of simian malaria.
A. letifer story was, however, not complete. Mosquito trapping activities in the canopy,
When a strain of P. cynomolgi was isolated from with monkey bait, and on the ground, with human
the swamp forest study area and exposed to A. bait, produced small catches both in number of
letifer from the same locality, it was found that individuals and number of species represented.
sporozoites developed readily in the salivary However, 2 species of anophelines, A. balabacensis
glands. Such highly specific strain relationships introlatus and A. leucosphyrus, were attracted to
between parasite and mosquito host must, on monkeys in the canopy and to man on the ground.
occasion, be very important in the epidemiology Thus, 2 possible mosquito liaisons between the
of monkey malaria. It is not known how malaria parasites of man and monkeys had been
frequently such specific relationships occur, but found. It also seemed significant that both of these
they could be an important consideration in any species belonged to the A. leucosphyrus group.
area where simian malaria exists in the absence Members of this species complex were known to be
of expected vectors or where expected vectors primarily jungle breeders, and had been seriously
cannot be specifically incriminated in the incriminated as vectors of human malaria in parts of
transmission. North Malaysia, Thailand, Cambodia, Indochina,
The epidemiology of simian malaria in the Burma, and Borneo. Specimens of both species,
fresh water swamp forest of Malaysia is with sporozoite positive salivary glands, were
confused by the high levels of natural mosquito collected at the rain forest site. The sporozoites
infections with P. traguli. It may be, that the from the naturally infected A. b. introlatus produced
same species of anopheline is carrying both an infection of P. inui when inoculated
simian and mouse deer malaria and it is intravenously into malaria-free rhesus monkeys
impossible, so far, to find the former in a (Wharton et al, 1962). Using similar techniques, a
mixture so dominated by the latter. At any rate, naturally infected A. leucosphyrus, trapped in the
the vector of simian malaria in this environment same area, was found to harbor an infection with P.
remains unknown. It is possible that the cynomolgi (Eyles et al, 1963).
monkeys become infected in another area, The fact that 2 members of the same species
because troops undergo a fairly constant complex were vectors of simian malaria was
migratory activity, and forested hills, with particularly interesting when it was considered that
potential breeding sites for proven vectors of the other species, in the same group, were believed to
22 PRIMATE MALARIAS

transmit human malaria in areas where simian Investigations to determine the vector of the
malaria was found, also. Studies to investigate monkey malaria were immediately successful. An
the possibility that a single species might be A. hackeri, collected at the base of a nipah palm
responsible for the transmission of both simian during a daytime-resting catch, was found to have
and human malaria, in the same environment, sporozoite positive salivary glands. The sporozoites
were undertaken in a forest area of Cambodia produced a P. knowlesi infection following
where the local authorities had experienced inoculation into a malaria-free rhesus monkey
serious difficulties in trying to prevent (Wharton and Eyles, 1961). Further elaboration of
transmission of human malaria by using this discovery proved frustrating. Sentinel monkeys,
chloroquinized salt and/ or residual insecticides. placed in cages near the mosquito daytime resting
Anopheles balabacensis balabacensis was sites and breeding areas, failed to become infected.
believed to be important in the transmission of Night-time trapping with both human- and monkey-
human malaria in the area. These investigations bait were singularly unsuccessful. Anopheles
revealed that in that area of Cambodia, A. b. hackeri continued to be plentiful and monkeys were
balabacensis is predominatly a forest mosquito, seen whenever the area was visited. Captured M.
attracted to monkeys in the canopy and to man fascicularis were always infected and 29 percent of
on the ground. Sporozoites from 13 wild caught the leaf-eating langur monkeys (Presbytis sp.)
mosquitoes were inoculated into malaria-free carried malaria parasites, too (Wharton et al, 1964).
rhesus monkeys, but no infections were Transmission continued to occur because infected
produced. Some of the naturally infected mosquitoes were still found at the resting sites. Still,
mosquitoes were collected in deep forest areas no A. hackeri came to any type of primate bait.
believed to be considerably beyond flight range At this juncture, a thorough review of the
from the human habitations. The authors investigative site and its surroundings was made.
concluded that the sporozoites were non-human The mosquito breeding sites were in cavities in the
species (possibly, gibbon parasites) not infective base of nipah palms left after the fronds had been
to rhesus monkeys (Eyles et al, 1964). cut for thatch, a major source of income for the
The confirmation of A. b. balabacensis as a people in the area. The land on which these trees
vector of monkey malaria came as a result of were grown had been reclaimed from a tidal area by
epidemiologic investigations in Northwest the use of a series of low bunds, or dams. Tidal
Malaysia. In this area, A. b. balabacensis was changes are not great along the Straits of Malacca,
considered to be a vector of human malaria. The so these dams did not have to be very high. The
study area was a monsoon forest where malaria area, where the mosquitoes were breeding and
transmission was believed to be highly seasonal. resting, had been under cultivation for some 20
Rainy and dry seasons occupied approximately years. Rubber trees, and some fruit trees, had been
equal parts of the year. Sporozoites from planted among the nipah palms, providing an area
naturally infected mosquitoes, trapped in the of deep shade and high humidity where the
forest, produced P. cynomolgi and P. inui rainwater would remain in catchments for long
infections in malaria-free rhesus monkeys periods.
(Cheong et al, 1965). Monkeys could be seen feeding and moving
Mangrove Forests. Early in the studies, it about in this cultivated area during the day but
was realized that some of the largest monkey toward the evening they moved to the tidal
populations lived in the extensive mangrove mangrove forest, a distance of some 500 yards, for
forests along the coast. Actually, it was in this the night. Platforms were constructed in the
habitat that M. fascicularis acquired one of its mangrove trees on the tidal side of the bund and
many local sobriquets, i.e., the crab-eating equipped with monkey-baited net traps; A. hackeri
macaque. Samples of blood from members of a were caught the first night. An extensive search
typical troop at Rantau Panjang, near Klang, on failed to locate mosquito breeding sites other than
the west coast of Malaysia, revealed that these those previously described, i.e., the bases of the
animals did indeed harbor malaria infections.
 ECOLOGY OF THE HOSTS IN RELATION TO THE TRANSMISSION OF MALARIA 23

nipah palms, several hundred yards from the specifically designed to investigate the
trapping area. This enigma was finally clarified epidemiology of the first reported natural infection
when marked mosquitoes (A. hackeri), released of man with a simian malaria (Chin et al, 1965).
near the breeding and resting sites, moved The infection with P. knowlesi had apparently been
purposefully, within a few hours, to monkey- contracted in a jungle area of Central Malaysia. In
baited net traps in the tidal mangrove. 1970, Warren et al reported the results of studies in
Ultimately, A. hackeri was proven to be a the jungle, where the human P. knowlesi infection
natural vector of P. coatneyi, P. cynomolgi, P. was probably acquired, and in nearby villages.
inui, and P. fieldi as well as P. knowlesi (Warren Human-baited net traps were operated in a village
and Wharton, 1963). These species of plasmodia near the forest and on the forest floor. Monkey-
all produce chronic long-lasting infections in the baited net traps were maintained on a series of
local monkeys and the density of A. hackeri did platforms constructed in the forest canopy. Total
not apparently vary greatly from one time of the mosquito collections were augmented by bare-leg
year to another. This mosquito is a member of catches. Two familiar members of the A.
the "jungle breeding" A. leucosphyrus group of leucosphyrus group, A. leucosphyrus and A. b.
mosquitoes. Its normal habitat is in the primary introlatus, were caught in both human- and
hill forests where it breeds in hollow logs and monkey-baited jungle traps, but neither species was
fallen bamboo. The man-made environment near found to invade the village. Anopheles maculatus
Rantan Panjang had provided a suitable breeding was collected in both village and jungle traps but in
site for the mosquito and there were plenty of such small numbers that it did not seem seriously
monkeys to satisfy their predilection for simian involved in the transmission of monkey malaria to
blood. However, a certain amount of adaptation man.
was necessary, for the mosquitoes had to fly a The forested hill on which these studies were
considerable distance, from their fresh-water conducted was surrounded by swamp forests and
breeding site, in order to feed on the sleeping some A. letifer were collected from the monkey-
monkeys. The adaptation was successful baited canopy traps reviving the old enigma of the
because 5 species of malaria were identified relationship between this mosquito and the
from the area and, as previously noted, 100 transmission of monkey malaria in nature.
percent of the M. fascicularis examined from the The investigators concluded, that the A.
area were found infected. leucosphyrus group of mosquitoes were essentially
Finding this "transmission laboratory" in a primate feeders. Since most of the species are forest
man-made environment demonstrated several dwellers and breeders, they maintain a high level of
pertinent points about the ecology of simian endemic malaria among the non-human primates
malaria in Malaysia. The primate-A. and are quite prepared to transmit the same
leucosphyrus group association is old and parasites to man if he enters the jungle under
apparently highly evolved. It emerges when the appropriate circumstances. It seemed highly likely
two prime elements are combined even in a that this was the manner in which the natural human
highly artificial situation. Not only do members infection with P. knowlesi was acquired. Since the
of this species complex transmit a variety of A. leucosphyrus group mosquitoes do not leave the
simian malarias, but A. balabacensis forest, in this particular area, at least, there is little
balabacensis is virtually a universal vector. In or no opportunity for the routine introduction of
short, this monkey malaria-mosquito system is simian malarias into nearby human populations.
flexible as demonstrated by its ability to function However, the chances of man becoming involved
in a variety of habitats under numerous with these simian malarias is much greater in areas
environmental stresses. where the A. leucosphyrus group of mosquitoes is
The most recent studies on the ecology of represented by A. b. balabacensis which maintains
simian malaria in Southeast Asia were also the jungle breeding activities of the group but also
conducted in West Malaysia. This project was readily invades villages to feed on man.
24 PRIMATE MALARIAS

Little is known about the epidemiology of distribution of A. leucosphyrus mosquitoes (see

simian malaria in the remainder of Asia, but the map, Fig. 2). They are conspicuously absent from
experience in Malaysia indicates that the central, northern, and western India, but present in
presence of A. leucosphyrus group mosquitoes is Assam and parts of East Pakistan. The
essential. The map, Figure 2, shows the representative of this species complex in southern
approximate distribution of this species complex India and Ceylon, where simian malaria is quite
in Southeast and South central Asia. Generally, common, is A. elegans which we predict will be
the known distribution of monkey malarias in identified as a natural vector of simian malaria in
Asia closely follows the distribution of these that area.
mosquitoes. Particular note should be made of Other ecological and epidemiological
the discontinuous distribution in India. Initially, factors obviously enter into the distribution of the
it was thought that the Indian rhesus monkey simian malarias in Southeast and South-central
was free of malaria. It was soon discovered, Asia. For example, P. knowlesi is highly pathogenic
however, that those trapped in Assam and to M. mulatta, following a rapid and almost
eastern parts of East Pakistan were sometimes invariably fatal course. Therefore, without severe
infected and, therefore, unsuitable for malaria modifications on one or both sides, this parasite and
research as non-infected animals. In contrast, the monkey could not survive in the same area.
thousands of M. mulatta from central, northern, There are probably other malaria parasites which
and western India have been examined, and no develop readily in a limited range of hosts. There
infections have been found. We believe, the are, for example, strains of malaria in Malaysian
explanation for this phenomenon lies in the leaf monkeys (Presbytis sp.) which grow only with

FIGURE 2.—Approximate distribution of Anopheles leucosphyrus group mosquitoes in Southeast and Southcentral Asia (modified
from Colless, 1956).
 ECOLOGY OF THE HOSTS IN RELATION TO THE TRANSMISSION OF MALARIA 25

difficulty in M. mulatta (Eyles et al, 1962). attracted to non-human primates. Garnham (1951)
However, these factors do not seem to contribute used monkeys as bait in his studies on the natural
to the distribution of the parasite at the same history of simian hepatocystis in Uganda. Platform
level as the limitations imposed by the vector and ground traps were used. Two A. coustani and 8
characteristics. The correlation between malaria A. implexus were caught; the author did not record
parasite distribution and the distribution of the whether these came from ground or from platform
A. leucosphyrus group mosquitoes is apparently traps. The paucity of information available on the
due to feeding preferences and biting habits of natural attraction of African non-human primates
the vectors rather than the susceptibility of the for anophelines is surprising considering the interest
mosquito to the parasite. Experimentally, the of many workers in the possibility that man and
simian malarias from Southeast Asia can be chimpanzees shared their malaria parasites, and that
transmitted by a large number of mosquito the apes might well serve as reservoirs for human
species (Warren and Wharton, 1963; Warren et malaria. Eventually, one species, P. malariae, was
al, 1963; Collins et al, 1965, 1966, 1967, 1967a, said to exist naturally in both man and the
1968, 1968a; Collins, 1969). In addition, it chimpanzee. Still, there was no concerted effort to
seems probable that in local situations, highly determine which mosquitoes were responsible for
specific relationships may have evolved, and maintaining the infections in the apes, nor if such
mosquitoes, other than members of the A. vectors were prone to feed on man as well.
leucosphyrus group, may be involved. There is scant information on the experimental
Anopheles letifer, in fresh water swamp forests susceptibility of a variety of anophelines to malaria
in Malaysia, is a prime candidate for such a role. parasites of African apes and monkeys. However,
However, it is our opinion that the relationships most of the work was carried out in Europe and the
between simian malaria and the A. leucosphyrus United States; the species of mosquitoes involved
group mosquitoes is so intimate that future were not African. Bray (1957) fed A. gambiae on
extensions in the geographic distribution of the chimpanzees infected with P. reichenowi and
simian malarias in Southeast and South central reported gut infections; the salivary glands did not
Asia will be associated with established become positive. The same investigator, (Bray,
populations of one or more members of this 1958) succeeded in transmitting P. schwetzi with A.
mosquito species complex. gambiae, but no sporozoites were seen in the
inoculum. He concluded, that this mosquito would
AFRICA be a poor natural vector since the sporozoites
survived only a short time in the salivary glands.
Information currently available on the Anopheles gambiae were also shown to be
epidemiology of non-human primate malaria in refractory to at least one strain of P. malariae
Africa is limited. Three species of Plasmodium isolated from the chimpanzee (Bray, 1960).
have been described from the great apes of There is no information available on possible
Africa, one of which may be shared with man vectors of P. gonderi. There are a great number of
(P. rodhaini). The other 2 species, P. reichenowi species of anophelines in Africa that are strictly
and P. schwetzi are closely related to the human forest breeders and biters but information on host
P. falciparum and P. vivax-P. ovale stocks. Only preferences is too incomplete at present to allow for
one true malaria parasite has been reported from speculation on their possible role in the
African monkeys, P. gonderi, although the transmission of non-human primate malaria.
related genus, Hepatocystis, is common in these Likewise, there is no information available on
animals. either natural or experimental vectors of the two
We are not aware of any studies that have malaria parasites described from Madagascan
been specifically directed toward identifying the lemurs (P. girardi and P. lemuris).
natural vectors of the ape and monkey malarias
in Africa. Gillies and De Meillon (1968) in their
comprehensive work on the anophelines south of
the Sahara do not record any species naturally
26 PRIMATE MALARIAS

AMERICA on the ground and on platforms, at various levels, in

the forest canopy. A variety of anophelines were
Information on the epidemiology of non- caught, but no single species was present in large
human primate malaria in the monkeys of South numbers. They found A. neivai in the forest canopy,
and Central America is more complete than for in an area near Panama City; a species Deane et al.
Africa. Deane and his colleagues in São Paulo (loc. cit.) considered a probable vector of P.
have been studying the distribution and brasilianum in the Manaus area. Undoubtedly, new
epidemiology of the simian malarias in Brazil information on the epidemiology of simian malaria
for a number of years. The results of ground and in Central America will be available soon, for this
platform mosquito catches, using monkey-bait, subject is now under investigation by Young and
implicate the bromeliad breeding A. cruzi as the his colleagues at the Gorgas Memorial Laboratory
mosquito responsible for the holoenzootic status in Panama.
of P. simium in Horto Florestal da Cantareira There is not enough information available to
near São Paulo. Similar results were obtained in allow for speculation on the influence of
other study areas in southeastern Brazil (Santa epidemiological factors on the distribution and
Catarina and Espiritu Santo) (Deane et al, 1969). prevalence of simian malarias in the Western
The situation becomes more complex in Hemisphere. Plasmodium brasilianum has adapted
Amazonas where a greater variety of mosquitoes well to a variety of genera and species of monkeys
invade the canopy to feed on monkeys. At Porto from southeastern Brazil to the jungles of Panama
Maria, where only one monkey was found to be and, possibly, even further north. During the same
parasite positive, a variety of anophelines were period, P. simium has remained confined to an area
found feeding in or near the canopy; A. of southern and eastern Brazil. However, we are of
mediopunctatus being particularly numerous. the opinion that there are many anophelines in the
The investigators felt that these mosquitoes were jungles of South America capable of transmitting P.
probably not important in the transmission of simium, and the limits in its present distribution are
monkey malaria since the same species was due to a lack of movement, after a recent
found in other areas where infections in introduction into an exotic environment; rather than
monkeys were nil. We would suggest that such to failure in many areas due to an unreceptive
areas should be monitored regularly, because the transmission system. Under such circumstances, it
simian malarias of South and Central America is possible that in areas where the sub-human
are still in the process of extending their host primates are now negative for malaria, future
and geographic range. The argument, that a investigations will reveal infections in the same
given species of anopheline is probably not primates. Therefore, we believe that the host(s),
involved in malaria transmission, because it is geographic distribution, and aspects of vector
abundant in an area where no vertebrate limitations of the simian malarias in South and
infections occur, may be debatable since it is Central America will remain confusing, unstable,
likely that all Western Hemisphere anophelines and subject to change until the parasites have tested
existed for thousands of years in the absence of all environmental niches in the available vertebrate
malaria. and invertebrate hosts.
Two species in the subfamily Anophelinae, It would be convenient, if investigators of
Anopheles neivai and Chagasia bonneae, were simian malaria ecology in Africa and South
found to be attracted to monkeys consistently in America were able to find a correlating factor as
the forest canopy near Manaus, and Deane et al. specific as the A. leucosphyrus group of mosquitoes
(loc. cit.) felt that these species were probably seems to be in Asia. However, as pointed out
responsible for the transmission of P. earlier, the relationship between the simian malarias
brasilianum in that area. There is less of Southeast and South-central Asia and the A.
information available on the vectors of P. leucosphyrus group of mosquitoes is probably a
brasilianum in other parts of South and Central very old and highly evolved one. It is our belief,
America. Galindo et al (1950), working in that the primate plasmodia have been living in
Panama, trapped mosquitoes, with human bait, Africa for a shorter period than in Asia and are only
recent arrivals in the New World. Therefore, it
 ECOLOGY OF THE HOSTS IN RELATION TO THE TRANSMISSION OF MALARIA 27

would seem that the chances of finding a single opportunity for such a specific relationship to have
species complex of mosquitoes, south of the developed in Central and South America is dim
Sahara, responsible for the transmission of non- indeed.
human primate malarias, is unlikely and the

REFERENCES
BRAY, R. S. 1957. Studies on malaria in chimpanzees. III. DEANE, L. M., FERREIRA NETO, J. A., OKUMURA, M., and
Gametogony of Plasmodium reichenowi. Ann. Soc. FERREIRA, M. 0., 1969. Malaria parasites of Brazilian
BeIge de Med. Trop. 37 : 169-174. monkeys. Rev. Inst. Med. Trop. São Paulo 11 : 71-86.
BRAY, R. S., 1958. Studies on malaria in chimpanzees. V. The EYLES, D. E., LAING, A. B. G., WARREN, McW., and
sporogonous cycle and mosquito transmission of SANDOSHAM, A. A., 1962. Malaria parasites of
Plasmodium vivax schwetzi. J. Parasit. 44 : 46-51. Malayan leaf monkeys of the genus Presbytis. Med. J.
BRAY, R. S., 1960. Studies on malaria in chimpanzees. VIII. The Malaya 17 : 85-86.
experimental transmission and pre-erythrocytic phase EYLES, D. E., WARREN, McW., GUINN, E., WHARTON, R.
of Plasmodium malariae, with a note on the host-range H., and RAMACHANDRAN, C. P., 1963.
of the parasite. Am. J. Trop. Med. & Hyg. 9 : 455-465. Identification of Anopheles balabacensis introlatus as a
CHEONG, W. H., WARREN, McW., OMAR, A. H., and vector of monkey malaria in Malaya, Bull. WId. Hlth.
MAHADEVAN, S., 1965. Anopheles balabacensis Org. 28 : 134-135.
balabacensis identified as vector of simian malaria in EYLES, D. E., FONG, Y. L., DUNN, F. L., GUINN, E.,
Malaysia. Science 150: 1314-1315. WARREN, McW., and SANDOSHAM, A. A., 1964.
CHIN, W., CONTACOS, P. G., COATNEY, G. R., and Plasmodium youngi n. sp., a malaria parasite of the
KIMBALL, H. R. 1965. A naturally acquired Malayan gibbon, Hylobates lar lar. Am. J. Trop. Med.
quotidian-type malaria in man transferable to monkeys. Hyg. 13 : 248-255.
Science 149 : 865. GALINDO, P., TRAPIDO, H., and CARPENTER, S. J., 1950.
COLLINS, W. E., JONES, F. E., and DOBROVOLNY, C. G., Observations on diurnal forest mosquitoes in relation to
1965. Transmission of the RO strain of Plasmodium sylvan yellow fever in Panama. Am. J. Trop. Med. 30 :
cynomolgi by A. stephensi, A. quadrimaculatus and A. 533-574.
labranchiae atroparvus. Mosq. News 25 : 389-392. GARNHAM, P. C. C., 1951. An attempt to find the vector of
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, Hepatocystis ( = Plasmodium) kochi (Levaditi and
J. R., 1966. Studies on the transmission of simian Schoen). Exp. Parasit. 1 : 94-107.
malarias. I. Transmission of two strains of Plasmodium GARNHAM, P. C. C., 1959. A new subspecies of Plasmodium
inui by Anopheles maculatus and A. stephensi. J. cynomolgi. Riv. di Parassit. 20 : 273-278.
Parasit. 52 : 664-668. GILLIES, M. T. and DE MEILLON, B., 1968. The anophelinae of
COLLINS, W. E., CONTACOS, P. G., and GUINN, E. G., 1967. Africa south of the Sahara (Ethiopian zoogeographical
Studies on the transmission of simian malarias. II. region). South African Inst. Med. Res., Johannesburg.
Transmission of the H strain of Plasmodium knowlesi Pub. 54, pp. 343.
by Anopheles balabacensis balabacensis. J. Parasit. 53 : HODGKIN, E. P., 1956. The transmission of malaria in Malaya.
841-844. Stud. Inst. Med. Res. Fed. Malaya. No.27 , pp.98.
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, WARREN, McW. and WHARTON, R. H., 1963. The vectors of
J. R., 1967a. Studies on the transmission of simian simian malaria: Identity, biology, and geographical
malaria. III. Infection and transmission of Plasmodium distribution. J. Parasit. 49 : 892-904.
coatneyi with Anopheles freeborni and A. balabacensis WARREN, McW., EYLES, D. E., WHARTON, R. H., and OW
balabacensis mosquitoes. J. Parasit. 53 : 1130-1134. YANG, C. K., 1963. The susceptibility of Malayan
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, anophelines to Plasmodium cynomolgi bastianellii. Ind.
J. R., 1968. Transmission of Plasmodium fieldi by J . Malariol. 17 : 81-101.
Anopheles maculatus, A. stephensi and A. balabacensis WARREN, McW., CHEONG, W. H., FREDERICKS, H. K., and
balabacensis. J. Parasit. 54 : 376. COATNEY, G. R., 1970. Cycles of jungle malaria in
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, West Malaysia. Am. J. Trop. Med. Hyg. 19 : 383-393.
J. R., 1968a. Some observations on the transmission of WHARTON, R. H. and EYLES, D. E., 1961. Anopheles hackeri, a
Plasmodium inui. J. Parasit. 54 : 846-847. vector of Plasmodium knowlesi in Malaya. Science 134
COLLINS, W. E., 1969. Some observations on the transmission of : 279-280.
simian malaria. Proc. 56th Ann. Meeting New Jersey
Mosq. Exterm. Assoc. pp. 152-158.
COLLESS, D. H., 1956. The Anopheles leucosphyrus group.
Trans. Roy. Ent. Soc. London 108 : 37-116.
28 PRIMATE MALARIAS

REFERENCES – Continued
WHARTON, R. H., EYLES, D. E., WARREN, McW., and WHARTON, R. H., EYLES, D. E., and WARREN, McW ., 1963. The
MOORHOUSE, D. E., 1962. Anopheles leucosphyrus development of methods for trapping the vectors of monkey
identified as a vector of monkey malaria in Malaya. malaria. Ann. Trop. Med. Parasit. 57 : 32-46.
Science 137 : 758. WHARTON, R. H., EYLES, D. E., WARREN, McW., and GHEONG,
W. H., 1964. Studies to determine the vectors of monkey
malaria in Malaya. Ann. Trop. Med. Parasit. 58 : 56-77.
 4
Life Cycle and the
Phenomenon of Relapse

A. LIFE CYCLE may be decidedly vacuolated, as in P. hylobati.

The growing trophozoite feeds on the
hemoglobin of the host red cell by phagotrophy.
IN the Primate Host. The cycle However, the hemoglobin is incompletely
of malaria in the primate host is initiated by the metabolized by the parasite and what remains,
inoculation of sporozoites by the female principally hematin, is malaria pigment. The
mosquito when she punctures the skin to obtain shape and color of these pigment particles vary
blood (Fig. 3). Instead of setting-up a cycle in from one species of malaria to another and,
the blood, as maintained by Schaudinn in 1902, consequently, often serve as an aid in species
and not disproved until 46 years later by Shortt diagnosis. Just prior to nuclear division, the
and Garnham (1948), the sporozoites soon leave vacuole disappears and the cytoplasm appears
the blood stream and enter the parenchyma cells more compact. The parasite nucleus now
of the liver, initiating the cycle of undergoes a series of mitotic divisions which
exoerythrocytic (EE) schizogony. As the ultimately produces a mature schizont. The
parasite grows, it assumes different shapes nuclei surrounded by a small amount of
(round, oval, or lobulate). It may contain cytoplasm are now termed merozoites. The
vacuoles, and from several to many flocculi. number of merozoites in a schizont varies
Normally, the EE body develops within a single according to the species of Plasmodium
parenchyma cell of the liver, displacing its although there is considerable variation even
nucleus. The nuclei of the parasite multiply by within a given species. Plasmodium eylesi
division, eventually increasing in number up to produces 20 to 34 merozoites, the average is 25,
as many as 40,000 per EE body; pigment is whereas P. malariae may produce 4 to 12
absent. When the schizont is mature, in 5 to 15 merozoites with an average number of 8. When
days, depending on the species, the merozoites the schizont is mature, the merozoites are
are released into the blood where they invade the released into the blood and, unless destroyed by
host's erythrocytes, and thereby initiate the the host's immune mechanism, invade other
schizogonic cycle in the blood. erythrocytes which initiates another asexual
The merozoites of some species display a cycle.
marked predilection to invade reticulocytes, The length of time required for
others prefer mature red cells and some are non- completion of the erythrocytic cycle ranges from
selective. The young asexual parasite, or approximately 24 hours (quotidian periodicity)
trophozoite, appears ring-shaped, the "stone" to approximately 72 hours (quartan periodicity).
being the nucleus. As the trophozoite grows, it Only one species of primate malaria, P.
may assume various shapes, amoeboid as in knowlesi, has a 24-hour cycle. Most of the
Plasmodium vivax, compact as in P. inui, or it primate malarias have a 48-hour

29
30 PRIMATE MALARIAS

FIGURE 3.—Diagrammatic presentation of the life cycle of the primate malaria parasite.

asexual cycle (tertian periodicity). The exact of erythrocytic schizogony, some parasites
interval varies; for example, different strains of develop into gametocytes. It is impossible in the
P. vivax require from 41.5 to 45.8 hours (Young, very early stages of development to differentiate
1944), whereas P. ovale requires 49 hours the young gametocytes from the developing
(Jeffery et al, 1954). The primate malarias trophozoites. However, as the gametocytes
known to have a 72-hour cycle are P. malariae, approach maturity, they are easily distinguished
P. brasilianum, and the various strains of P. from the mature trophozoites (see individual
inui. The parasites in subsequent chapters will chapters for specific differences). The exact
be referred to as having developmental cycles of origin of these forms is unknown. They must
24, 48, and 72 hours. arise as a product of schizogony, but whether
During the growth of the parasite, the host certain schizonts are predestined to produce
erythrocyte often exhibits secondary changes; asexual parasites and others sexual forms is one
the most prominent are enlargement (as with P. of several mysteries in the life-cycle of malaria.
vivax and P. cynomolgi) and the presence of Their tendency to be produced in waves would
stippling. The latter is made visible by staining suggest that the host's immune response may be
the air-dried blood film with Romanowsky associated with gametocyte production.
stains. These important diagnostic features will
be readily seen in the illustrations of the
different species. After one or more generations
 LIFE CYCLE AND THE PHENOMENON OF RELAPSE 31

However, certain strains have become The microgametes are produced by a process
gametocyteless, such as the Santee Cooper strain termed exflagellation. Soon after ingestion, the
of P. falciparum (Jeffery, 1951), which suggests nucleus of the microgametocyte divides, giving
that the host may have little, if any, influence on rise to 8 nuclei. These migrate to the periphery
the initiation of gametocyte production. Two and enter long cytoplasmic processes which
forms are produced: micro- and macro- project from the surface of the parasite. The
gametocytes, commonly referred to as male and microgametes lash about vigorously and soon
female gametocytes. In general, the latter separate from the parent body. The microgamete
predominate. In some cases, notably P. enters the macrogamete to form the zygote.
falciparum, gametocytes will continue to Shortly thereafter, the ookinete is formed. This
circulate in the peripheral blood long after their vermiculate body moves actively and forces its
initial production. However, their infectivity way into an epithelial cell of the host's mid-gut.
appears to be short-lived and there is some A cyst wall is formed and this stage, the oocyst,
evidence to suggest that their infectivity may be now commences its growth lying between the
associated with a certain period of the day basement membrane of the gut wall and the
(Hawking et al, 1968). cells, and projecting into the haemocoele of the
In the Mosquito. The cycle in the peripheral mosquito. Oocysts are only found on the mid-
blood exists only, in terms of species survival, gut and are usually more concentrated toward
for the production of sexual forms, the the posterior end. Meiotic division occurs within
gametocytes. The gametocytes of the primate the young oocyst, usually commencing about 48
malarias can only complete their destiny in an hours after the blood meal (Bano, 1959).
anopheline mosquito. Succeeding mitotic divisions produce nuclei
The female anopheline seeks a primate host with the particular haploid number of
because she is hungry, a fact clarified by the late chromosomes for that species. As the oocyst
Dr. Robert W. Burgess in the early 1940's. Her grows (sometimes reaching a diameter of 100 µ)
hunger is satisfied by biting and in the process the number of nuclei increases; vacuoles appear
she takes blood containing gametocytes and as the cytoplasm divides into sporoblasts. From
other blood-stages of the parasite into her these arise the sporozoites. Oocysts in which the
digestive tract. In the mid-gut, or mesenteron, sporoblasts and developing sporozoites are
the mature gametocytes shed their red cell visible under light microscopy will be referred
envelopes and transform into gametes, which we to, in subsequent chapters, as differentiated.
now know to be the mature sexual forms. Each sporozoite contains a single nucleus and
Laveran (1880) and many other workers had the number of sporozoites produced per oocyst
seen the 'strange' forms in the blood, but it was a has been estimated as about 10,000 (Pringle,
3rd year American medical student, William 1965). Upon maturity, the time depending on the
George MacCallum, who discovered their sexual temperature and humidity conditions, the oocyst
nature. MacCallum (1897), during his summer ruptures and the sporozoites are released into the
vacation in Canada, studied the malaria parasites haemocoele of the mosquito. The sporozoites
in crow blood and deduced that the non- migrate to all parts of the body but some enter
segmenting forms were male and female the acinal cells of the salivary glands. When the
parasites. Upon his return to Johns Hopkins infected mosquito begins to feed, the sporozoites
Medical School in the fall, he confirmed his enter the salivary duct and are thereby
suspicions while studying blood from a woman introduced into the blood stream of the primate
infected with P. falciparum. He watched the host, initiating the infection in a new
process of exflagellation whereby the male environment.
(micro-) gametocyte throws out thread-like
processes which lash about and soon separate B. RELAPSE
from the parent body. He observed one of these
thread-like bodies, saw it penetrate and thus The phenomenon of relapse has intrigued
fertilize the rounded-up female (macro-) gamete. malariologists for decades. As early as 1893,
32 PRIMATE MALARIAS

Golgi suggested that parasites of human malaria et al (1968) for P. knowlesi, Voller and Rossan
may develop in endothelial cells not affected by (1969) for P. cynomolgi, and by ourselves for P.
antimalarial drugs and that the protected brasilianum.
parasites could be the source of relapses. The first experimental evidence of rhythm
In 1897, almost at the time that Ronald in relapse activity came in 1900 when Sir
Ross was completing his basic studies on the Patrick Manson allowed infected mosquitoes to
transmission of malaria, Thayer published a bite his son, P. Thurburn Manson, then a young
series of lectures with several references to medical student. The younger Manson came
relapse in vivax malaria. In his speculations to down with vivax malaria and the infection was
explain latency, which must obtain preceding a treated with quinine. Following treatment, he
relapse, he postulated that there must be an continued in good health until 9 months later
undiscovered form of the parasite. He wrote "the when he had a typical relapse which he himself
organism may remain perhaps within the cell reported in detail in 1901. Another volunteer, in
body of certain phagocytes for long periods of the same time period, was Major C. F. Fearnside
time, only to be set free again as a result of some who had a relapse experience similar to that of
insult, the nature of which is not as yet young Manson which he reported in 1903. In
appreciable to us." More than 70 years later, we retrospect, these early reports set a pattern which
still do not know the exact nature of these was later recognized as a common phenomenon
phenomena, but it is generally accepted that: 1) in many strains of P. vivax.
fixed-tissue forms of the true relapsing malarias After Schaudinn's description of the direct
(i.e., Plasmodium vivax, P. ovale, P. cynomolgi, invasion of a red blood cell by the sporozoite of
P. fieldi, and P. simiovale) release young forms P. vivax (1902), attention was concentrated,
periodically which parasitize the erythrocytes almost entirely, on this stage of the parasite.
and thereby initiate the relapse (Fig. 4) and 2) However, as information accumulated,
along with the above type of reactivation of the especially the data derived from the study of
infection is the one arising from renewed induced malaria in the treatment of general
activity, after a period of quiescence, in the paresis, it became apparent that all human
conventional red cell cycle. This renewed malarias did not have the same relapse potential,
activity, which we call a recrudescence, is due, and that there was a difference in the response to
possibly, to the development of new antigenic therapy in blood-induced and sporozoite-
variants of the parasite as was shown by Brown induced infections (Yorke, 1925;

FIGURE 4.—Diagrammatic presentation of the parasitologic and clinical cycle in the vertebrate host of those malarias which
have a true relapse mechanism.
 LIFE CYCLE AND THE PHENOMENON OF RELAPSE 33

Yorke and Macfie, 1924). In 1931, James indirect evidence. The direct evidence came in
suggested that sporozoites, after being injected the same year, with the description by Shortt and
by the mosquito, are carried to the internal Garnham (1948) and Shortt, Garnham, and
organs where they enter reticulo-endothelial Malamos (1948) of the exoerythrocytic stages of
cells and go through a cycle of development P. cynomolgi in the liver of an experimentally
with the eventual production of merozoites infected rhesus monkey. Soon, similar
which parasitize red blood cells. This proposal preerythrocytic forms were demonstrated in the
was based primarily on the fact that therapeutic livers of human volunteers infected with P.
regimens known to be effective against malaria vivax (Shortt et al, 1948) and P. falciparum
could not cure an infection when administered (Shortt et al, 1949; Jeffery et al, 1952).
during the incubation period. It was reasoned, With the demonstration of the
that if sporozoites entered directly into the red exoerythrocytic stages of avian and primate
blood cells and became trophozoites and malarias, a whole new developmental phase of
schizonts, they would have been destroyed by the parasites could be examined. Various
the drugs and no active infection could have proposals were advanced to explain the
resulted. Meanwhile, research in the avian phenomenon of relapse in malaria. The hiding
malarias had been developing rapidly, and in place of the parasite, during long periods when
1935, Huff and Bloom clearly demonstrated the patients were clinically and parasitologically
exoerythrocytic stages to be a fundamental part negative, had been debated for many years. The
of the life cycle of P. elongatum. discovery of the exoerythrocytic stages of
As data about fixed tissue parasites from primate malarias seemed to have revealed their
birds accumulated, mainly the work of James hiding place. With the report of an EE schizont
and Tate (1937) and the brilliantly executed in the liver of a monkey 3½ months after
studies of Huff and his co-workers (1943 to sporozoite inoculation (Shortt and Garnham,
1948), it became abundantly clear that such a 1948), most workers in the field came to believe
cycle must also occur in primate malarias. In that a direct relationship existed between these
1946, Sapero, in his Craig Lecture (Sapero, fixed tissue stages and true relapses.
1947) presented presumptive evidence for the The relationship of relapse of primate
link between fixed tissue stages and relapse in malarias to an exoerythrocytic stage of the
such lucid language that their actual parasite was not new. Various workers, involved
demonstration seemed an easy task. in experimental therapy of malaria, had
In 1945, Fairley et al had shown that predicted that an exoerythrocytic stage of the
sporozoites, following their introduction, parasite was responsible for long term relapses
disappeared from the circulating blood during in P. vivax. The absence of a persistent tissue
the first hour and no evidence of infection was to phase in P. falciparum was also proposed
be found until 6 days later in falciparum (Shannon and Earle, 1945; Fairley et al, 1947)
infections and 8 days later in vivax infections, prior to the actual demonstration of EE schizonts
when blood transfusions from the infected of P. cynomolgi by Shortt and Garnham (loc.
individuals produced infection in the recipients. cit.). The persistent tissue phase as the source of
The evidence in support of the existence of the parasites in typical P. vivax relapses was
preerythrocytic stages in primate malarias was firmly supported by Shannon and Earle (1945)
now so strong that it seemed only a matter of and Fairley et al (1947), and at least tentatively,
time until they were demonstrated. Coatney and by Huff (1947). The evidence to support this
Cooper (1948) summarized the information hypothesis was primarily based on the extensive
available on drugs with apparent action against work on EE stages in avian malarias by a variety
preerythrocytic stages of avian and primate of workers in the 30's and 40's and the striking
malarias. These investigators reported that 8- differences in response to therapy in blood-
aminoquinolines and certain biguanides were induced and sporozoite-induced infections with
active against the presumed exoerythrocytic P. vivax. Coatney and Cooper (1948a), based on
forms of human and simian malarias, the data from studies with the St. Elizabeth strain of
presence of which had been deduced from P. vivax, not only supported the EE stage theory
34 PRIMATE MALARIAS

as the mechanism of relapse, but proposed a way related to the exoerythrocytic stages of the
concept of latency which we will reintroduce parasite. This investigator insisted that "not a
later in this discussion. single fact" went against the initial opinion of
It is clear that a large number of workers in Bignami (1910) that relapses were related to the
the field of malaria were convinced, not only of persistence of endoerythrocytic parasites.
the existence of EE stages in primate malarias, It is appropriate, at this point, to discuss
but of their association with relapses. Therefore, these two theories concerning the origin of
the results of the remarkable series of relapses in malaria inasmuch as the controversy
experiments by Shortt and his group which has remained essentially unchanged since the
conclusively demonstrated the EE schizonts of early 50's. Corradetti accepts the existence of EE
P. cynomolgi, P. vivax, and P. falciparum were stages of primate malarias, but still (1966),
generally sympathetically received. When the believes that relapses are due entirely to the
work of Coatney and Cooper (1948a), showing persistence of erythrocytic stages of the parasite;
that massive blood transfusions during latency, Corradetti's position has not changed
following treatment of the initial attack, failed to appreciably since 1950 (Corradetti and Verolini,
produce infections in recipients, although the 1950). Bray (1967) produced an excellent
donors' infections relapsed later, was combined review of his position on the relationships
with the demonstration of an EE schizont of P. between EE stages and relapse mechanisms in
cynomolgi 3½ months after sporozoite which he considered the individual points in
inoculation, most workers considered the relapse Corradetti's thesis. Bray's arguments generally
story to be complete (Shortt and Garnham, reflect the contemporary opinion of most
1948a). The specific mechanism proposed was malariologists. There are several aspects of this
that merozoites from mature EE schizonts enter controversy which warrant a more detailed
red blood cells, producing the familiar clinical consideration.
and parasitological features of malaria. Other Corradetti (1965) presented a number of
merozoites, possibly from the same schizont, well reasoned points and his strongest argument
enter normal liver cells and continue the cycle of rests with the fact that blood-induced infections,
exoerythrocytic schizogony. This latter process of quite long duration, have been observed with
would continue indefinitely, and, when the P. cynomolgi, P. malariae, and P. falciparum. It
active immunity of the host was, for any reason, is also agreed, that the blood may be negative
reduced, some of the merozoites would invade for malaria parasites by routine microscopic
the red blood cells to produce a relapse. This examination for periods of varying duration
concept of Shortt and Garnham (loc. cit.) was during any of these extended infections.
widely but not universally accepted as the most Actually, work from our laboratory indicates
likely explanation for the production of relapses that even stronger support for his thesis would
in certain species of both human and simian be found with blood-induced infections of P.
malaria. inui which are known to persist in monkeys for
It has been noted that acceptance of the years. This we consider to be a well reasoned
exoerythrocytic stage of malaria as the source of argument and could well support Corradetti's
relapses was not universal. Huff (1948) and Huff case if it were taken no further. However, all of
and Coulston (1948) were convinced of the the evidence must be considered. It is true that
existence of the exoerythrocytic stages in blood-induced infections with P. cynomolgi
primate malarias, but Huff (1950) expressed persist for long periods, but such infections can
serious reservations concerning the parasitic be quickly and totally eradicated with
nature of the bodies described by Shortt. Fairley schizonticidal drugs, such as chloroquine or
(1949) did not entirely agree with the continuing quinine. Sporozoite-induced infections, with the
exoerythrocytic cycle, as described by Shortt same parasite, can be cleared of parasites in the
and Garnham, either. In 1950, Corradetti peripheral circulation with the same drugs, but
expressed categoric disagreement with the idea the infection returns with a frequency which is
that relapses in mammalian malaria were in any dependent upon the strain of parasite used.
 LIFE CYCLE AND THE PHENOMENON OF RELAPSE 35

This evidence is, of course, not new. Differences strain parasite, in the same person. This
in the response of sporozoite- and blood-induced experiment clearly indicated that the absence of
infections of P. vivax to schizonticidal drugs parasites from the peripheral circulation was real
have been recognized for many years (Yorke and not a suppression to very low levels by
and Macfie, 1924). Corradetti (1966) did not immunologic activities. We consider these
include data from chemotherapeutic studies of P. experiments to be conclusive, but Corradetti
vivax and P. cynomolgi in his discussion of (1966) felt that it would be necessary to transfer
relapse mechanisms. every red blood cell without producing an
The extreme duration of infections with P. infection, before one could know that no
malariae naturally occupies an important parasites were present. In the same paper, it is
position in any discussion of relapse noted that Corradetti and Verolini (1950)
mechanisms in malaria. There are reports of subinoculated blood during negative periods
persistence of infection for 30 to 40 years and from monkeys with blood-induced P. cynomolgi
recrudescences are generally expected through a infections with negative results. We have
period of from 5 to 8 years. Ciuca et al (1964) carefully reviewed this reference and cannot find
followed the course of a blood-induced infection the account of a subinoculation, with negative
and found erythrocytic parasites 525 days after results, of whole fresh blood from an infected,
inoculation. The same workers reported that but negative donor, that at a later time
schizonticides were radically curative in demonstrated a patent parasitemia. Even a single
sporozoite-induced P. malariae infections, and, result of this nature would be interesting; if any
they concluded, that this parasite possessed no parasite could consistently produce this
secondary exoerythrocytic stages. Corradetti response, it would be quite important.
called attention to this finding, a point which The final evidence in support of the direct
was well taken since radical cures with involvement of exoerythrocytic stages in
schizonticidal drugs had been interpreted by malarial relapses which has been challenged is
others to be associated either with blood-induced the demonstration of the parasites themselves.
infections or with sporozoite-induced infections Corradetti was skeptical because of the low
of malarias not possessing a relapse potential. number of late EE schizonts that have been seen.
More recent data from our laboratory suggest Actually, as Bray (1967) pointed out, the
that P. malariae has no true relapse mechanism. number of liver stages seen 30 days or more
Furthermore, schizonticidal drugs have been after sporozoite inoculation is considerably
found to produce radical cures of sporozoite- greater than that noted by Corradetti. However,
induced P. inui infections, the quartan parasite the questions posed by the latter investigator
of Asian monkeys. concerning: 1) how such stages are known to be
One of the strongest pieces of evidence to secondary and not the result of some form of
support the exoerythrocytic source of malaria latency and 2) why 'nests' of parasites are not
parasites in relapse situations is the total absence found in the area where an earlier schizont
of these parasites from the peripheral blood matured, have not been answered satisfactorily.
during negative intervals. These negative The accumulation of evidence relating long
intervals may occur naturally or may be induced term relapses to a persistent tissue stage of the
by the use of schizonticidal drugs. The absence parasite seems to us to be absolutely conclusive.
of parasites during these periods was established There remain a number of gaps in our
by the inoculation of massive amounts of whole knowledge and a number of areas in which
blood from infected to malaria-free volunteers generally accepted concepts do not provide all of
without producing infections (Cooper et al, the answers. We consider the theory of a
1949). Cooper et al (1947) demonstrated the continuing cycle of exoerythrocytic schizogony
susceptibility of volunteers during the long to be an inadequate explanation from two points
negative interval in St. Elizabeth strain P. vivax of view. It seems inescapable that the
infections by successfully superimposing a mechanism responsible for the long term relapse
blood-induced infection, with the homologous
36 PRIMATE MALARIAS

in the St. Elizabeth strain of P. vivax is also generally believed that the immune mechanisms
involved in the delayed patency phenomenon of the malaria-infected vertebrate do not reach
seen with other strains of P. vivax (Tiburskaya, the liver stages but are only effective against the
1964). The delayed primary attacks of P. ovale erythrocytic stages of the parasite. If
reported by Chin and Contacos (1966) and exoerythrocytic schizogony were a continuing
Trager and Most (1963) were apparently due to process, it would follow that, once established,
the use of suppressive drugs at the time of the infection would be terminated only by the
infection. There seems to be no doubt that each death of the host.
of these infected individuals was susceptible Garnham (1967) has reviewed this subject
during the long period between infection and the and agrees that the process of continuous
primary clinical attack. Therefore, there is no secondary exoerythrocytic schizogony has not
immediately available explanation why an been established. In this publication, he
earlier erythrocytic multiplication would not reintroduced the concept of latency or a dormant
have occurred if parasites were continually stage of the parasite as a possible explanation for
being released into the blood from maturing the relapses seen in certain species of primate
liver schizonts. The same question can be asked malaria and noted that "if such an idea could be
concerning the delayed patency phenomenon in proved, then many puzzling features of relapses
certain strains of P. vivax (Tiburskaya, loc. cit.). and latency would be explained." Shute (1946)
This weakness in the Shortt and Garnham pointed out that sporozoites could survive in the
(1948b) theory of continuing exoerythrocytic salivary glands of mosquitoes for considerable
schizogony was considered much earlier by periods of time and suggested that they might
Fairley (1949) and by Coatney et al (1950). also be able to do the same thing in the human
The second area in which the cyclic host. It is interesting that Corradetti (1966)
maturation of fixed tissue schizonts might be suggested that the late tissue forms described by
challenged is in the relationship between Bray (1957) might have been derived directly
declining immune levels and the eventual from sporozoites whose development had been
reappearance of parasitological and clinical delayed by some "unfavorable condition."
relapse. The original concept, as introduced by However, the same author apparently never
Shortt and Garnham (1948a), was that entertained the possibility that the same delayed
merozoites continually entering the blood from development could be responsible for relapses.
maturing tissue schizonts would be destroyed by There is recent experimental evidence to
the immune mechanisms of the host. Eventually, support the concept of a latent stage as an
when the level of immune activity was essential part of the mechanism of relapse in
sufficiently low, the parasites would be able to malaria. Warren et al (1970) have found that P.
multiply and thereby produce a relapse. Cooper cynomolgi sporozoites from mosquitoes exposed
et al (1947) were able to superinfect volunteers to X-irradiation immediately prior to dissection
with sporozoite-induced infections of the St. and inoculated into malaria-free monkeys
Elizabeth strain of P. vivax by inoculating whole produced infections which appeared to be
blood infected with the homologous strain normal, although profound damage was
during the long interval between primary attack observed in EE schizonts from the same
and initial relapse. It is therefore clear, that the animals. However, monkeys infected with
subject was susceptible to blood-stage parasites irradiated sporozoites demonstrated fewer
from an extraneous source--why not, then, from relapses than seen in the controls which had
tissue schizonts? It is difficult to understand how received comparable numbers of sporozoites.
the immune response in sporozoite-induced The authors concluded, that their findings were
infections would ever drop to the level the result of a random destruction of sporozoites
postulated by Shortt and Garnham (1948b) with with the concomitant elimination of specific,
the constant antigenic stimulus that would be predetermined relapses.
present with merozoites entering the blood Experiments, more specifically designed to
continually from exoerythrocytic schizonts. It is
 LIFE CYCLE AND THE PHENOMENON OF RELAPSE 37

demonstrate this point, were carried out by to be transmitted to new non-immune hosts. We
Warren et al (manuscript) in which a series of believe that the need for making gametocytes
monkeys were inoculated with varying numbers available in the peripheral blood of appropriate
of sporozoites of P. cynomolgi. Prepatent hosts is of such biological urgency that two
periods were only slightly delayed in animals mechanisms for such an activity have evolved
which received low numbers of sporozoites. and have been maintained by the primate
Infections followed the expected course in all plasmodia.
animals and no differences were noted until after We would surmise that a dormant or latent
treatment with chloroquine. Once relapses began tissue phase of the parasite was the first to
to occur, there was a clear-cut reduction in their evolve and was probably brought forward from
number directly related to the number of an ancient coccidian ancestor. Sufficient
sporozoites received. These authors concluded information is not available at the present time
that the results strongly challenged the concept to establish the exact developmental stage of the
of a continuing cycle of histiotrophic merozoites parasite which becomes dormant, but one must
in the liver being responsible for relapses in certainly suspect that either the sporozoite or an
malaria, since, by this theory, the number of intermediary stage, between the sporozoite and
relapses should not be influenced by the number the exoerythrocytic schizont, possesses the
of sporozoites in the original infecting inoculum; capacity to settle down and remain quiescent for
one sporozoite should be capable of initiating long periods of time. The extent of this period
the complete sequence. In Figure 5 we have would seem to be dependent upon the species
presented the hypothetical course of infection and even the strain of parasite in question.
for malarias with different potentials for relapse It is clearly evident that there are many
and/or recrudescence. lacunae in our knowledge of the relapse
The survival value of a long term relapse mechanism. Eventually these gaps will be filled
mechanism to a malaria parasite is undoubtedly in but until then, the complete picture of the
great and contributes significantly to its ability relapse phenomenon is denied to us.

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38 PRIMATE MALARIAS

FIGURE 5.— Hypothetical infections of malaria with different relapse potentials. Infections A-G. Relapse and recrudescence
Potential of hypothetical infections with Plasmodium cynomolgi.
LIFE CYCLE AND THE PHENOMENON OF RELAPSE 39
40 PRIMATE MALARIAS

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HUFF, C. G., 1947. Life cycle of malarial parasites. Ann. Rev. Plasmodium vivax. Brit. Med. Jour. 1 : 547.
Microbiol. 1 : 43-60. SHORTT, H. E., FAIRLEY, N. H., COVELL, G., SHUTE, P. G.,
HUFF, C. G., 1948. Exoerythrocytic stages of malarial parasites. and GARNHAM, P. C. C., 1949. The pre-erythrocytic
Am. J. Trop. Med. 28 : 527-531. stage of Plasmodium falciparum. A preliminary note.
HUFF, C. G., 1950. Pre-erythrocytic stages of simian and human Brit. Med. Jour. 2 : 1006-1008.
malarial parasites. (Letter to the Editor, Trop. Med. SHUTE, P. G., 1946. Latency and long-term relapses in benign
News). 7 : 22-23. tertian malaria. Trans. Roy. Soc. Trop. Med. & Hyg. 40 :
HUFF, C. G. and BLOOM, W., 1935. A malaria parasite infecting all 189-200.
blood and blood-forming cells of birds. J . Infect. Dis. 57 : THAYER, W. S., 1897. Lectures on malarial fevers. D. Appleton &
315-336. Co., New York. pp. 326.
HUFF, C. G., COULSTON, F., and CANTRELL, W., 1943. Malarial TIBURSKAYA, N. A., 1964. Classification of P. vivax strains into
cryptozoites. Science 97 : 286. groups by type of incubation. Med. Parazit., Moscow 33 :
HUFF, C. G. and COULSTON, F., 1944. The development of 204-216.
Plasmodium gallinaceum from sporozoite to erythrocytic TRAGER, W. and MOST, H., 1963. A long-delayed primary attack
trophozoite. J. Infect. Dis. 75 : 231-249. of ovale malaria. Am. J. Trop. Med. & Hyg. 12 : 837-839.
HUFF, C. G., COULSTON, F., LAIRD, R. L., and PORTER, R.J., VOLLER, A. and ROSSAN, R. N., 1969. Immunological studies
1947. Pre-erythrocytic development of Plasmodium with simian malaria. I. Antigenic variants of Plasmodium
lophurae in various hosts. J. Infect. Dis. 81 : 7-13. cynomolgi bastianellii. Trans. Roy. Soc. Trop. Med. &
HUFF, C. G. and COULSTON, F., 1948. Symposium on Hyg. 63 : 46-56.
exoerythrocytic forms of malaria parasites. II. A search WARREN, McW., POWERS, K., GARNHAM, P. C. C., and
for pre-erythrocytic stages of P. vivax and of P. SHIROISHI, T ., 1970. The influence of X-irradiation and
cynomolgi. J. Parasit. 34 : 264-274. dilution of sporozoites on relapse patterns of infections
JAMES, S. P., 1931. The use of plasmoquine in the prevention of with Plasmodium cynomolgi. (in press)
malaria infections. Proc. Roy. Acad. Amsterdam 34 : YORKE, W., 1925. Further observations on malaria made during
1424-1425. treatment of general paralysis. Trans. Roy. Soc. Trop.
JAMES, S. P. and TATE, P., 1937. New knowledge of the life-cycle Med. & Hyg. 19 : 108-122.
of malaria parasites. Nature 139 : 545-549. YORKE, W. and MACFIE, J. W. S., 1924. Observations on malaria
JEFFERY, G. M., 1951. Observations on a gametocyteless strain of made during treatment of general paralysis. Trans. Roy.
Plasmodium falciparum. J. Nat. Mal. Soc. 10 : 337-344. Soc. Trop. Med. & Hyg. 18 : 13-33.
JEFFERY, G. M., WOLCOTT, G. B., YOUNG, M.D., and YOUNG, M. D., 1944. Studies on the periodicity of induced
WILLIAMS, D. JR., 1952. Exo-erythrocytic stages of Plasmodium vivax. J. Nat. Mal. Soc. 3 : 237-247.
Plasmodium falciparum. Am. J. Trop. Med. & Hyg. 1 :
917-926. (NS) = Not seen.
JEFFERY, G. M., YOUNG, M. D., and WILCOX, A., 1954. The
Donaldson strain of malaria. 1. History and characteristics
of the infection in man. Am. J. Trop. Med. & Hyg. 3 :
628-637.
LAVERAN, A., 1880. Un nouveau parasite trouve le sang des
malades atteints de fievre origine parasitaire des accidents
de l'impaludisme. Bull. et Memoires Soc. Med. Hopitaux
de Paris. (2 ser.) 17 : 158-164.
MACCALLUM, W. G., 1897. On the flagellated form of the malarial
parasite. Lancet 2 : 1240.
MANSON, P. T., 1901. Experimental malaria: Recurrence after nine
months. Brit. Med. Jour .2 : 77.
PRINGLE, G., 1965. A count of the sporozoites in an oocyst of
Plasmodium falciparum. Trans. Roy. Soc. Trop. Med. &
Hyg. 59 : 289-290.
 SECTION 2
Vivax-Type Parasites
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 5
Plasmodium vivax (Grassi and Feletti, 1890)
Grassi and Feletti, 1890, gave the name vivax for
SYNONYMS: Haemamoeba the human tertian parasite under the genus
malariae Feletti and Grassi, 1890, partim; Haemamoeba. In 1885, Marchiafava and Celli
Haemamoeba vivax Grassi and Feletti, 1890; had proposed plasmodium as the genus name for
Plasmodium malariae tertianae Celli and the malaria parasites with the result that the
Sanfelice, 1891; Plasmodium malariae tertianae combination Plasmodium vivax (Grassi and
Kruse, 1892; Haemamoeba laverani var. Feletti, 1890) came into general use but lacked
tertiana Labbe, 1894 (?) ; Haemosporidium official status until the International Commission
tertianae Lewkowicz, 1897; Haemamoeba on Zoological Nomenclature, after much soul
malariae tertianae Laveran, 1901; Plasmodium searching, made the historic decision, opinion
camarense Ziemann, 1915. 283, to validate the names of the human malaria
The first person to see and describe the true parasites in common use (see Hemming, 1954).
malaria parasite of man was the French army Vivax malaria has a worldwide distribution
surgeon, Louis Alphonse Laveran. There is little but makes its greatest inroads in temperate
doubt that during his investigations of 1880 and climates. The disease is more or less confined to
1881 he saw the human tertian parasite in the the lowlands, coastal areas, marshes, lake
blood of patients at Contantine, Algeria. He margins, and reclaimed sea beds. There are no
made no attempt to attach names other than known strains of the parasite which can
calling the organisms Oscillaria malariae complete their sporogonous development at
referring, undoubtedly, to the fine hair-like temperatures below 15° C (59°F) and
projections he saw develop from a pigmented consequently vivax is stopped north and south of
spherical body in fresh blood from a patient with the equator, by summer isotherms of 15° or
malaria. 16° C.
The parasite of tertian malaria was well In the tropics, P. vivax may predominate
recognized by Golgi (1886, 1889). In the first over P. falciparum, as in the lower Amazon, but
paper, he mentioned that a tertian parasite must the absolute prevalence in any area may not be
have a developmental cycle different from known because of the rapid build-up of
quartan. He then proceeded to describe the immunity to all strains of the parasite, in areas of
course of the disease during four tertian attacks high transmission, with consequent suppression
(giving Prof. Reva credit for allowing him to use of parasitemia. This situation was well
his patient), including the morphology of the demonstrated by Missoroli (1932). The highest
parasites in relation to the paroxysm. In the more incidence under present conditions is probably
extensive paper of 1889, he re-affirmed his in Asia where it extends as a wide belt across the
earlier findings and presented figures of the entire continent.
development of the parasite in the red cell with In 1949, Brumpt mentioned what he called
amazing accuracy. Even though he clearly the "benign tertian mystery" in writing about the
separated tertian from quartan malaria, he did situation in Liberia, Gabon, Lagos, and
not name the parasite of either one. In an Stanleyville where, in the presence of efficient
addendum to a paper on the malaria of birds, vectors of P. vivax, no vivax malaria occurs.

43
44 PRIMATE MALARIAS

Garnham (1966) enlarged upon this by including American Negroes who acquired vivax malaria
"most of West Africa in the belt between the in Korea and in whom the natural history and
Congo and Mauritania." The explanation may be clinical attacks appeared no different than the
that the indigenous population is highly same infections in Caucasians (Hankey et al,
refractory to this parasite. This phenomenon was 1953). It is interesting that on the same day the
ably investigated by Young et al (1955) who above episode was enacted at Fort Benning, the
studied many different strains of P. vivax during late Dr. Alf Alving had the exact same
some 20 years, and found that only 23 percent of experience at Fort Dix, N.J.
American Negroes came down with developed Beginning in 1900, many investigators have
infections as against 96 percent of Caucasians; studied different strains of P. vivax and among
even when massive numbers of parasites were these, the ones which received the most attention
introduced, the susceptibility rate remained the were the Madagascar strain (James, 1931 and
same. James et al, 1936), the Dutch strain (Schüffner
The rule of thumb is that pronounced et al, 1929), the McCoy strain (Boyd, 1940;
resistance to P. vivax is confined to the true Boyd and Kitchen, 1944), and the New Guinea
Negro but like many such rules, there are strain (Fairley et al, 1945). In our own work, we
exceptions, as the senior author learned when he have undertaken studies on many different
encountered a very sick true Negro in the strains from various parts of the world, but have
malaria ward at Fort Benning, Ga. in 1951. The concentrated our efforts on the St. Elizabeth (see
record showed that the patient was infected with Coatney and Young, 1941; Coatney and Cooper,
P. vivax acquired in Korea. This was questioned 1948; Coatney et al, 1950a), and the Chesson
with some impatience; whereupon, a fresh smear strain (Ehrman et al, 1945; Coatney and Cooper,
was made, stained, and examined by the senior 1948). The last two, along with certain others,
author. It was P. vivax! This was one of many will be discussed more fully later in this chapter.
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46 PRIMATE MALARIAS
 PLASMODIUM VIVAX 47

Cycle in the Blood pigment granules come together to form larger

chunks and then they coalesce into a single
PLATE I yellowish-brown lump. The maturing schizont
occupies almost the whole red cell with the
The young merozoite enters the red blood stippling forced into a rim-area. The host cell is
cell, generally a reticulocyte, as first reported by enlarged and its cytoplasm appears depleted (Figs.
Craik (1920) and later verified by Kitchen (1938) 19-27).
and others, where it appears with a deep red The young gametocytes are easily separated
nucleus and a whisp of cytoplasm (Fig. 2). As the from the asexual parasites because they are
parasite grows, a loop of cytoplasm forms compact, lack a vacuole, are not amoeboid, and
enclosing a vacuole, with the circular nucleus at have a large nucleus with, sometimes, the
the anchor point (Fig. 3). Further growth produces suggestion of a halo. The enlarged host cell shows
a larger parasite with a distinct vacuole and pronounced Schüffner's stippling. The immature
sometimes with an accessory chromatin dot. macrogametocyte stains a deep blue. Dark
Following this development, the red cell increases pigment granules are scattered in the cytoplasm
in size and displays Schüffner’s stippling. The (Fig. 28). The mature macrogametocyte stains a
cytoplasm of the parasite is increased in amount, lighter blue than the developing form and
becomes decidedly amoeboid, and exhibits very occupies most of the host cell. Its nucleus is
small granules of light brown pigment. Multiple generally eccentric and may show a darker portion
invasion of the host red cell is not uncommon inside the main body. Dark pigment grains are
(Figs. 4-10). The remainder of the development of scattered in the cytoplasm. The host cell is
the trophozoite consists of concentrating the enlarged, its cytoplasm appears pale and depleted
cytoplasm with loss of the vacuole, the nucleus (Fig. 29). The young microgametocyte resembles
becomes larger and may assume grotesque shapes. the immature macrogametocyte but as it
Where only hours before, the trophozoite approaches maturity, the staining of the cytoplasm
occupied a large part of the host cell, it now is toward bluish-gray, the nucleus is larger,
becomes compact, pigment granules become occupying about half or more of the parasite, and
prominent, and stippling more intense (Figs. 11- takes a reddish-purple stain. The dark pigment is
18). The first stage of schizogony results in 2 confined to the area of the cytoplasm, leaving the
nuclei and, then, in rather rapid succession, other nucleus free. The stippling of the host cell is
divisions occur, to deliver up to 24 nuclei; the forced toward its periphery (Fig. 30).
usual number is 16, although certain strains have The asexual cycle takes 48 hours.
18 or more consistently. With the process of
nuclear division other changes occur, too. The

PLATE I. – Plasmodium vivax

Fig. 1. Normal red cell. Figs. 22, 23. Developing schizonts.
Fig. 2-5. Young trophozoites. Figs. 24-27. Nearly mature and mature schizonts.
Fig. 6-16. Growing trophozoites. Figs. 28-29. Nearly mature and mature
Figs. 17, 18. Mature trophozoites. macrogametocytes.
Figs. 19-21. Early schizonts. Fig.30. Mature microgametocyte.
48 PRIMATE MALARIAS
 PLASMODIUM VIVAX 49

Sporogonic Cycle mosquitoes incubated at a temperature of 25° C

(Table 1).
PLATE II In A. b. balabacensis, the oocysts, at day 4,
ranged from 9 to 15 µ in diameter with a mean of
Bano (1959) observed two small dot-like and 12 µ. The oocysts continued to grow so that by
two rod-like chromosomes in 49-hour oocysts of day 11, the size ranged from 18 to 59 µ with a
P. vivax in Anopheles stephensi mosquitoes. The mean of 46 µ. Sporozoites were present in the
haploid number of chromosomes was determined salivary glands by day 12. In A. freeborni, the
to be two. The size of oocysts at this time ranged mean oocyst diameters were generally greater
from 11 to 14 µ in diameter. Although many than seen in the A. b. balabacensis; at day 11, the
workers have observed the oocysts of P. vivax, the size of the oocysts ranged from 25 to 67 µ with a
studies of Shute and Maryon (1952) more nearly mean of 46 µ; sporozoites were present in the
approximate our observations. These workers salivary glands. The oocyst diameters in the other
observed the development of oocysts of this species of mosquitoes fell within the range of
parasite, at a temperature of 25°C, in A. these 2 mosquitoes. Sporozoites were present in
atroparvus mosquitoes. They reported that the 50 the salivary glands of A. maculatus and A.
to 100 greenish-brown pigment granules did not stephensi by day 11 and in the salivary glands of
form a pattern in the very young oocysts. By days A. quadrimaculatus by day 12.
4 and 5, however, there was a tendency toward In this, and in subsequent chapters, the
chain formation, often in 3 lines. By day 6, the growth of the oocysts is compared with those of
pigment was practically obscured and by day 7, P. cynomolgi. In each instance, the comparison is
only a few grains were observable. The oocysts made from measurements made on the same
were 10 to 46 µ in diameter. From day 3 to 6, the species of mosquito. In this case, the comparison
daily increase in diameter was about 7 µ; between is between the growth curves of P. vivax and P.
the 6th and 7th day, however, growth became cynomolgi in A. freeborni mosquitoes (Fig. 6).
more rapid. Sporogony was completed in 9 days. Plasmodium cynomolgi has a larger mean oocyst
In our studies, observations were made on diameter than P. vivax and much larger maximum
the oocyst development of the Chesson strain of oocyst diameter. Both of the parasites completed
P. vivax, in A. b. balabacensis A. freeborni, A. their development, as measured by the presence of
maculatus, A. stephensi, and A. quadrimaculatus sporozoites in the salivary glands, by day 11.

PLATE II
FIGURES 1 – 13. – Developing oocysts and sporozoites of Plasmodium vivax in Anopheles maculates, A. freeborni, A.
quadrimaculatus, and A. b. balabacensis mosquitoes. X 580.
Fig. 1. 4-day oocysts showing peripheral pigment. Fig. 8. 10-day oocyst.
Fig. 2. 5-day oocyst Fig. 9. 10-day oocyst showing early formation of
Fig. 3. 6-day oocysts. sporoblasts.
Fig. 4. 7-day oocyst showing pigment and small vacuoles. Fig. 10. 11-day oocysts showing prominent vacuolation.
Fig. 5. 8-day oocyst. Fig. 11. 12-day oocyst showing differentiation.
Fig. 6. 9-day oocyst. Fig. 12. Fully differentiated 12-day oocyst.
Fig. 7. 9-day oocysts showing concentration of vacuoles. Fig. 13. Sporozoites present near salivary gland tissue
12 days after feeding.
 50 PRIMATE MALARIAS

FIGURE 6.—A comparison of the mean oocyst diameter curve and ranges in oocyst diameters of Plasmodium vivax and P.
cynomolgi in Anopheles freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

TABLE 1.-- Oocyst diameters of Plasmodium vivax in Anopheles b. balabacensis, A. freeborni, A. maculatus, A. stephensi, and A.
quadrimaculatus mosquitoes.

A. b. balabacensis A. freeborni A. maculatus A. stephensi A. quadrimaculatus

Days after
Infection Mean Mean Mean Mean Mean
No. Range No. Range No. Range No. Range No. Range
* * * * *
4 100 9-15 12 100 11-15 13 100 11-17 13 56 11-15 13 102 11-14 12
5 100 12-19 16 76 12-20 16 53 11-19 15 100 12-18 14 36 12-18 15
6 100 12-26 22 92 15-28 23 17 18-26 22 100 14-27 22 64 13-25 22
7 100 13-32 25 100 17-39 30 100 15-35 27 100 18-42 31 100 18-31 25
8 100 18-40 32 100 18-50 36 100 ‡ 34 100 24-51 40 100 21-48 36
9 100 18-42 31 100 27-60 48 100 ‡ 40 100 30-59 14 100 18-52 41
10 100 18-61 45 100 21-60 50 100 ‡ 47 100 31-67 53 100 24-59 45
11 100 18-59 46† 95 25-67 46†** 100 26-66 47†** 100 24-65 48†** 100 21-59 49†
12 †** †** 100 27-59 47†**

Totals 800 9-61 763 11-67 670 11-66 756 11-67 802 11-59

* Measurements expressed in microns.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
‡ Ranges in oocyst diameters not available.
 PLASMODIUM VIVAX 51

FIGURE 7—A comparison of the mean oocyst diameter curves of Plasmodium cynomolgi, P. vivax, P. falciparum, and P. ovale
in Anopheles freeborni mosquitoes.

A comparison of the oocyst growth curves of and Garnham (1948). These tissue stages were
the 3 human tertian malarias (P. vivax, P. found in a 7-day liver biopsy taken from a human
falciparum, and P. ovale) with P. cynomolgi (Fig. volunteer upon whom approximately 1,728
7) shows several distinct differences. Although P. anopheline mosquitoes, infected with P. vivax,
vivax has a smaller mean diameter than P. were allowed to bite on 2 successive days. In
cynomolgi, the shape of its growth curve is addition, 200 pairs of salivary glands were
strikingly similar to that of P. cynomolgi. In dissected out from the same mosquitoes, and
contrast, P. falciparum and P. ovale have more of inoculated intravenously. Schizonts, considered to
a straight line growth curve. Whereas sporozoites be 6- and 7-day forms, were described from the
are present in the salivary glands of mosquitoes liver tissue taken at biopsy. The 6-day forms were
infected with P. vivax and P. cynomolgi by day ovoid masses similar to those seen in P.
11, P. falciparum and P. ovale require 12 and 14 cynomolgi except that they were larger, about 42
days, respectively for their development. µ in their greatest dimension. The various shapes,
The sporozoites are narrow, elongate bodies, the vacuolation, the absence of tissue reaction,
either straight or slightly curved, with one end and the staining characteristics of the cytoplasm
more blunted than the other. The length of the and chromatin were also similar to what they had
living sporozoite is 14 µ and in dried preparations, first observed in P. cynomolgi. The 7-day stage,
is 1 to 2 µ less (Garnham, 1966). Using electron reported by these authors, consisted of a single,
microscopy, Garnham et al (1963) gave detailed fully or nearly mature form, since merozoites
descriptions of the structures present in this form were seen escaping from it.
of the parasite which displays a complicated The youngest P. vivax tissue stage was
morphology. described by Rodhain in 1956 in liver biopsy
We have readily transmitted the infection to material from a chimpanzee which had been
man via the bites of A. freeborni, A. inoculated with sporozoites of vivax malaria 7 and
quadrimaculatus, A. b. balabacensis, and A. 4 days earlier. These 4-day forms (possibly 7-day
maculatus mosquitoes. tissue stages) ranged in size from 24 µ in diameter
up to 47.7 by 35.2 µ. The nuclei, or chromatin
masses, measured slightly over 2 µ and were
Cycle in the Tissue rather sparsely distributed. Rodhain considered
The first tissue stages of a human malaria these forms to be similar to those described for
parasite, P. vivax, were demonstrated by Shortt
52 PRIMATE MALARIAS

man by Shortt and Garnham (1948). diminution in the number of tissue stages per
Bray (1957) described 8- and 15-day tissue measure of liver as the infection continues. Bray
stages seen in liver biopsy material from a demonstrated this in his study of P. vivax in
chimpanzee inoculated with sporozoites of P. chimpanzees. With the inoculum used by him,
vivax from A. gambiae mosquitoes. The 8-day P. Bray estimated a decrease from 20,000 primary
vivax schizonts were similar to, but larger than, exoerythrocytic forms (8-day biopsy) per mm3 of
the 7-day forms demonstrated by Shortt and liver to 500 secondary exoerythrocytic forms in
Garnham in 1948. He described two distinct the 15-day biopsy material.
stages of immature schizonts. The EE bodies There can be no question that the liver cycle
averaged 52 by 44 µ and were almost always oval of vivax malaria persists for upwards of several
in shape. The immature stage contained 1 to 2 years since many strains of vivax continue to
large vacuoles, cytoplasm was abundant and, at exhibit true relapses through a minimum of 2 to 3
times, collected into darker staining aggregates. years. Ciuca et al (1955) observed relapses up to 8
Occasionally, many small vacuoles were present, years. It is doubtful that these latter were true
arranged around most of the periphery. Nuclei relapses. Professor Gh. Lupascu, one of the late
were relatively sparse. No clefts were observed. Dr. Ciuca's colleagues, has recently written one of
Bray also described premature and mature us (PGC) that there was a good possibility of
schizonts. In the premature stage, nuclei had reinfection in their patients. Many of them had
completed their final division but with little or no been released from the hospital for varying
change in their shape or size; the vacuoles had intervals and could easily have been reexposed to
disappeared. The nuclei are elongate or form bars infection because malaria was at that time
or loops; the cytoplasm breaks up and condenses endemic to the area.
upon the nuclei. This process of merozoite Before we leave the discussion of the P.
formation is completed in a very short time. The vivax cycle in the fixed tissue, mention should be
mature schizont is ovoid without any appreciable made of Fairley's monumental work (1947) on the
increase in size. They are packed with spherical observations of various phases of the vivax life
merozoites which measure 1 to 1.4 µ in diameter. cycle. In this work, he subinoculated large
When the mature schizont ruptures, the volumes of blood (200 ml.) from individuals
merozoites are released. The size of the free heavily exposed to infection with vivax malaria
merozoite is 0.8 to 1.2 µ. by mosquito bites. He observed that
Bray observed no patterns of nuclear subinoculation during the first 30 minutes after
arrangement or septal formation of the cytoplasm exposure to infection resulted in eventual patent
in any of the 8-day tissue stages of vivax malaria. infection in the subinoculee. After 30 minutes and
There was no tissue reaction surrounding the EE through 7 days, subinoculations were non-
bodies and no hypertrophic changes in the host- infective. On the 8th day subinoculation was
cell (hepatic) nuclei. infective, indicating that the tissue schizonts had
The tissue stages found in chimpanzee liver liberated merozoites into the circulating blood on
by Bray 15 days after exposure to infection day 8.
appeared similar to the 8-day schizonts. There Course of Infection
were no features which allowed for distinguishing
these EE bodies as second generation forms.
Fairley's experiments (1947) showed that the
Secondary exoerythrocytic tissue stages were also
earliest prepatent period (i.e., the time from
found and described from a chimpanzee 9 months
infection, day 0, to the time parasites become
after inoculation with vivax sporozoites by
microscopically detectable in the circulating
Rodhain (1956a). Three of the 4 late tissue stages
blood) could be as early as 8 days; this has been
were elongate, irregular, or regular shaped ovals
observed, but only rarely. In the last 10 years, we
measuring 81, 61, and 49 µ in greatest dimension
have encountered only one prepatent period of 8
while the fourth form was lobulated.
days (Coatney et al, 1963).
The concensus is that there is a decided
 PLASMODIUM VIVAX 53

In addition, Boyd and Kitchen (1937) and Ciuca and suggested, that strains of this type would have
et al (1937) reported one 8-day prepatent period greatly enhanced chances for survival in
each in a large series of patients whose infections temperate zones.
were induced by bites of infected mosquitoes. Yorke (1925) observed relapses in patients 6
Putnam et al (1947) reported a 7-day prepatent to 13 months after exposure to infection. James
period. However, this would be an 8-day period (1931) and James et al (1936) observed late
according to the manner in which all other relapses with the Madagascar strain of vivax
investigators calculate the prepatent period. malaria. Schüffner et al (1929) observed
Kitchen (1949) reported 5 prepatent periods of 8 prolonged "latency" in the form of delayed
days in work with 9 different strains of vivax. primary attacks in 8 of 8 patients exposed to
Generally, the duration of the prepatent infection with the Netherlands strain of vivax
period reported for many strains of Plasmodium malaria. Boyd and Kitchen (1944) and Shannon et
vivax (Chesson, St. Elizabeth, Madagascar, al (1948) observed the bimodal activity pattern
McCoy, New Guinea, Roumanian, South (late relapses) with experimentally induced
Vietnam, Scanlon, West Pakistan, and infections with the McCoy (U.S.) strain of vivax
Venezuelan) have ranged from 8 to 27 days, with malaria. The Korean strain also exhibited the
medians or means ranging from 10.5 to 19 days bimodal pattern of clinical activity and a period of
(Boyd and Kitchen, 1937; Ciuca et al, 1937; long-term latency (Hankey et al, 1953).
Fairley et al, 1947; Coatney et al, 1950; Coatney Proof that such a consistently long period of
et al, 1950a; Contacos and Coatney, 1963; time obtained between primary and relapse
Winckel, 1955). activity is common was furnished by studies of
Other strains of vivax malaria exhibit delayed Coatney et al (1950) in volunteers who showed
prepatent periods, sometimes called protracted delayed relapse activity approximately 9 months
incubation periods, but probably more accurately after exposure to infection. They showed also that
described as delayed primaries. the time characteristic was independent of the
The St. Elizabeth strain of vivax malaria season by exposing volunteers to infection during
(Coatney et al, 1950) is usually characterized by a 9 months of the year. Relapses appeared only as a
short prepatent period of 11 to 18 days after function of time with median periods of latency
infection. Only 3 volunteers out of 123 exhibited between 194 and 300 days.
delayed primary attacks at 298 to 319 days after Coatney and Cooper (1948) stated that
exposure to infection. Coatney and Cooper (1948) morbidity statistics during World War II clearly
reviewed and described the characteristic of showed that the vivax malarias from the Solomon
bimodal activity (parasitic and/or clinical) of a Islands, New Guinea, and other areas of the
large number of vivax strains. They related the Southwest Pacific did not exhibit consistent
transmission studies of Sir Patrick Manson (1900) patterns of prolonged latency in their relapse
who exposed his son, P. Thurburn Manson, to mechanisms. Fairley (1945), working with vivax
infection with vivax malaria by bites of strains from New Guinea, reported no patterns of
mosquitoes sent from Italy. A primary attack of delayed relapse.
malaria was experienced after 2 weeks and the Coatney and Cooper (1948) summarized
attack was treated with quinine. Some 9 months their studies with 2 strains of vivax malaria, used
later, a relapse of vivax malaria was experienced in drug evaluation studies in prisoner volunteers,
by Sir Patrick's son. There then followed many the St. Elizabeth (U.S.A.) and the Chesson (New
reports relating to 8- to 9-month intervals between Guinea-South Pacific). The activity pattern of the
primary and relapse activity, fitting more or less St. Elizabeth strain is consistent with a relatively
into a pattern for many strains of vivax in many short prepatent period, between 7 and 14 days.
countries. Hackett (1937) pointed out how this This is followed by a period of many months
characteristic provided an explanation for spring when fixed-tissue stages remain quiescent, and
malaria and for the overwintering of the parasite, then, by a late period of activity, with repeated
54 PRIMATE MALARIAS

spaced invasions of red blood cells which may as 29 days; the latest was at day 609. These results
continue for upwards of 2 years, sometimes, would tend to indicate that long term relapses are
longer. not confined to temperate zone strains.
The Chesson strain pattern of activity is On the basis of life-pattern, Nikolaiev (1949)
radically different from that of the St. Elizabeth differentiated 2 subspecies of vivax malaria. One
strain. The former is characterized by fairly was characterized by short incubation periods of 7
regular reinvasions of the blood stream, after the to 23 days (from the southern part of U.S.S.R.),
original attack, from fixed-tissue stages, gradually which he designated P. vivax vivax. The other
becoming more widely spaced. In heavy subspecies (from northern and central areas of
infections, relapses continue for upwards of 18 U.S.S.R.), characterized by long incubation
months, occasionally, longer. periods (from 253 to 381 days), he called P. vivax
The authors in commenting on the hibernans. These designations failed to receive
differences between the Chesson and the St. wide acceptance. Another Russian study in the
Elizabeth strains stated that the differences in same vein was that of Tiburskaya (1961) who
relapse activity between the 2 strains was in described a strain isolated in 1953 from a patient
keeping with the presumed tropical origin of the who had never left Moscow. The author passed
Chesson and the temperate zone origin of the St. the strain for 5 years via Anopheles labranchiae
Elizabeth strain. atroparvus mosquitoes and in 103 infections the
Winckel (1955) presented material which incubation periods were short (9 to 20 days), but
appears to be somewhat paradoxical; namely, that in 13 they were extended (216 to 327 days). These
the Netherlands strain under natural conditions data are somewhat reminiscent of our studies with
produced delayed primary attacks, usually at St. Elizabeth vivax.
about 8 months after infection. When infections Most of the strains of vivax malaria studied
were induced experimentally, however, the to date have short prepatent periods. The greatest
majority (51 out of 87) had early primary attacks or most significant differences appear to lie in
(less than 21 days). However, on the basis of their relapse patterns; namely, a very short latent
reports by Schüffner et al (1929) (obviously not period or a very long latent period, between the
their own data), Winckel stated that there are 3, primary attack and the first relapse.
not 2, types of vivax strains which can be We have purposefully omitted details of
separated by virtue of their life pattern; the clinical human malaria because this work is
tropical Chesson and Madagascar strains which concerned with the biology and parasitology of
have early primary attacks with frequent and early the primate malarias and, too, such information is
relapses; the temperate zone St. Elizabeth strain well covered in numerous textbooks. We have
which has early primary attacks but late relapses; included, however, the more general and pertinent
and the Netherlands temperate zone strain which aspects of human malaria.
displays delayed primary attacks followed by Vivax malaria infections are considered to
frequent relapses. have relatively benign characteristics.
More recent studies tend to suggest that this Observations of large numbers of vivax
classification of tropical versus temperate zone infections, allowed to continue until terminated
malaria is not all-inclusive. For example, we have spontaneously, suggest that instances of death due
studied a Central American (Panama), and to vivax malaria, in otherwise healthy adults, must
certainly "tropical", strain of vivax and observed indeed be very rare (Kitchen, 1949).
latent periods of approximately 5 months after Whorton et al (1947) reported that prodromal
exposure to infection or 4 months after treatment symptoms before the primary attack in cases of
of the primary attack. In similar studies involving Chesson vivax were almost never observed.
a Venezuelan strain of P. vivax, 5 of 6 volunteers Kitchen (1949) stated that prodromal symptoms
who experienced relapse activity had their first are usually manifest in persons most susceptible
relapse 110 to 335 days after exposure to to malaria. In our experience, with several strains
infection. Only one of the 6 had a relapse as early of vivax malaria in non-immune volunteers,
 PLASMODIUM VIVAX 55

prodromal symptoms have not been commonly intermittent fever has been tertian as frequently as
observed; but, when present, consisted mainly of it has been quotidian. The characteristic of the
headache and sometimes generalized malaise. fever pattern is a reflection of: 1) a single highly
In non-immune individuals, the onset of the synchronous brood of parasites which produces
primary attack is usually characterized by a slight, sharp fever spikes, 2) an asynchronous single
rigorless paroxysm. Kitchen (1949) stated that the brood which produces plateau-like fever, and 3)
course, or sequence of events, of the uninterrupted multiple broods which produce daily paroxysms.
attack of vivax is characterized by remittent fever, If the infection is allowed to run, it is not unusual
followed by intermittent fever, and then to for multiple broods to get-in-step resulting in a
remission or spontaneous termination of the tertian fever pattern; likewise, tertian patterns,
primary attack. During the period of remittent sometimes, become quotidian for a time, only to
fever, which lasts generally from 2 to 5 days, the revert to tertian again.
continuous fever curve is characterized by In a series of mosquito-induced infections
quotidian fever spikes. These daily fever spikes with P. vivax, the mean duration of the primary
exhibit progressively higher values reaching a attack was observed to be 19.3 days (Boyd et al,
maximum shortly after the appearance of 1936a). Paroxysms accompanied by chills are
intermittent fever. James (1926) reported that indicative of a more severe paroxysm and usually
fever at the onset of primary attacks of Indian and attended by a greater degree of fever than those
Madagascar strains may be continuous or without chills. The mean maximum temperature
remittent over a period ranging from 1 to 3 days for 654 paroxysms, with chills, was 104.2° F and
and that, if the early fever was intermittent, it was for 346 paroxysms, without chills, was 102.5° F
usually quotidian, rarely tertian. Kitchen (1949) (Kitchen, 1949). Maximum paroxysmal
emphasizes that this intermittent fever in the temperatures are usually attained during the early
completely susceptible person is also quotidian. part of the second week.
During the earliest intermittent paroxysms, certain Kitchen and Putnam (1946), in a large series,
characteristics are conspicuous: 1) the parasite noted that chills introduced up to 71 percent of all
density is mounting; 2) the paroxysms exhibit paroxysms. Chills were observed to last an
gradually increasing peak temperatures; 3) the average of 50 to 55 minutes. They stated that the
duration of the paroxysm is protracted; and 4) a temperature at the beginning of the chill was, as a
rigor does not introduce the first few intermittent rule, not in the febrile range; i.e., under 100° F
paroxysms. and that, as a rule, fever appeared shortly after the
Whorton et al (1947) reported that in patients onset of the chill, with an average increase of over
infected with Chesson strain vivax, 80 percent had 4° F (maximum increase was 7° F).
remittent fever at the onset. Of the 20 percent who Boyd (1941) stated that chills are not
had intermittent fever at the beginning, 84 percent observed at the onset of primary vivax infections
were quotidian and only 16 percent exhibited and may not be evident for one or even 2 weeks.
tertian fever spikes. The duration of remittent He reported that chills did occur in their patients
fever in that strain varied from 24 to 184 hours. during the period of remittent fever but were
They suggested that remittent fever, at the onset infrequent (only 26 percent of 144 patients). In a
of the primary attack was probably related directly total 158 patients, only 46 percent had chills
to irregular segmentation of the parasite. The during any stage of the primary attack, with the
more typical intermittent febrile paroxysms are first chill being observed as early as the 8th day.
directly related to the maturation and However, the incidence of chills might have been
segmentation of one brood of parasites. During higher had primary attacks been allowed to
the initial remittent fever, segmentation occurs at continue.
irregular intervals (James, 1926; Boyd, 1941). Coatney and Young (1942) reported on
In our experience, with wholly susceptible detailed studies of 338 paroxysms in 21 infections
volunteers infected with Chesson vivax, the
56 PRIMATE MALARIAS

with the St. Elizabeth strain of vivax malaria in the asexual parasites and that the peak of
Caucasian patients. They observed chills in only segmentation precedes the fever peak. It follows
201 (59 percent) of the paroxysms. They then, that the time between fever peaks
suggested that the absence of chills in as many as (paroxysms) is a measure of the length of the
41 percent of all paroxysms indicated that the asexual cycle which in a one-brooded infection of
term "chills and fever" does not adequately vivax is said to be 48 hours. However, when
describe a malarial paroxysm. They found that the precise measurements of periodicity were carried
average temperature at the onset of each chill was out by Young (1944), it was discovered that the
100.6° F. In addition, the average duration of the length of the paroxysmal interval for the St.
chill was 39 minutes; the average temperature rise Elizabeth strain was 43 hours, 25 minutes; with a
was 2.3° F; the average time from onset of fever New Hebrides strain the interval was 45 hours, 46
(100° F) to peak fever was 3 hours, 52 minutes; minutes; and with a Baltimore strain the interval
and the average duration of temperature 100° F was 41 hours, 31 minutes. Also, according to
and above, was 10 hours, 10 minutes. They Young (loc. cit.) the fever charts published by
observed that the average rate of fever rise was Marchiafava and Bignami (1894) showed
3.3 times faster during the chill (1° F in 17 intervals of less than 48 hours. Kitchen (1949)
minutes) than during any other period of fever rise was concerned with the total paroxysm-picture in
(1° F in 56 minutes). In the chill accompanied contrast to Young's precise measurements and
paroxysms, fever peaks averaged 0.7° F higher showed that only 28.9 percent were isochronous,
and duration of fever averaged 2 hours shorter 31.6 percent occurred more than 48 hours after the
than paroxysms without chills. It is generally preceding paroxysm, and that 39.6 percent
thought that during this period, the patient is cold occurred less than 48 hours after the preceding
because he is shaking; but in truth, he is getting paroxysm. In his experience, paroxysms less than
hotter all the time as can be seen in Figure 8. It is 48 hours apart were more conspicuous during the
well recognized that the rise in a patient's early primary attack.
temperature is associated with the maturation of

FIGURE 8.—The temperature curve in relation to the chill in 201 paroxysms in Plasmodium vivax infections (after Coatney
and Young, 1942).
 PLASMODIUM VIVAX 57

Whorton et al (1947) reported the mean enlargement (Kitchen, 1949).

duration of fever over 103° F rectally in 235 Whorton et al (1947) found the subjective
paroxysms of Chesson vivax was 7.7 hours (only symptoms of Chesson vivax to be similar to those
the first 3 paroxysms of the primary attack were described for other strains. During the initial stage
measured). The maximum fever observed by them of remittent fever, malaise was almost universal.
was 108.2° F rectally which occurred on the 10th The other complaints were similar to those
day of a primary attack. mentioned above. Headache was strikingly
Fever in the experiences with vivax malaria frequent, often very severe and persistent. They
reported by Kitchen (1949) frequently was found observed that the spleen can become palpable as
to exceed 106° F; temperatures of 107° F were early as the second day of illness. In most
uncommon. Kitchen (1949) reported an instance instances, the maximum parasite density is
of a temperature recording of 107.6° F in a patient attained between the 7th and 14th days and
who reacted to this high temperature by maximum parasitemia rarely exceeds 50,000
convulsion--a rare occurrence. parasites per mm3 and usually remains below
As stated earlier, vivax infections are 25,000 per mm3. Kitchen (1949) reported one
generally considered to have benign vivax infection with a maximum parasite count of
characteristics. In some patients, a pronounced 96,000 per mm3 which was not attended by any
exaggeration of one or more of the usual signs alarming symptoms.
and/or symptoms may be evident and this Figure 9 shows the minimum and maximum
phenomenon is probably related more to the parasitemia curves for 20 infections with the St.
variability of the host rather than to the parasite. Elizabeth strain of P. vivax in our Caucasian
Symptomatology generally includes headache, patients. In addition, there are median parasitemia
anorexia, backache, nausea with or without curves for blood-induced and sporozoite-induced
vomiting, myalgia, abdominal pain, and infections. It can be seen that the median peak
generalized malaise. Hepatomegaly and parasitemia for blood- and sporozoite-induced
splenomegaly (up to 7 cm. below the left costal infections was reached on day 9 and 10 with
margin) with or without tenderness are not maximum median counts of 8,365 and 6,775,
uncommon during the primary attack. Rarely, the respectively. After attaining peak parasitemia, the
spleen may extend into either the lower left or median curves gradually decreased so that by days
right quadrant. Tenderness is quite variable and 20, 30, 40, and 60, the median counts for the
not always proportional to the degree of sporozoite-induced infections were approximately

FIGURE 9.—Minimum and maximum parasitemias and median parasitemias curves for blood- and sporozoite-induced
infections with St. Elizabeth strain Plasmodium vivax in Caucasian patients.
58 PRIMATE MALARIAS

3,000, 2,000, 1,000, and 100 parasites per mm3, An unexplained racial insusceptibility or
respectively. This difference is evident in the resistance to infection with many strains of vivax
figure at approximately 50 days. The parasitemia malaria has been repeatedly reported for the
in blood-induced infections similarly decreased Negro. Mayne (1932) first reported that American
except that at days 40 and 60 the median counts Negroes were relatively insusceptible to infection
were approximately 2,000 and 1,000 parasites per with vivax malaria. In 1933, Boyd and Stratman-
mm3, respectively. The maximum parasite counts Thomas also reported Negroes to be generally
for the blood-induced infections were 38,673 on refractory to vivax malaria. Boyd (1934) showed
day 11 and 23,232 on day 12 for the sporozoite- the Negro was more immune to vivax than
induced infections. falciparum or malariae malaria. Young et al
Figure 10 shows the parasitemia curves (1946, 1955) showed Negroes were more resistant
(minimum, median, maximum) for infections in than Caucasians to sporozoite induced vivax
10 Negro patients with either the Chesson, St. malaria. Whorton et al (1947a) observed that 6 of
Elizabeth, or Korean strain of vivax malaria. 8 Negroes inoculated with heavily parasitized
Exposure to infection was by the inoculation of blood failed to develop patent parasitemias. The
parasitized blood or by sporozoites. It can be seen other 2 showed evidence of partial refractoriness.
that the median peak parasitemia (6,850 parasites Young et al (1955) reported that only 23 percent
per mm3) obtained on day 7 and that the median of American Negroes developed patent infections
parasitemia then very slowly but steadily with vivax malaria as compared to a 96 percent
declined. One of the most interesting facts evident infection rate in Caucasians. West African
in this figure is the maximum parasitemia of Negroes (Liberians) were found to be susceptible
45,844 per mm3 observed in one patient on day 7. to infection with the Madagascar strain of vivax
Even more interesting, is the fact that this patient malaria in only 3.3 percent (1 out of 30) of the
had experienced previous infections with ovale attempts (Bray, 1958).
and malariae malaria. Six of the 10 Negro patients Rhesus monkeys and other macaques have
infected with vivax malaria had maximum not been found susceptible to P. vivax either after
parasitemias greater than 10,000 parasites per the intravenous inoculation of heavily parasitized
mm3. Three of the 10 Negro patients had blood or large numbers of sporozoites and
experienced previous malaria infections. malariologists despaired of finding any of the
monkeys susceptible to human malaria.

FIGURE 10.—Minimum, median, and maximum parasitemia curves in Negro patients infected with either Chesson, St.
Elizabeth, or Korean strain vivax malaria.
 59

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60 PRIMATE MALARIAS
 PLASMODIUM VIVAX 61

However, in 1966, Young et al and Porter and trivirgatus monkeys resulted in a more rapid rise
Young reported that the owl or night monkey, in the median parasitemia curve to a maximum
Aotus trivirgatus, from Central America was level of approximately 10,000 per mm3 by day 23.
susceptible to infection with Plasmodium vivax. At the end of the 30-day observation period, the
In our studies involving New World median parasitemia was approximately 1500 per
monkeys with human malarias, we have been able mm3.
to establish 7 strains, or isolates, of vivax malaria The parasitemia curve for an A. trivirgatus
in owl monkeys. In fact, 6 of the 7 strains were monkey infected with a strain of P. vivax from
established in intact non-splenectomized animals. West Pakistan is presented in Figure 12. This
(Plate III depicts the appearance of Plasmodium animal was splenectomized prior to the
vivax in the blood of this animal.) Once detectable intravenous inoculation of parasitized blood. On
parasites were present, the parasitemias rose fairly the 14th day after inoculation, daily feeding of
rapidly (Fig. 11) to a level of approximately 800 Anopheles freeborni mosquitoes was initiated.
per mm3 by day 11. After a drop in the median Even though this was the first day gametocytes
parasitemia by day 14, to approximately 100 per were found in the peripheral blood film, they were
mm3, the parasitemia rose to a level of highly infectious to the mosquitoes. This high
approximately 1500 per mm3 by day 18 and level of infection was maintained for the next 9
slowly declined thereafter. At the end of the 30- days followed by a gradual drop in the infection
day observation period, the median parasite count rate until day 32, when no mosquito infections
was approximately 500 per mm3. Subsequent were obtained.
passage of the infections into splenectomized A.

FIGURE 11.—Median parasitemia curves for primary (from man to monkey) and secondary (monkey to man) infections
of Plasmodium vivax in Aotus trivirgatus monkeys.

PLATE III.—Plasmodium vivax in the night monkey, Aotus trivirgatus.

Fig. 1 Normal red cell. Figs. 12-17. Developing schizonts.

Figs. 2-5. Young trophozoites. Figs. 18-21. Nearly mature and mature schizonts.
Figs. 6-9. Growing trophozoites. Figs. 22, 23. Developing and mature macrogametocytes.
Figs. 10, 11. Mature trophozoites. Figs. 24, 25. Nearly mature and mature microgametocytes.
62 PRIMATE MALARIAS

FIGURE 12.—Parasitemia and infectivity rates of Anopheles freeborni mosquitoes after feedings on an Aotus trivirgatus monkey
(AO-91) infected with the West Pakistan strain of Plasmodium vivax.

This was followed by a second period of lower- infection was that of a typical vivax showing that
level infectivity of 9 days. The animal died 49 the parasite had not been altered by its sojourn in
days after inoculation. The fact that an infection the chimpanzee.
of P. vivax in the Aotus trivirgatus monkey could Cadigan et al (1968) were able to infect a
be infectious to mosquitoes over an extended gibbon with P. vivax even though only transient
period of time (in this case, 30 of 35 feeding infection obtained.
days), indicates a great potential for their use in
experiments necessitating large numbers of Host Specificity
infected mosquitoes.
Rodhain (1956) and Bray (1957) have shown
Man is apparently the only natural host for
that chimpanzees are partially susceptible to P.
Plasmodium vivax. Experimental infections have
vivax, in that the liver of the chimpanzee would
been induced, however, in chimpanzees (Mesnil
support development of exoerythrocytic parasites
and Roubaud, 1917, 1920; Garnham et al, 1956)
but did not support the development of the
and in gibbons, Hylobates lar, (Cadigan et al,
erythrocytic stages well. However, when the
1968). In 1966, Porter and Young reported the
animal, harboring the Madagascar strain, was
successful infection of owl monkeys, Aotus
splenectomized, a high parasitemia appeared in a
trivirgatus, and Geoffroy's tamarin, Saguinus
short time. Prior to splenectomy, no erythrocytic
geoffroyi, by the inoculation of parasitized blood
parasites had been observed in smears of the
from man. In addition, Young et al (1966) and
peripheral blood. Subsequently, exposure of 2
Porter and Young (1966) reported the successful
splenectomized chimpanzees to infection with this
transmission of P. vivax from the owl monkey to
same strain of P. vivax quickly resulted in patent
man via the bites of Anopheles albimanus
infections which developed high parasitemias.
mosquitoes. These findings prompted intensive
In 1956, Garnham et al infected a
study of P. vivax in South American monkeys.
chimpanzee with fresh blood parasitized by the
Deane et al (1966) reported the experimental
Madagascar strain of vivax. The parasite did not
infection of splenectomized squirrel monkeys,
grow well in the chimpanzee but well enough so
Saimiri sciureus, with P. vivax and Young and
that A. stephensi mosquitoes could be infected.
Porter (1969) reported the infection of spider
After a suitable incubation period, the mosquitoes
monkeys, Ateles fusiceps and A. geoffroyi, along
were allowed to bite one of the authors. He
with the white-faced monkey, Cebus capucinus,
became infected. Parasitemia appeared on day 10
with P. vivax from A. trivirgatus donors.
and when the patient was taken to the hospital on
Baerg et al (1969) reported transmission of
day 14, his temperature was 41.2° C. The
P. vivax from Aotus trivirgatus and Ateles fusiceps
 PLASMODIUM VIVAX 63

to Ateles fusiceps, A. trivirgatus, and S. geoffroyi feeding of this species and A. stephensi, the
via the bites of A. albimanus mosquitoes, but only standard had 10 oocysts per gut, the A. stephensi
with some difficulty. However, Ward et al (1969) would have had 13.19 oocysts per gut. By this
showed that the Chesson strain of P. vivax in the method it is possible to determine the relative
Aotus trivirgatus and the chimpanzee could be susceptibility of all the mosquitoes to each other
transmitted with relative ease to Aotus trivirgatus by their relationship to the standard.
and the chimpanzee via the bites of A. b.
balabacensis, A. stephensi, and A. Immunity and
quadrimaculatus mosquitoes.
The almost worldwide distribution of P. Antigenic Relationships
vivax is indicative of the large number of
mosquitoes capable of transmitting the parasite. In 1924, Yorke and Macfie demonstrated the
Of the 5 species of Anopheles routinely used in existence of homologous strain immunity against
our laboratory (Table 2), A. stephensi was the Plasmodium vivax by showing that after the
most susceptible to infection with it, followed by development of acquired immunity individuals
A. b. balabacensis, A. freeborni, A. maculatus, were refractory to superinfection by the same
and, finally, A. quadrimaculatus. strain. Homologous strain immunity against vivax
In this and subsequent chapters, the relative malaria has since been confirmed repeatedly by
susceptibility of a species of Anopheles to Boyd and Stratman-Thomas (1933a), Boyd et al
infection with a particular species of Plasmodium (1936), Boyd and Matthews (1939), Boyd (1942),
is based on the determination of the average Boyd and Kitchen (1943,1946), and Jeffery
number of oocysts per 100 guts (Gut Infection (1956). These investigators were able to show that
Index) for a standard mosquito, either A. the development of the immunity obtained in
freeborni, A. b. balabacensis, or A. maculatus, fed subjects infected either by sporozoites or by
simultaneously with the species being compared. parasitized blood. The phenomenon was manifest
The Gut Infection Index ratios are determined by in the form of decreased parasite density, shorter
the relationship of the GII of the standard duration of patent parasitemia, and/or decreased
mosquito to that of the test mosquito; the GII of clinical manifestations.
the standard mosquito is then given an arbitrary Boyd and Kitchen (1943, 1946) stated that
rating of 100. In Table 2, A. freeborni is given the abundant evidence, gathered by themselves and
arbitrary rating of 100. If in a simultaneous others, existed to show that persons convalescing

TABLE 2.--Comparative infectivity of Plasmodium vivax to Anopheles freeborni, A. stephensi, A. b. balabacensis, A. maculatus,
and A. quadrimaculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison tests ratio
Standard Other Standard Other
F-1 100
F-1 : St-1 6 38 44 86.8 84.1 131.9
F-1 : Bal 11 88 61 68.2 82.0 109.3
F-1 : Mac 24 271 272 47.6 42.3 47.0
F-1 : Q-1 9 82 108 86.6 66.7 41.2

* F-1 = Anopheles freeborni; St-1 = A. stephensi; Bal = A. b. balabacensis; Mac = A. maculatus; Q-1 = A. quadrimaculatus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of
A. freeborni to another species where the GII of A. freeborni = 100.
64 PRIMATE MALARIAS

from an infection with vivax malaria had acquired subsequent infections. They concluded that
potent immunity to the strain of parasite which superinfections with heterologous strains of vivax
produced their attack. While recovery from an malaria result in clinical attacks of milder
attack of vivax malaria is indicative of acquired intensity than the original ones.
immunity to the strain which excited the attack, Boyd et al (1939) reported that an absence of
the same individual may be expected to exhibit a cross-immunity between species of malaria (P.
subclinical parasitemia subsequent to a vivax and P. falciparum) was observed whether
reinoculation with the same strain of parasite. superinfection occurred during the incubation
Boyd and Kitchen (1946) showed that the level of period, during the acute primary attack, or shortly
immunity in convalescing malarial infections after recovery from the acute attack.
could be increased by a series of subsequent Whorton et al (1947a) in their studies with
reinoculations with the same strain of parasite. the Chesson strain of vivax malaria discussed
Oftentimes, a level of immunity (hyperimmunity) innate or natural immunity as well as acquired
could be attained where the individual was able immunity. They reported that of 8 Negroes
not only to withstand, but also to promptly inoculated intravenously with blood parasites of
destroy, large inocula of parasites, indeed, many the Chesson strain, all were partially or
million times greater than the minimal number completely refractory to infection. Four of the
required to infect a non-immune individual. patients developed neither patent parasitemia nor
Boyd, Stratman-Thomas, and Kitchen (1936) fever. This phenomenon has been reported by
reported that an effective homologous immunity many investigators. For example, Young et al
to the McCoy strain of vivax malaria could persist (1955) in their studies of induced human malaria
for more than 3 years. Boyd and Matthews (1939) in patients reported that Negroes generally
reported that 2 patients still showed signs or demonstrated a refractoriness to infections with
evidence of immunity 7 years after their primary many domestic and foreign strains of vivax
experience with the homologous strain. The signs malaria under conditions in which Caucasian
of immunity were diminished parasitemia, patients were wholly susceptible.
increased clinical tolerance, and accelerated Tobie and Coatney (1961) were the first to
activation of immune mechanisms. report that antisera to P. vivax and to P. cynomolgi
Boyd and Kitchen (1943) made 2 attempts to would cross-react with the heterologous antigens.
transfer the hyperimmune state passively through Voller (1962) showed that such cross-reactions
the transfusion of large volumes of blood. Both would also occur between P. vivax and P.
attempts failed to prevent infection or modify the bastianellii (= B strain P. cynomolgi), P. gonderi,
course of infection in susceptible persons. and P. osmaniae (= OS strain P. inui). Further
Jeffery (1956) reported that patients studies by Tobie et al (1962) indicated that
reexposed to homologous strain infection of although considerable cross-reaction was
Chesson vivax, by bites of infected mosquitoes, obtained, the maximum antibody titer tended to be
were found to usually experience infections with a the homologous antigen. Diggs and Sadun (1965)
shorter and milder course, and, after treatment, a studied the cross-reactivity of sera from
single relapse ensued which was usually volunteers infected with P. falciparum and others
asymptomatic. infected with P. vivax, using the IFA technique,
Nicole and Steel (1926) reported that and found that sera from patients with P.
immunity may also exist between heterologous falciparum had geometrical mean reciprocal titers
strains of vivax malaria. Boyd (1942) confirmed of 1:28.2 against the homologous antigen in
this observation with American strains of vivax contrast to 1:6.3 against the P. vivax antigen. In a
malaria. Ciuca et al (1937) reported a relative reverse study, sera from patients with P. vivax
cross-immunity in vivax malaria between infections had homologous geometrical mean
imported strains, and strains indigenous to titers of 1:17.2 and heterologous mean titers of
Roumania. 1:9.3.
Boyd et al (1934), based on their studies with
induced vivax malaria, reported that previous
infections with malaria do reduce the severity of
 PLASMODIUM VIVAX 65

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BAERG, D. C., PORTER, J. A., JR., and YOUNG, M. D., 1969. BRUMPT, E., 1949. The human parasites of the genus Plasmodium.
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Panamanian primates. Am. J. Trop. Med. & Hyg. 18 : Saunders Co., Phila.
346-350. CADIGAN, F. C.,JR.; WARD, R. A., and PUHOMCHAREON, S.,
BANO, L., 1959. A cytological study of the early oocysts of seven 1968. Transient infection of the gibbon with Plasmodium
species of Plasmodium and the occurrence of post- vivax malaria. Trans. Roy. Soc. Trop. Med. & Hyg. 62 :
zygotic meiosis. Parasitology 49 : 559-585. 295-296.
BOYD, M. F., 1934. Observations on naturally induced malaria. CELLI, A. and SANFELICE, F., 1891. Sui parassiti del globulo
Southern Med. Jour. 27 : 115-159. rosso nell'uoms et negli animali. Quar. Ist. Igene Sper.
BOYD, M. F., 1940. On strains or races of the malaria parasites. Am. Univ. Roma, 1 (N.S.) 33-63 and Ueber die Parasiten des
J. Trop. Med. 20 : 69-80. Rothen Blutkorpercheres im Menschen und in
BOYD, M. F., 1941. The infection in the intermediate host: Thieren.Fortsch. Med. 9 : 581-586.
symptomatology, general considerations. Am. Assoc. CIUCA, M., BALLIF, L., CHELARESCU, M., and LAVRINENKU,
Adv. Sci. (No.15), 163-182. M., 1937. A l'etude de l'infection experimentale au
BOYD, M. F., 1942. Criteria of immunity and susceptibility in Plasmodium vivax. Arch. Roum. de Pathol. Exper. et
naturally induced vivax malaria infections. Am. J. Trop. Mikrobiol. 10 : 217-265.
Med. 22 : 217-226. CIUCA, M., CHELARESCU, M., SOFLETEA, A., CONSTAN-
BOYD, M. F. and STRATMAN-THOMAS, W. K., 1933. Studies on TINESCU, P., TERITEANU, E., CORTEZ, P.,
benign tertian malaria. 4. On the refractoriness of Negroes BALANOVSCHI, G., and ILIES, M., 1955.
to inoculation with Plasmodium vivax. Am. J. Hyg. 18 : Experimentale a l'etude de l'immunite dans le paludisme.
485-489. Editions De L'academie de al Republique Populaire
BOYD, M. F. and STRATMAN-THOMAS, W. K., 1933a. Studies Roumaine (Bucharest), 61-108.
on benign tertian malaria. I. On the occurrence of COATNEY, G. R. and COOPER, W. C., 1948. Recrudescence and
acquired tolerance to Plasmodium vivax. Am. J. Hyg. 17 : relapse in vivax malaria. Proc. 4th Int. Cong. Trop. Med.
55-59. & Mal. 1 : 629-639.
BOYD, M. F. and MATTHEWS, C. B., 1939. Further observations COATNEY, G. R. and YOUNG, M. D., 1941. The taxonomy of the
on the duration of immunity to the homologous strain of human malaria parasites with notes on the principal
Plasmodium vivax. Am. J. Trop. Med. 19 : 63-67. American strains. Am. Assoc. Adv. Sci. (No.15), 19-24.
BOYD, M. F. and KITCHEN, S. F., 1937. A consideration of the COATNEY, G. R. and YOUNG, M. D., 1942. A study of the
duration of the intrinsic incubation period in vivax paroxysms resulting from induced infections of
malaria in relation to certain factors affecting the Plasmodium vivax. Am. J. Hyg. 35 : 138-141.
parasites. Am. J. Trop. Med. 17 : 437-444. COATNEY, G. R., COOPER, W. C., RUHE, D. S., YOUNG, M. D.,
BOYD, M. F. and KITCHEN, S. F., 1943. On attempts to and BURGESS, R. W., 1950. Studies in human malaria.
hyperimmunize convalescents from vivax malaria. Am. J. XVIII. The life pattern of sporozoite-induced St.
Trop. Med. 23 : 209-225. Elizabeth strain vivax malaria. Am. J. Hyg. 51 : 200-215.
BOYD, M. F. and KITCHEN, S. F., 1944. Renewed clinical activity COATNEY, G. R., COOPER, W. C., and YOUNG, M. D., 1950a.
in naturally induced vivax malaria. Am. J. Trop. Med. 24 Studies in human malaria. XXX. A summary of 204
: 221-234. sporozoite-induced infections with the Chesson strain of
BOYD, M. F. and KITCHEN, S. F., 1946. An attempt at active Plasmodium vivax. J. Natl. Mal. Soc. 9 : 381-396.
immunization with Plasmodium vivax killed in vivo. Am. COATNEY, G. R., CONTACOS, P. G., LUNN, J. S.,
J. Trop. Med. 26 : 749-752. KILPATRICK, J. W ., and ELDER, H. A., 1963. The
BOYD, M. F., STRATMAN-THOMAS, W. K., and KITCHEN, S. effect of a repository preparation of the dihydrotriazine
F., 1936. On the duration of acquired homologous metabolite of chlorguanide, CI-501, against the Chesson
immunity to Plasmodium vivax. Am. J. Trop. Med. 16 : strain of Plasmodium vivax in man. Am. J. Trop. Med. &
311-315. Hyg. 12 : 504-508.
BOYD, M. F., STRATMAN-THOMAS, W. K., and MUENCH, H., CONTACOS, P. G. and COATNEY, G. R., 1963. The relapse
1934. Studies on benign tertian malaria. 6. On activity of a Venezuelan strain of Plasmodium vivax.
heterologous tolerance. Am. J. Hyg. 20 : 482-487. Unpublished data.
BOYD, M. F., KITCHEN, S. F., and MUENCH, H., 1936a. Seasonal CRAIK, R., 1920. A note on the erythrocytes in malaria. Lancet 1 :
variations in the characteristics of vivax malaria. Am. J. 1110-1112.
Trop. Med. 16 : 589-592. DEANE, L. M., NETO, J. F., and SILVEIRA, I. P. S., 1966.
BOYD, M. F., KITCHEN, S. F., and MATTHEWS, C. B., 1939. Experimental infection of a splenectomized squirrel-
Consecutive inoculations with Plasmodium vivax and monkey, Saimiri sciureus, with Plasmodium vivax. Trans.
Plasmodium falciparum. Am. J. Trop. Med. 19 : 141-150. Roy. Soc. Trop. Med. & Hyg. 60 : 811-812.
BRAY, R. S., 1957. Studies on malaria in chimpanzees. II. DIGGS, C. L. and SADUN, E. H., 1965. Serological cross reactivity
Plasmodium vivax. Am. J. Trop. Med. Hyg. 6 : 514-520. between Plasmodium vivax and Plasmodium falciparum
BRAY, R. S., 1958. The susceptibility of Liberians to the as determined by a modified fluorescent anti- body test.
Madagascar strain of Plasmodium vivax. J. Parasit. 44 : Exp. Parasit. 16 : 217-223.
371-373. EHRMAN, F. C., ELLIS, J. M., and YOUNG, M. D., 1945.
66 PRIMATE MALARIAS

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GARNHAM, P. C. C., 1966. Malaria parasites and other Bailliere et Fils. 104 pp.
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GARNHAM, P. C. C., LAINSON, R., and GUNDERS, A. E., 1956. endoglobulaires on haemocytozoa. C. R. Soc. BioI. 53 :
Some observations on malaria parasites in a chimpanzee, 798.
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GARNHAM, P. C. C., BIRD, R. G., and BAKER, J. R., 1963. MANSON, PATRICK, 1900. Experimental proof of mosquito-
Electron microscope studies of motile stages of malaria malaria theory. Brit. Med. Jour. 2 : 949-951.
parasites. IV. The fine structure of the sporozoites of four MARCHIAFAVA, E. and BIGNAMI, A., 1894. Malaria. London,
species of Plasmodium. Trans. Roy. Soc. Trop. Med. & New Syndenham Society, VoI. CL.
Hyg. 57 : 27-31. MARCHIAFAVA, E. and CELLI, A., 1885. Nuove ricerche sulla
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Ospital 7 : 419-422. MAYNE, B., 1932. Note on experimental infection of Anopheles
GOLGI, C., 1889. SuI ciclo evolutive dei parassiti malarici nella punctipennis with quartan malaria. Pub. Hlth. Rep. 47 :
febre terzana; diagnosi qifferenziale tra i parassiti 1771-1774.
endoglobulari malarici della terzana et quelli della MESNIL, F. and ROUBAUD, E. , 1917. Sur la sensibilite du
quartana. Arch. Sc. Med. 13: 173-195. chimpanze au paludisme humain. C. R. Acad. Sc., Paris
GRASSI, B. and FELETTI, R., 1890. Parasites malariques chez les 165 : 39-41.
oiseaux. Arch. Ital. de Biologie 13 : 297-300. MESNIL, F. and ROUBAUD, E., 1920. Essais d'inoculation de
HACKETT, L. W., 1937. Malaria in Europe; an ecological study. paludisme au chimpanze. Ann. Inst. Pasteur 34 : 466-480.
Oxford University Press, London. MISSOROLI, A., 1932. Tipi epidemici delle febbri malariche. Riv.
HANKEY, D. D., JONES, R., JR., COATNEY, G. R., ALVING, A. di Malariologia 2 : 1-24.
S., COKER, W. G., GARRISON, P. L., and DONOVAN, NICOLE, J. E. and STEEL, J. P., 1926. Further report on acquired
W. N., 1953. Korean vivax malaria. I. Natural history and immunity to malarial inoculation. J. Trop. Med. & Hyg.
response to chloroquine. Am. J. Trop. Med, & Hyg. 2 : 29 : 48-49.
958-988. NIKOLAIEV, B. P., 1949. Subspecies of the parasite of tertian
HEMMING, F., (Ed.), 1954. Opinions and declarations rendered by malaria (Plasmodium vivax). Dokl. Akad. Navk., USSR.
the International Commission on Zoological 67 : 201-210 (NS).
Nomenclature. Opinion 283, Vol. 7, London. PORTER, J. A., JR., and YOUNG, M. D., 1966. Susceptibility of
JAMES, S. P., 1926. Report on the first laboratory work in malaria in Panamanian primates to Plasmodium vivax. Mil. Med.
England. League of Nations Health Org., Malaria Comm., 131 : 952-958.
Geneva. PUTNAM, P., BOYD, M. F., and MEAD, P. A., 1947. Periodic or
JAMES, S. P., 1931. Some general results of a study of induced cyclically recurring phenomena of vivax malaria
malaria in England. Trans. Roy. Soc. Trop. Med. & Hyg. infections. Am. J. Hyg. 46 : 212-247.
24 : 477-525. RODHAIN, J., 1956. Les formes preerythrocytaires du Plasmodium
JAMES, S. P., NICOL, W. D., and SHUTE, P. G., 1936. Clinical and vivax chez le chimpanze. Ann. Soc. BeIge Med. Trop. 36
parasitological observations on induced malaria. Proc. : 99-103.
Roy. Soc. Med. 29 : 879-894. RODHAIN, J., 1956a. Paradoxical behaviour of Plasmodium vivax in
JEFFERY, G. M., 1956. Relapses with Chesson strain Plasmodium the chimpanzee. Trans. Roy. Soc. Trop. Med. & Hyg. 50 :
vivax following treatment with chloroquine. Am. J. Trop. 287-293.
Med. & Hyg. 5 : 1-13. SCHUFFNER, W. A. D., KORTEWEG, P. C., and SWELLEN-
KITCHEN, S. F ., 1938. The infection of reticulocytes by GREBEL, N. H., 1929. Experimental malaria with
Plasmodium vivax. Am. J. Trop. Med. 18 : 347-359. protracted incubation period. Proc. Roy. Acad. Amst. 32 :
KITCHEN, S. F., 1949. Vivax malaria. Malariology, Vol. II, edited 903-911.
by Mark F. Boyd. W. B. Saunders Co., Phila. SHANNON, J. A., EARLE, D. P., JR., BERLINER, R. W., and
KITCHEN, S. F. and PUTNAM, P., 1946. Observations on the TAGGART, J. V., 1948. Studies on the chemotherapy of
character of the paroxysm in vivax malaria. J. Natl. Mal. the human malarias. I. Method for the quantitative assay
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KRUSE, W., 1892. Der Gegenwartige Stand Unserer Kenntnisse von Clin. Invest. 27 : 66-74 (Part 2).
den Parasitaren Protozoen. Hyg. Rdsch. 2 : 357-380 and
453-500.
 PLASMODIUM VIVAX 67

REFERENCES – Continued
SHORTT, H. E. and GARNHAM, P. C. C., 1948. The pre-
erythrocytic development of Plasmodium cynomolgi and EICHEL-BERGER, L., and COULSTON, F. , 1947a. The
Plasmodium vivax. Trans. Roy. So c. Trop. Med. & Hyg. Chesson strain of Plasmodium vivax malaria. IV.
41 : 785-795. Immunity. J. Infect. Dis. 81 : 1-6.
SHUTE, P. G. and MARYON, M., 1952. A study of human malaria WINCKEL, C. W. F., 1955. Long latency in Plasmodium vivax
oocysts as an aid to species diagnosis. Trans. Roy. Soc. infections in a temperate zone. Doc. Med. Geog. Trop. 7 :
Trop. Med. & Hyg. 46 : 275-292. 292-298.
TIBURSKAYA, N. A., 1961. Features specific to the Moscow strain YORKE, W., 1925. Further observations on malaria made during
of P. vivax. Medskaya Parazit. 30 : 446-454. (NS) treatment of general paralysis. Trans. Roy. Soc. Trop.
TOBIE, J. E. and COATNEY, G. R., 1961. Fluorescent antibody Med. & Hyg. 19 : 108-122.
staining of human malaria parasites. Exp. Parasit. II: 128- YORKE, W. and MACFIE, J. W. S., 1924. Observations on malaria
132. made during treatment of general paralysis. Trans. Roy.
TOBIE, J. E., KUVIN, S. F., CONTACOS, P. G., COATNEY, G. R., Soc. Trop. Med. & Hyg. 18 : 13-33.
and EVANS, C. B., 1962. Fluorescent antibody studies on YOUNG, M. D., 1944. Studies on the periodicity of induced
cross reactions between human and simian malaria in Plasmodium vivax. J. Natl. Mal. Sac. 3 : 237-240.
normal volunteers. Am. J. Trop. Med. & Hyg. 11 : 589- YOUNG, M. D. and PORTER, J. A., JR., 1969. susceptibility of
596. Ateles fusiceps, Ateles geoffroyi and Cebus capucinus
VOLLER, A., 1962. Fluorescent antibody studies on malaria monkeys to Plasmodium vivax. Trans. Roy. Soc. Trop.
parasites. Bull. Wld. Hlth. Org. 27 : 283-287. Med. & Hyg. 63 : 203-205.
WARD, R. A., RUTLEDGE, L. C., and HICKMAN, R. L., 1969. YOUNG, M. D., ELLIS, J. M., and STUBBS, T. H., 1946. Studies
Cyclical transmission of Chesson vivax malaria in sub- on imported malarias: 5. Transmission of foreign
human primates. Nature 224 : 1126-1127. Plasmodium vivax by Anopheles quadrimaculatus. Am. J.
WHORTON, C. M., YOUNT, E., JR., JONES, R., JR., ALVING, A. Trop. Med. 26 : 477-482.
S., PULLMAN, T. N., CRAIGE, B., JR., and EICHEL- YOUNG, M. D., EYLES, D. E., BURGESS, R. W., and JEFFERY,
BERGER, L., 1947. The Chesson strain of Plasmodium G. M., 1955. Experimental testing of the im munity of
vivax malaria. III. Clinical aspects. J. Infect. Dis. 80 : 237- Negroes to Plasmodium vivax. J. Parasit. 41 : 315-318.
249. YOUNG, M. D., PORTER, J. A., JR., and JOHNSON, C. M., 1966.
WHORTON, C. M., PULLMAN, T. N., KIRSCHBAUM, W. R., Plasmodium vivax transmitted from man to monkey to
JONES, R., JR., ALVING, A. S., CRAIGE, B., JR., man. Science 153 : 1006-1007.
ZIEMANN, H., 1915. Ueber eingenartige Malariaparasiten-formen.
Zentralbl. Bakt. 1 Abt. Orig. 76: 384-391.

(NS) = Not seen.

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 6
Plasmodium cynomolgi Mayer, 1907

Inoki et al (1942) termed the parasite found in

IN 1907, Martin Mayer found a Macaca cyclopis, the Taiwan macaque, P. inui
malaria in some Macacus cynomolgus (= M. cyclopis. In 1951, Inoki et al added information
fascicularis) monkeys imported into Germany on its periodicity, 48 hours, which marked it as a
from Java and, following a brief description of tertian parasite rather than a quartan. In line with
the parasite, he named it Plasmodium this information, Garnham (1959) referred to it
cynomolgi. The next year (1908) he described as P. cynomolgi cyclopis and Hsieh (1960) as P.
the blood parasites in more detail and was cynomolgi var. cyclopis. Bray (1963) gave it
careful to point out the differences between the specific status as P. cyclopis. The main point of
new parasite and P. inui and P. pitheci, each difference seems to be its virulence which is
described by Halberstaedter and Prowazek in hardly sufficient reason for specific rank. We
1907 from the same general area, and between propose to wait for more evidence before
P. vivax endemic in the same region. In the making a decision regarding the Inoki parasite.
ensuing years, up to 1935, when Mulligan again Plasmodium cynomolgi ceylonensis was
described the parasite and returned it to its described by Dissanaike et al, (1965) from the
original status of Mayer, 1907, the parasite has Ceylon toque monkey, Macaca sinica. The
been reported without a name or under various points of difference of this parasite and the
designations including P. inui, P. inui var. neotype are slight. In our studies, we have
cynomolgi. However, irrespective of the elected to deal with this parasite as the C strain
designation or non-designation, the main of P. cynomolgi. In the same year, Dissanaike et
characteristics (tertian periodicity, amoeboidity, al (1965) mentions a P. cynomolgi from the grey
and Schüffner's stippling, etc.) stand out so langur (Presbytis entellus thersites). In our
clearly as to cause little doubt as to the species records, we have carried this parasite as the
involved. langur strain of P. cynomolgi.
It is indeed fortunate that the strain from In addition, we have studied 6 other isolates
Malaya studied by Mulligan has been of P. cynomolgi:
maintained through the years, and although not
Berok-isolated from a naturally infected M.
the type strain, as pointed out by Eyles (1963), it
nemestrina monkey trapped in the southern part
is the neotype and the one designated as the TC
of the State of Perak, Malaysia. Although the
strain (Eyles, 1960a), the M strain (Coatney et
animal was taken in 1961, the isolation was not
al, 1961; Schmidt et al, 1961), and the
made until 1964.
Rockefeller strain (Garnham, 1959). In 1959,
PT-isolated from a naturally infected M.
Garnham gave the subspecific name bastianellii
nemestrina monkey trapped in 1960 in the area
to a parasite, also from Malaya, which he
of Port Dickson in the State of Negri Sembilan,
thought was enough different to warrant the
Malaysia.
designation. We have elected to call this the B
strain (Coatney et al, 1961; Schmidt et al, 1961). RO--isolated from a M. mulatta monkey
Each of these strains is discussed more fully imported into the U.S. from the Assam-Burma
later in this chapter. area in December, 1960. (Schmidt et al, 1961a

69
70 PRIMATE MALARIAS

and personal communication from Dr. L. H. bodia in October, 1962 (Bennett and Warren,
Schmidt). 1965).
Gombak--isolated by Eyles et al (1963) Smithsonian--isolated by us from a
from an infected Anopheles balabacensis naturally infected stump-tailed macaque, M.
introlatus mosquito taken at Ulu Gombak, near speciosa (=arctoides) monkey housed in the
Kuala Lumpur in the State of Selangor, National Zoological Park, Washington, D. C.
Malaysia. Further data on these strains of P.
Cambodian--isolated from a M. irus (= cynomolgi are presented later in this chapter.
fascicularis) monkey taken in west-central Cam-
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 PLASMODIUM CYNOMOLGI 73

divisions as in P. vivax, when at maturity the

Cycle in the Blood merozoites, which are randomly distributed, may
PLATE IV number 14 to 20; the usual number is 16. The
pigment becomes condensed either peripherally
The young ring forms appear in the or centrally in one or several masses (Figs.) 6-
circulating blood with a spherical nucleus and 23).
sometimes with only a whisp of cytoplasm (Fig. The mature gametocytes are round,
3). The nucleus is generally single but may be resemble P. vivax forms, and are located in
double and of unequal size (Fig. 4). Double enlarged host cells. The cytoplasm of the macro-
infection of the red cell is not uncommon (Figs. gametocyte is relatively compact. The pigment
5, 7, 13). When the young parasite has grown to is heavy and scattered. The nucleus is compact,
a diameter of about half that of the host cell, located eccentrically, and may have a dense,
enlargement of the erythrocyte becomes evident; centrally located deep staining portion (Fig. 24).
Schüffner's stippling and pigment appear (Fig. The microgametocytes may take one or two days
6). The older trophozoites look like the P. vivax longer to develop to maturity than the distaff
parasites of man. The Schüffner's stippling is parasites. They stain a reddish-purple in contrast
more prominent and the cytoplasm of the to the light blue of the macrogametocytes. The
parasite becomes amoeboid (Figs. 8-10); nucleus is diffuse and takes up most of the
pigment is in small granules, yellowish-brown, parasite. It may exhibit a more compact portion
and scattered in the cytoplasm. The host cell is which stains a deep reddish-black. Pigment is
now further enlarged. At maturity, the located in the non-nuclear area of the parasite
trophozoite almost fills the erythrocyte, its and is scattered (Fig. 25).
nucleus has increased in size, the parasite
appears more compact, and pigment is abundant The asexual cycle occupies 48 hours.
(Figs. 14, 15). Schizogony proceeds with nuclear

PLATE IV.—Plasmodium cynomolgi

Fig. 1. Normal red cell. Figs. 14, 15. Nearly mature and mature trophozoites.
Figs. 2-7. Young trophozoites. Figs. 16-20. Developing schizonts.
Figs. 8-13. Growing trophozoites, note double Figs. 21-23. Nearly mature and mature schizonts.
infection Fig. 13. Fig. 24. Mature macrogametocyte.
Fig. 25. Mature microgametocyte.
74 PRIMATE MALARIAS
 PLASMODIUM CYNOMOLGI 75

(1932) was apparently the first to describe the

Sporogonic Cycle development of P. cynomolgi in the mosquito. In
PLATE V Anopheles kochi, 8 days after feeding, the
oocysts had an average diameter of 47 µ; on the
Garnham (1966) stated that the micro- 9th day, the average diameter was 77 µ and
gametocyte of P. cynomolgi exflagellates very oocyst differentiation was apparent. Sporozoites
readily, producing about 8 microgametes. The were present in the salivary glands by day 14.
nuclei of the male and female elements fuse to Extensive studies on the growth of P.
form the zygote within 8 hours of fertilization cynomolgi in different mosquitoes were made by
producing a very condensed body measuring Bennett et al (1966a, 1966b). Employing 5
only 4 µ in diameter. The zygote assumes a different strains of the parasite, they
vermiculoid shape and thus becomes an ookinete demonstrated that there were strain differences
at about 16 hours. The hind portion is swollen in the average size of the mature oocysts as well
and the front portion is narrowed almost to a as in the time required for the sporozoites to
point. At 24 hours, it is 16 µ long. The dark reach the salivary glands. Such differences
golden brown pigment grains are collected in occurred with the same strain of the parasite in
strands at the broad end, while a small vacuole is different species of mosquitoes and were,
present near the tip. Penetration of the gut therefore, considered characteristic of the
epithelium to become the oocyst is completed in plasmodium and not dependent on the host
40 to 42 hours after the infective feeding. mosquito. Only the success of the oocysts
Using phase-contrast microscopy and time maturing and of the sporozoites reaching the
lapse photography, Freyvogel (1966) observed salivary glands were controlled by the species of
that the ookinete of P. cynomolgi travels with a mosquito. Using the median oocyst size, on the
spiral-type motion. The ookinete is able to day of maturation, as a basis for comparison of
anchor itself to a surface with its posterior the strains, the M strain was the largest (60 to 70
(blunt) end. µ), followed by B (50 to 60 µ), Gombak (45 to
In describing the early changes in the 50 µ), Berok (40 to 45 µ), and, finally,
nucleus, Bano (1959) noted that before 48 hours, Cambodian (35 to 50 µ). When a comparison of
the nucleus is in a resting stage. The spireme the strains was made with regard to the day
then gives rise to eight diploid chromosomes, sporozoites were first present in the salivary
two pairs of which are large and filamentous and glands, they found that the M strain took the
two pairs are small and bead-like. The binuclear longest (9.5 to 10.5 days) followed by B (9.5
oocyst is formed by the 50th hour when the days), Gombak (9.5 days), Berok (7.5 to 8.5
oocyst is about 14 µ in diameter. The pigment days), and the Cambodian (7.5 days).
granules are scattered in a linear pattern Sporozoites were found in the salivary glands in
throughout the oocyst. After 72 hours, the the shortest time in the parasite producing the
nuclei, the majority of which are at the resting
phase, number 10 to 12 in each oocyst. Green

PLATE V
FIGURES 1-12.—Developing oocysts and sporozoites of Plasmodium cynomolgi (B strain) in Anopheles b. balabacensis
mosquitoes X 580 (Except Figures 1, 3, and 12).
Fig. 1. 4-day oocysts showing scattered pigment. X 740. Fig. 8. 9-day differentiating oocyst.
Fig. 2. 5-day oocysts. Fig. 9. 9-day oocyst showing more advanced stage of
Fig. 3. 6-day oocyst. X 740. differentiation.
Fig. 4. 7-day oocyst. Fig. 10. 10-day differentiated oocyst.
Fig. 5. 7-day oocysts. Fig. 11. 10-day fully differentiated oocyst.
Fig. 6. 8-day oocyst showing first signs of differentiation. Fig. 12. Sporozoites near salivary gland tissue. X 930.
Fig. 7. 8-day oocyst showing early differentiation.
 76 PRIMATE MALARIAS

smallest oocysts. These workers concluded that larger and that the sporozoites appeared in the
studies on the sporogony of the different species salivary glands one day sooner than in A.
of malaria and isolates might contribute to a freeborni. This conflicts slightly with the
better understanding of the relationships findings of Bennett et al (1966a) who found that
between species and between different isolates, the sporozoites were in the salivary glands
too. sooner with the parasite producing the smaller
Differences in oocyst diameters between oocysts.
different strains of P. cynomolgi were also According to Bennett et al (1966a), the
emphasized by Dissanaike et al (1965) when sporozoites of the Cambodian strain of P.
they indicated that by day 7, oocysts of the B cynomolgi averaged 9 to 10 µ in length and 1 to
and C strains had an average size of 50 µ 2 µ in width. The nuclei showed a variety of
whereas the M strain had a mean size of only forms which they divided into three categories:
27 µ. 1) the nucleus diffuse, occupying one-third to
In our studies, only the B strain was studied one-half of the sporozoite, 2) the nucleus in two
in any detail (Table 3). This parasite was readily or three compact, dense, adjoining fragments,
infectious to mosquitoes and sporogony was and 3) the nucleus in a single, compact, dense
normal in each of the 5 species of Anopheles mass occupying one-fifth to one-fourth of the
tested. In A. freeborni, the oocysts 4 days after body and situated about midway in the
feeding had a mean diameter of 14 µ with a sporozoite. The average length of the
range of 9 to 18 µ. The oocysts continued to sporozoites gradually increased between the first
grow so that by day 10, the mean oocyst and seventh day of presence in the salivary
diameter was 61 µ with a range of 41 to 89 µ. glands. Category 3 forms, were predominant
Sporozoites were present in the salivary glands throughout the period of observation although
by day 11. category 1 forms were well represented on the
The oocyst diameters in the other 4 species first 2 days and category 2 forms on the first 3
were within the range found in A. freeborni. days. By day 7 to 9, 98 to 100 percent of the
Sporozoites were present in the salivary glands sporozoites belonged to the third category.
of the A. b. balabacensis and the A. stephensi on Employing electron microscopy, Garnham
day 10, and in the A. maculatus and the A. et al (1963) showed that the pellicle of the
quadrimaculatus on day 11. A comparison of sporozoite is about 25 mµ thick and appears as
the oocyst growth curves (Fig. 13) in A. two electron-dense layers separated by a less
freeborni and A. b. balabacensis shows that in dense zone.
the A. b. balabacensis the oocysts were slightly

TABLE 3.--Oocyst diameters of Plasmodium cynomolgi (B strain) in Anopheles freeborni, A. b. balabacensis, A. maculatus, A. stephensi and A.
quadrimaculatus.

A. freeborni A. b. balabacensis A. maculatus A. stephensi A. quadrimaculatus

Days after
Infection
No. Range Mean* No. Range Mean No. Range Mean No. Range Mean No. Range Mean

4 100 9-18 14 100 8-18 14 107 8-17 13 100 11-18 14 105 12-17 14
5 118 9-19 14 116 12-19 15 127 11-19 14 133 11-21 15 131 12-25 19
6 115 12-35 23 113 14-31 22 138 12-27 21 114 12-30 22 120 12-30 22
7 105 18-53 35 147 22-55 38 130 12-38 25 111 12-37 25 107 14-47 30
8 127 17-59 42 125 27-61 47 103 18-53 38 124 24-66 45 135 18-65 43
9 149 24-83 53† 100 30-84 57† 125 21-84 55† 125 21-74 52† 120 35-83 62†
10 103 41-89 61† 123 30-94 68†‡ 118 24-67 50† 100 30-74 57†‡ 125 29-92 64†
11 95 28-89 56†‡ 134 20-92 60†‡ 122 24-77 48†‡ 105 30-77 56†‡ 126 24-94 52†‡

* Measurements expressed in microns; incubation temperature 25° C.

† Oocyst differentiation.
‡ Ranges in oocyst diameters not available.
 PLASMODIUM CYNOMOLGI 77

FIGURE 13.—A comparison of the mean oocyst diameter curve and ranges in oocyst diameters of Plasmodium cynomolgi in
Anopheles b. balabacensis and A. freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the
salivary glands).
 PLASMODIUM CYNOMOLGI 79

infections. The evidence was clear, that

Cycle in the Tissue wherever the sporozoites and subsequent
PLATE VI schizogonies occurred, it was not in the
peripheral blood. Similarly, our own studies
The discovery of an exoerythrocytic stage carried out in March of 1945 (Cooper, Ruhe, and
in primate malaria was announced by Shortt and Coatney, 1949) showed that with the St.
Garnham on 24 January, 1948. Prior to the Elizabeth strain of P. vivax the blood is not
actual discovery it was agreed generally that infectious during the latent period in patients
contrary to Schaudinn's convincing "dream", of whose infections relapsed subsequently. These
the sporozoite going directly into the host red authors transferred 250 ml. of blood from
cell, there had to be a fixed tissue stage to patients 181 days after the primary infection to
account for prepatent periods and for relapses. In other volunteers. Only one of 6 recipients
fact, in speaking of the latter, Thayer (1897) in a developed malaria and his donor experienced a
series of lectures on human malaria, at the Johns relapse 7 days after blood was drawn for
Hopkins University, agreed with earlier transfer. The other 5 volunteers were shown to
investigators that there must be some be susceptible to infection when homologous
undiscovered form of the parasite; he wrote "the strain parasitized blood was inoculated into
organism may remain perhaps within the cell them. These and similar experiments did not
body of certain phagocytes for long periods of prove the presence of EE stages in primate
time, only to be set free again as a result of some malaria but as indirect evidence the stage was
insult, the nature of which is not yet appreciable set for the Shortt and Garnham discovery.
to us." Fifty-one years later, it did become The initial announcement told of finding 6-
"appreciable to us." In the interim, pre- and 7-day EE bodies and subsequent papers
erythrocytic stages of many of the bird malarias through 1954 (Shortt and Garnham, 1948,
were described, mainly by the Huff school of 1948a; Shortt, Garnham, and Malamos, 1948;
investigators (see Huff and Coulston, 1944 and Hawking, Perry, and Thurston, 1948; Shortt,
Huff, 1947). Also, during this period, there were 1948; Shortt and Garnham, 1948b; and Shortt,
various doubtful records of a corresponding Bray, and Cooper, 1954) filled in the primary
stage in human malaria (see Angelini, 1947 and exoerythrocytic schizogony from day 2 through
Huff, 1947). day 12 and certain older forms including one at
Fairley et al (1945, 1947) transferred from day 104.
200 to 500 ml of blood from patients bitten by As shown by Fairley et al (loc. cit.) the
infected mosquitoes to clean test subjects. sporozoite leaves the peripheral blood shortly
Recipients became infected when blood was after it is introduced into the host and probably
taken within 30 minutes of mosquito biting but enters a parenchyma cell of the liver. The
after that, all results were negative until day 7 in
falciparum infections, and day 8 in vivax

PLATE VI.—Exoerythrocytic bodies of Plasmodium cynomolgi in liver of Macca mulatta monkeys. X 240. Figs. 1-5, 11, 12, 14-
16 are B strain; Figs. 6, 10, and 13 are M strain; Figs. 7 and 9 are RO strain; Fig. 8 is PT strain. Figs. 1-15 EE bodies
stained by Giemsa-Colophonium technique; Fig. 16 EE body stained with Hematoxylin-Eosin.
Fig. 1. 5 day body. Fig. 10. 12 day body.
Fig. 2. 6 day bodies showing prominent flocculi. Fig. 11. 14 day body.
Fig. 3. 6 day body showing host cell nucleus. Fig. 12. 17 day body.
Fig. 4. 7 day body. Fig. 13. 18 day body.
Fig. 5. 7 day body. Fig. 14. 60 day body.
Fig. 6. 8 day body. Fig. 15. 105 day body.
Fig. 7. 9 day body. Fig. 16. 143 day body; this figure courtesy of
Fig. 8. 10 day body showing numerous nuclei. Dr. Leon Schmidt.
Fig. 9. 11 day body.
80 PRIMATE MALARIAS

earliest stage seen, a 2-day form, is a spherical to lack of complete synchronicity, remaining first
ovoid body about 2.3 to 2.45 µ in diameter. The generation schizonts continue their development.
cytoplasm stains blue; the nucleus is single and These slower-growing forms are compact and
stains red. (The authors were not unmindful of entire. The merozoites appear as if composed
the difficulties connected with recognizing this entirely of chromatin but there is surely a
minute stage and that of day 3, also; however, cytoplasmic envelope, too. Bray (1957) found
their illustrations appear convincing.) The 3-day these forms measured 46 µ in diameter and
form is spherical or ovoid and measures 4.5 to Eyles (1960a) found them to measure about 49.8
5.9 µ in diameter. It occupies a more or less µ with extremes of 35 to 62.5 µ. Ten day forms
peripheral position in the cell. The cytoplasm generally contain differentiated merozoites.
stains blue and there are 8 or more nuclei which Ruptured forms, when seen, appear to have lost
stain reddish-violet. The 4-day forms are about their compact regular outline. Older first
twice the size of those of the previous day, about generation schizonts have been seen but except
10 µ in diameter. The cytoplasm stains a pastel for their large size, up to 108 µ, they appear
blue. The nuclei have increased to about 20 and about like the 8 to 10 day forms.
stain a reddish-mauve. The periphery of the Secondary schizonts have been seen at 60
schizont is marked by a fine membrane. By the and 105 days (see Plate VI, Figs. 15 and 16) 143
next day, the 5th, the schizont is about 14 µ in days by Schmidt and at 378 days by Warren.
diameter and houses up to 70 nuclei. The host Secondary schizonts is an arbitrary designation
cell nucleus is pushed to one side but otherwise for there is no proof that these older forms,
the host cell shows no sign of being invaded; probably responsible for relapse, arise from the
staining is typical. rare parasite of the first or succeeding
Up to this point, the development of the generations and, for reasons of their own, re-
tissue parasite is more or less routine but by the enter a liver cell. Certainly these liver schizonts
6th day there is a decided change. The size is do not originate from circulating blood forms--
now up to 29 µ in diameter, the host cell is much for if they did, blood-induced infections would
enlarged, and its nucleus is pushed toward the relapse, also. They do not. Relapse, in the true
periphery. The cytoplasm of the parasite stains a sense, is a part of the life-pattern of P.
pastel blue. The nuclei are difficult to count but cynomolgi as it is of P. vivax, P. fieldi, and
the number is over 100. Vacuoles are a new certain others, but the process responsible for
development and become more prominent as producing the tissue schizonts which initiate it is
growth proceeds. Seven day forms may assume still a matter for speculation. It is not impossible
various shapes, other than the characteristic that these continuing EE bodies are derived from
oval, due to tissue resistance, but irrespective of the original sporozoites which lie dormant for a
the shape the internal structure is constant. The time, and then, through some urge, not
cytoplasm stains light-blue or mauve and is appreciable to us, are moved to comple te their
somewhat granular. The chromatin particles destiny. A fuller discussion is found in an earlier
stain magenta, tend to be spherical, rod- or boat- section (Chapter 4).
shaped and measure about 0.75 µ in diameter. The secondary schizonts, found at 60 days
The 8-day EE bodies average about 38 µ in and/or later, resemble the initial forms in all
diameter. They are spherical to oval. The respects including active division. This latter
tendency for vacuolization seems to be a feature characteristic shows that they are not latent or
connected with parasites from some monkeys retarded forms. Garnham (1966) believes that
and absent from others. The cytoplasm stains these forms can be separated from earlier forms
blue as in the younger forms, the nuclei red. because their contour is generally "markedly
Some of the schizonts are mature by this time convoluted." This has not been our experience as
because parasites may appear in the peripheral may be seen by examining Figs. 15 and 16 (60-
blood. By the 9th day, the maturation of the EE and 105-day parasites) and the same is true for
bodies is definitely in force but since there is a the 143- and the 378- day forms. As the
 PLASMODIUM CYNOMOLGI 81

evidence stands now we doubt that the contour membrane which lies close to the cytoplasm of
of the parasite can be relied upon as an the parasite. Mutliple irregularly shaped nuclei,
indication of the age of the EE body. 1.1 x 1.6 µ, are scattered throughout the body.
There were no reports on the ultrastructure The nucleus is homogenous and granular with a
of the EE bodies of any of the primate malarias clear space surrounding it. The cytoplasm of the
until the paper by Sodeman et al (1970) on 7- EE body contains densely packed free
day forms of the B strain of Plasmodium ribosomes. Some of these appear in a linear
cynomolgi (Plate VII). They studied 4 of these fashion. Mitochondria are present.
bodies which had an average size of 28 x 17µ. There are 2 types of vacuoles: Type 1 is 0.3
The liver cell border, with its organelles, to 3.5 µ, round, and with a distinct membrane.
surrounds the EE body completely. The host cell They appear empty but some contain membrane-
is enlarged and its nucleus is pushed to one side. like structures; Type 2 vacuoles are smaller,
There appear to be no degenerative changes in homogeneous, and more electron dense. They
the cell as a result of the parasitism. are generally round, but may be crescent-shaped.
The lobulated EE body has a two-layered It is hoped that these authors will continue

PLATE VII.—Midsection of a 7-day-old exoerythrocytic body of Plasmodium cynomolgi in rhesus monkey liver. The parasitized
hepatic cell surrounds the EE body. A wavy outer membrane and a thin inner membrane enclose the EE body.
Several areas of linear arranged ribosomes are seen (R). Nuclei (N) have a clear space surrounding them. (X 4600).
Electron-photomicrograph courtesy of Dr. Thomas Sodeman.
82 PRIMATE MALARIAS

their investigations so that we may learn more or by intravenous and/or intrahepatic injection.
about the ultrastructure of this and other primate The prepatent periods ranged from 7 to 16 days
malaria EE bodies. with a mean of 9.8 days. In general, slightly
shorter prepatent periods were obtained when
large numbers of sporozoites were inoculated
Course of Infection intravenously and/or intrahepatically than were
Plasmodium cynomolgi is readily obtained by mosquito bite.
transmitted by a number of species of The two strains of P. cynomolgi which have
mosquitoes. In our studies, there was a total of received the greatest attention are the Mulligan
132 successful transmissions out of 136 attempts or M strain and the subspecies bastianellii or the
(Table 4). These transmissions were initiated by
the inoculation of sporozoites, by mosquito bite,

TABLE 4.—Summary of transmissions for seven strains of Plasmodium cynomolgi using six species of Anopheles.

Transmissions/Attempts Total Prepatent Period (days)

Strain
trans.
Fre* Qua Ste Mac Atr Bal Range Mean

B† 10/10 23/23 1/1 44/47 8/8 86/89 7-16 9.8

M 2/2 4/4 6/6 8-11 10.0
Camb 2/2 1/1 3/3 9-13 11.0
Berok 1/1 6/6 2/2 9/9 8-12 9.7
Gombak 1/2 1/1 1/1 2/2 1/1 6/7 8-14 10.0
RO 2/2 11/11 1/1 1/1 15/15 8-12 9.4
C 3/3 1/1 1/1 2/2 7/7 8-16 10.7

Totals 19/20 45/45 4/4 54/57 1/1 9/9 132/136 7-16 9.8

* Fre = Anopheles freeborni, Qua = A. quadrimaculatus, Ste = A. stephensi, Mac = A. maculatus, Atr = A. atroparvus,
Bal = A. b. balabacensis.
† B = Bastianellii strain, M = Mulligan strain, Camb = Cambodian strain, Berok = Berok strain, Gambok = Gambok strain,
RO = Rossan strain, C = Ceylonensis strain.

FIGURE 14.—Parasitemia curve for 1 Macaca speciosa and median parasitemia curves for 102 M. mulatto monkeys infected
with the B strain of Plasmodium cynomolgi.
 PLASMODIUM CYNOMOLGI 83

B strain. The histories of these two isolates were count after 60 days was only 100 per mm3. The
ably discussed by Eyles (1963). inoculation of one M. speciosa monkey resulted
Our efforts have been primarily directed in a peak parasitemia of 200 per mm3, 4 days
towards study of the B strain, the results of after inoculation. This was followed by a drop to
which are shown in Figure 14. In 8 an undetectable level. Two recrudescenses were
splenectomized M. mulatta monkeys infected by observed 28 and 35 days after inoculation.
inoculation of parasitized blood, the median In order to examine the normal parasitemias
parasitemia reached a level of approximately of infections with both the M and the B strains,
200,000 per mm3 by day 8, followed by rises to Dr. Leon Schmidt of the Southern Research
approximately 400,000 and 300,000 per mm3 on Institute, Birmingham, Ala., supplied the data,
days 13 and 22, followed by a sudden drop in from 174 M. mulatta monkeys, used in preparing
parasite levels. Maximum parasitemias as high Figures 15 through 19.
as 1,200,000 were obtained in some animals The B strain infections, induced by the
during the 26 day observation period. In 44 inoculation of parasitized blood (Fig. 15), gave a
intact M. mulatta monkeys similarly inoculated, mean peak parasitemia, by the 8th day of
the peak median parasitemia of approximately patency, of approximately 1,000 per 10,000
200,000 per mm3 was obtained on day 8, also. RBC. After the peak, the parasite level rapidly
However, in these animals, the parasitemia fell declined to approximately 80 per 10,000 RBC
rapidly to a level of 18,000 per mm3 by day 13 by day 12. This was followed by successive
and after several succeeding fluctuations, decreasing waves in the parasite count until day
reached a level of approximately 500 per mm3 45 when the parasitemia had fallen to 2.6 per
by day 60. In 50 intact M. mulatta monkeys, 10,000 RBC. Thereafter, the parasitemia rose
infected by inoculation of sporozoites, the peak and was 10 per 10,000 RBC at the end of the 60
median parasitemia was about the same as that day observation period. In the monkeys infected
encountered in the blood-induced infections. by inoculation of sporozoites (Fig. 16), the peak
However, the parasitemia dropped to a lower mean parasitemia was again, on the 8th day of
level by day 35 (50 per mm3) followed by a rise patency, at a level of approximately 700 per
in parasite level by day 47 and, then, by another 10,000 RBC. The parasite level then declined
drop in the parasitemia. The median parasite

FIGURE 15.—Mean parasitemia curve, with minimum and maximum parasite counts, for 29 Macaca mulatta monkeys infected
with B strain Plasmodium cynomolgi by the inoculation of parasitized blood. (Data courtesy Dr. Leon Schmidt).
84 PRIMATE MALARIAS

rapidly to approximately 90 per 10,000 RBC on RBC. The parasite level declined rapidly to a
day 14. This was followed by a declining much lower level than encountered with the B
parasite count to a level of approximately 5 per strain, approximately 4 per 10,000 RBC by day
10,000 RBC by day 60. 23. The mean parasitemia remained at this low
The M strain infections, initiated by the level for the remainder of the 60-day observation
inoculation of parasitized blood (Fig. 17) period. In monkeys infected with the M strain,
resulted in a peak mean parasitemia on day 9 by sporozoite inoculation (Fig. 18), the peak
with a count of approximately 600 per 10,000 mean parasitemia was on day 10 at a level of

FIGURE 16.—Mean parasitemia curve, with minimum and maximum parasite counts, for 60 Macaca mulatta monkeys infected
with B strain Plasmodium cynomolgi by inoculation of sporozoites. (Data courtesy Dr. Leon Schmidt).

FIGURE 17.—Mean parasitemia curve and minimum and maximum parasite counts, for 25 Macaca mulatta monkeys infected
with M strain Plasmodium cynomolgi by the inoculation of parasitized blood. (Data courtesy Dr. Leon Schmidt).
 PLASMODIUM CYNOMOLGI 85

approximately 500 per 10,000 RBC. The strain maintains a consistently higher level of
parasite level then declined to approximately 1.4 parasitemia throughout the 60-day observation
per 10,000 RBC by day 30. After a subsequent period. That this characteristic is attributable to
rise, the parasitemia eventually fell to this particular strain is evidenced by the fact that
approximately 1 per 10,000 RBC by day 60. the same difference in the parasitemia occurred
As shown in Figure 19, sporozoite-induced with animals infected by blood inoculation. Only
infections with the B strain peak slightly sooner in the period between day 35 and day 50, when
than do those with the M strain parasite. The the curve of the blood induced infections with
main difference between the 2 isolates lies in the the B strain dropped, was there a joining of the
subsequent parasite levels. After day 15, the B curves for the 2 strains. It appears clear that the

FIGURE 18.—Mean parasitemia curve and minimum and maximum parasite counts for 60 Macaca mulatta monkeys infected with
M strain Plasmodium cynomolgi by inoculation of sporozoites. (Data courtesy of Dr. Leon Schmidt).

FIGURE 19.—Mean parasitemia curves for infections of B strain Plasmodium cynomolgi and M strain P. cynomolgi in Macaca
mulatta monkeys.
86 PRIMATE MALARIAS

B strain parasitemias were consistently or recrudescenses of the infection). Of

maintained at a higher level than were those of considerable interest was the presence of a
the M strain. pattern of every-other-day infectivity which
It has long been established that P. persisted often for many days; for example,
cynomolgi is a relapsing malaria in the true between days 26 and 37, days 42 and 63, and
sense. In monkeys infected with the M strain, days 78 and 89. This pattern of infectivity to
via mosquito bite, during a one-year period of mosquitoes was also seen in similar studies with
observation, there were from 2 to 17 relapses other P. cynomolgi infections. Since the highest
(Fig. 20). In one monkey (T-445), the level of infection was correlated with the
appearance of relapses was frequent and, in predominance of very young trophozoite forms
some ways, predictable; in another (T-495), in the blood, it is postulated that the gametocytes
relapses occurred only twice, 40 and 120 days mature at approximately the same time as the
after exposure to infection. The total number of schizonts; and, that they are more infectious in
relapses which an animal, such as T-445, is this early period than 24 hours later. It appears
capable of having would appear to be large since that gametocytes lose their infectivity fairly
there appeared to be little diminution in rapidly. Whether this infectivity is subsequently
frequency of relapses at the end of the restored is doubtful since a day of high level
observation period. infectivity (for example day 34) may be
We have been concerned for a long time followed by several days (in this case 7 days) of
about the most propitious time for feeding low level infectivity. Restoration of infectivity is
mosquitoes in order to obtain good infections. associated with a rise in parasitemia. The
Studies have been carried out on both blood- and infectivity to mosquitoes 113 days after
sporozoite-induced infections. The latter is the exposure to infection, or, after 103 days of
natural mode of infection and, for that reason, almost continuous patent parasitemia, indicates
the results of one of the studies is presented in that infectivity can continue for an extended
Figure 21. The initial feeding on day 5 of period of time with P. cynomolgi in M. mulatta
parasitemia (15 days after sporozoite monkeys.
inoculation) revealed that mosquito infection In man. Up to 1960, the attitude among
was already taking place. This continued malariologists generally was: "Monkey malaria
through day 36 followed by 5 days in which is for monkeys, and human malaria is for
mosquitoes failed to become infected. humans." That attitude took a shattering blow,
Subsequent mosquito infections appeared to be on 5 May 1960 when the senior author (GRC),
correlated with rises in the parasitemia (relapses on answering a telephone call from Dr. Don E.

FIGURE 20.—Frequency of relapse activity in 4 Macaca mulatta monkeys infected with M strain of Plasmodium cynomolgi by
the inoculation of sporozoites via the bites of Anopheles maculatus mosquitoes. (Drug regimen: 300 mgm. quinine x
5 days with each appearance of parasites).
 PLASMODIUM CYNOMOLGI 87

Eyles, head of the Section on Cytology, effect of drugs on the exoerythrocytic stages of
Laboratory of Parasite Chemotherapy, NIB, in the B strain of P. cynomolgi (Eyles and Coatney,
Memphis, Tennessee, heard him say, "Bob, I 1962). Because it was thought, "man could not
have monkey malaria." I was incredulous--had it be infected with monkey malaria" and because
really happened? The remainder of the the object was to get sporozoites from as many
conversation went about as follows: mosquitoes as possible during a single day, Dr.
GRC: If you have monkey malaria, don't Eyles and his technician (Mrs. N.C.O.) paid
take any drugs. scant attention to the occasional mosquito that
DEE: I thought you would say that so I escaped into the room. Two days after Dr. Eyles
took chloroquine before I placed this call to you. recognized the cause of his illness, 7 May, the
GRC: I hope you drew blood for technician (N.C.O.) became ill with fever. Five
inoculation into a clean rhesus. ml. of her blood was given to a clean rhesus
DEE: I did, and the blood has been given to monkey; parasites were present 10 days later,
the monkey. Now all we have to do is wait for and a normal infection ensued. On the same day,
the monkey to come down. another 20 ml. of blood was drawn from N.C.O.
(It did-8 days later: Eyles et al, 1960). and divided between two inmate volunteers.
This was the first recognized instance of a Each of the volunteers developed clinical
simian malaria infection transmitted to man by malaria, but neither one had anything but
natural means and, therefore, what happened minimal parasite counts.
during the next two months was a prelude to the At the time of the subinoculations, there
developments of the next 10 years. was no proof that the parasite involved was P.
Dr. Eyles' illness was due to an accidental cynomolgi and, if it were P. cynomolgi, that it
infection in connection with the study of the was transferred by mosquito bite. To answer

FIGURE 21.—The infectivity of Anopheles quadrimaculatus mosquitoes during the course of a sporozoite-induced infection
with B strain Plasmodium cynomolgi, with its parasitemia curve, in a Macaca mulatta monkey.
88 PRIMATE MALARIAS

these points, two staff members allowed (Coatney et al, 1961, and Schmidt et al, 1961),
mosquitoes (A. freeborni) infected with P. and when initially successful, it produced a mild
cynomolgi to bite them. H.A. became ill after 11 disease. The B strain, however, possibly due to
days but parasites could not be demonstrated. its recent isolation, was more readily accept-
Blood was taken from him and injected into a able to the human host. Also, the vector may
clean monkey. The animal was positive for the have played an important role. Anopheles
infection 6 days later. The second man (C.S.S.), quadrimaculatus, the old standby in this
also bitten by infected A. freeborni mosquitoes, country, is a relatively poor vector, but A.
was positive for the infection 14 days later. It freeborni is highly efficient. Interestingly
was thus proved that P. cynomolgi can be enough, similar accidents have been few or
transmitted to man by mosquito bite. absent during the last 10 years, probably due to
Surprisingly, almost at the same time, R.G. in more careful handling of infected mosquitoes.
Dr. Leon Schmidt's laboratory at the Christ This brace of accidents, coupled with the
Hospital, in Cincinnati, Ohio, came down with intentional natural infections in man, indicated a
an accidental infection. The infecting parasite zoonosis of unknown proportions, but one which
was again the B strain of P. cynomolgi (see might have a profound effect on the emerging
Eyles et al, 1960; Schmidt et al, 1961). world-wide program of malaria eradication,
After this rash of accidental infections, it toward which the U.S. was appropriating some
was evident that mosquitoes infected with this 60 million dollars per annum. It was considered
parasite should be handled with the same respect imperative that we have information on this
accorded those infected with the human subject without delay. To obtain such
malarias. We did not expect further accidents, information, studies would have to be carried
but in this we were mistaken. In mid-May, Dr. out in the area where P. cynomolgi is endemic,
Schmidt sent mosquitoes infected with the B namely-Malaya.
strain cynomolgi parasites to Dr. Clay Huff at The logical one to inaugurate these studies
the Naval Medical Research Institute in was Dr. Eyles. When offered the chance to carry
Bethesda, Maryland. Mrs. D.M., with wide out a study of simian malaria in depth, he
experience in handling infected mosquitoes, accepted with enthusiasm. He arrived in Kuala
took charge of them. On 4 July, she became ill Lumpur, Malaya on 17 August 1960 to head the
with a high fever and was taken to a local Far East Research Unit (FERU), LPC, NIH, and
hospital. On the second day following to work cooperatively with personnel of the
admission, when the attending physician was not Malayan Institute of Medical Research
sure of the diagnosis, D.M. suggested to him (IMR).The productivity of Eyles and his
that she might have monkey malaria. The coworkers was prodigious. Their work and that
physician had never heard of monkey malaria, of the LPC workers in this country is the basis
and, because she had a high fever, he assumed for this monograph.
she was delirious. The patient was confident that With the knowledge that P. cynomolgi
she had simian malaria, a fact soon confirmed. infects and produces disease in man, it was felt
Mrs. D.M. was transferred to the NIH Clinical desirable to embark on a more intensive study
Center where the infection was treated and the involving both the M and B strains of the
patient made an uneventful recovery. parasite, with special emphasis on
Faced with this remarkable series of parasitological and clinical aspects in man. As a
accidents so close together, one is moved to result of this effort, three papers appeared in
inquire as to why they occurred and why they rapid succession: Beye et al, 1961, Schmidt et
had been absent previously. The explanation al, 1961, and Contacos et al, 1962. In addition,
possibly lies with the fact that prior to 1959 Schneider (1961) in France, and Garnham et al
work had been limited to the M strain, isolated (1962) in England, reported less extensive
by Mulligan in 1935, and maintained, generally studies with the B strain of P. cynomolgi.
by blood-inoculation, in widely scattered lab- The results of our effort which involved
oratories here and abroad. The M strain was some 56 patients (34 B strain, 22 M strain)
transmitted to man with some difficulty
 PLASMODIUM CYNOMOLGI 89

infected via mosquito bite (A. freeborni or A. (6) The first fever appeared between 16 and
quadrimaculatus) or by the inoculation of 19 days.
parasitized blood, can be summarized about as (7) The maximum temperature was 105.2°
follows: F.
(1) Negroes are refractory to infection with (8) Tertian fever patterns were not the rule
this parasite as are some Caucasians. but prominent in some patients.
(2) Monkey to man, man to man, and man Clinical symptoms in patients infected with
to monkey transmission of the infection via either strain consisted of cephalgia, anorexia,
mosquito bite is not only possible, but, at times, myalgia, and nausea, in that order. The
relatively easy to accomplish. The bite of a symptoms were usually present only during
single infected mosquito resulted in a patent febrile episodes, were of moderate severity, and
infection in one of 3 volunteers. The prepatent easily controlled by simple medications. The
period was 19 days. The patient experienced 4 most prominent physical findings were
tertian fever cycles with a maximum splenomegaly and hepatomegaly.
temperature of 103° F. (Contacos and Coatney, We have produced infections in many
1963). Also, the same authors showed that the volunteers with P. cynomolgi and from them we
infection in a monkey (PT strain) brought have selected 29 B strain infections (13
directly from the field, in contrast to the long sporozoite- and 16 blood-induced) (Fig. 22), and
laboratory-residence of the M strain and the 3 26 M strain infections (11 sporozoite- and 15
year laboratory-residence of the B strain, could blood-induced) (Fig. 23), none of which
be transferred to man by mosquito bite at the received treatment, whose parasite counts were
first attempt. The prepatent period was 20 days. known for the first 50 days (an arbitrary cutoff
(3) The prepatent period with either strain point) of their infection. Perusal of these figures
is about 19 days, with a range of 15 to 20 days will show that the parasitologic picture coincides
for the B strain and 16 to 37 days, with one with what was described earlier. The clinical
exception of 82 days, for the M strain. manifestations in these patients were in the same
(4) The maximum parasitemia is somewhat vein.
higher with the M strain, about 300 parasites per Schneider (loc. cit.) had 3 patients, one
mm3 as against 150 per mm3 for the B strain. infected by sporozoites and the other 2 by the
(One M strain patient, infected by blood- inoculation of parasitized blood. He reported
inoculation, developed a parasite count of 8,300 mild fever episodes, none higher than 100° F,
per mm3.) accompanied by very low parasitemias.
(5) There were no differences in the The following year, Garnham et al (loc.
duration of parasitemia in the 2 strains.

FIGURE 22.—Individual parasite counts and median parasitemia curve for 29 infections of B strain Plasmodium cynomolgi in man
(13 sporozoite- and 16 blood-induced).
90 PRIMATE MALARIAS

cit.) reported on sporozoite-induced infections in ceylonensis (= our C strain) and P. fragile. None
14 patients (8 by mosquito bites and 6 by of the patients evidenced infection during an
intravenous injections of sporozoites). The observation period of 30 days. Here, and
maximum temperature recorded was 104° F. probably in the earlier cases, too, the observation
The earliest prepatent period was 11 days and period was only 30 days. It is not unlikely that a
the average incubation period was 13 days. The longer period of observation would have turned
infections exhibited the typical behavior of up an infection in some of the recipients.
relatively severe symptoms with low Bennett and Warren (1965) using a strain of
parasitemias. All the infections were treated P. cynomolgi isolated from an M. irus monkey
early, but there is little doubt that had they been taken in Cambodia found it infective to man via
allowed to continue, they would have exhibited the bites of A. maculatus mosquitoes. The
the usual pattern of the untreated infection. prepatent period was 21 days. In 1970, Cheong
These studies of the early sixties convinced and Coombs transmitted P. cynomolgi to man by
the most skeptical that P. cynomolgi was a mosquito bite.
zoonosis of unknown potential. In fact, the In our own studies we have transmitted the
senior author had gone so far as to predict that P. Gombak strain to one of 2 men exposed on one
cynomolgi would be the first field-acquired occasion, the prepatent period was 52 days; and
simian malaria in man; it was not to be (see the Smithsonian to each of two men, the
Chapter 26). prepatent periods were 25 and 26 days,
Later attempts to infect man with P. respectively.
cynomolgi experimentally embrace work in 2
different areas of the Orient. Dissanaike et al
(1965) gave blood parasitized with P. cynomolgi Host Specificity
ceylonensis (= our strain C) to each of 4
Plasmodium cynomolgi naturally infects
patients; 2 other patients were bitten by heavily
Macaca irus (= fascicularis), M. nemestrina
infected A. atroparvus mosquitoes. (The primate
(Eyles et al, 1962), M. radiata (Prakash and
host was not given.) None of these patients
Chakrabarti, 1962), M. cyclopis (Inoki et al,
became infected. The length of the observation
1951), M. sinica (Dissanaike, 1963), M. mulatta
period was not given. In the same year,
(Schmidt personal communication), Presbytis
Dissanaike (1965) reported giving parasitized
cristatus (Eyles et al, 1962a), and P. entellus
blood to 4 other patients; 2 received P.
(Dissanaike et al, 1965).
cynomolgi and P. shortti (= our OS strain P.
Experimentally, infections have been
inui) and the other 2 got P. cynomolgi

FIGURE 23.—Individual parasite counts and median parasitemia curve for 26 infections of M strain Plasmodium cynomolgi
in man (11 sporozoite- and 15 blood-induced).
 PLASMODIUM CYNOMOLGI 91

obtained in M. mulatta, Cercopithecus aethiops and transmitted by a wide variety of

(Huff and Coulston, 1944), Cebus capucinus coindigenous and exotic species of mosquitoes.
(Garnham, 1959), Papio papio (Garnham, Because of this, it has been used extensively in
1959), and in man (Eyles et al, 1960; and experimental studies. In Table 5, we have listed
others). those anopheline mosquitoes which have been
Plasmodium cynomolgi is considered to be tested for their susceptibility to infection with
an oligoxenous parasite since it is infective to this parasite. In addition, Mansonia

TABLE 5.—Anopheline mosquitoes tested for susceptibility to infection with Plasmodium cynomolgi and those shown to be
vectors experimentally.

Level of
Mosquito species Transmission References
susceptability

Anopheles annularis High – Mulligan, 1935

A. annularis Moderate Ind. Res. Fund. As., 1947
–
A. aconitus Low – Warren, et al, 1963
A. aconitus Low Bennett et al, 1966
+
A. albimanus Low – Eyles, 1960b
A. albimanus Low – Omar, 1968
A. albimanus Low Omar, 1968a
–
A. argyropus Low Bennett et al, 1966
–
A. aztecus High + Garnham and Lainson, 1957
A. aztecus High – Garnham, 1959
A. aztecus High Dissanaike et al, 1965
–
A. atroparvus Refractory – Mayer, 1908
A. atroparvus Unknown – Weyer, 1937
A. atroparvus High + Rodhain and van Hoff, 1940
A. atroparvus High + Hawking et al, 1948
A. atroparvus High + Shortt and Garnham, 1948
A. atroparvus High + Collins et al, 1965
A. atroparvus High – Dissanaike et al, 1965
A. atroparvus High Omar, 1968
+
A.b. balabacensis High Collins, 1969
+
A.b. introlatus Moderate – Bennett et al, 1966
A.b. introlatus Low Warren et al, 1963
–
A. barbirostris High – Ind. Res. Fund. As., 1947
A. barbirostris Refractory – Warren et al, 1963
A. barbirostris Low – Warren and Wharton, 1963
A. barbirostris Moderate Bennett et al, 1966
–
A. baezai Refractory – Warren et al, 1963
A. baezai Refractory Bennet et al, 1966
–
A. campestris Moderate – Bennett et al, 1966
A. campestris Low Warren et al, 1963
–
A. crawfordi Low – Warren et al, 1963
A. crawfordi Low Bennett et al 1966
–
A. culicifacies High – Mulligan, 1935
A. culicifacies High Ind. Res. Fund. As., 1947
92 PRIMATE MALARIAS

Level of
Mosquito species Transmission References
susceptability
A. donaldi Low – Warren et al, 1963
A. donaldi Low – Bennett et al, 1966

A. elegans High + Choudhury et al, 1963a

A. freeborni High + Schmidt et al, 1948,1961,1963,1966,1970

A. freeborni High + Eyles, 1960, 1960a
A. freeborni High + Eyles et al, 1960
A. freeborni High + Eyles and Coatney, 1962
A. freeborni High + Beye et al, 1961
A. freeborni High + Coatney et al, 1961
A. freeborni High + Contacos et al, 1962
A. freeborni High + Rossan et al, 1964
A. freeborni High + Collins, 1969

A. fluviatilis Moderate – Ramakrishnan and Mohan, 1962

A. fluviatilis High – Choudhury et al, 1963

A. gambiae High – Bray and Garnham, 1964; Garnham, 1966

A. gambiae Moderate – Omar, 1968

A. hackeri High – Warren et al, 1963

A. hodgkini Low – Warren et al, 1963

A. hodgkini Low – Bennett et al, 1966

A. hyrcanus Moderate – Ind. Res. Fund. As., 1947

A. indiensis Low – Warren et al, 1963

A. indiensis Low – Bennett et al, 1966

A. kochi High + Warren et al, 1963

A. kochi High + Bennett et al, 1966
A. kochi Moderate – Green, 1932

A. lesteri Moderate + Bennett et al, 1966

A. letifer Moderate + Warren and Wharton, 1963

A. letifer Moderate + Bennett et al, 1966

A. leucosphyrus High – Warren et al, 1963

A. leucosphyrus Low – Bennett et al, 1966

A. maculatus Low – Green, 1932

A. maculatus High – Mulligan, 1935
A. maculatus High + Warren et al, 1963
A. maculatus High + Bennett et al, 1966
A. maculatus High + Collins, 1969

A. peditaeniatus Low – Warren et al, 1963

A. peditaeniatus Low + Bennett et al, 1966

A. philippinensis Moderate + Warren et al, 1963

A. philippinensis Moderate + Bennett et al, 1966

A. pujutensis Refractory – Warren et al, 1963

 PLASMODIUM CYNOMOLGI 93

Level of
Mosquito species Transmission References
susceptability
A. quadrimaculatus High + Coggeshall, 1941
A. quadrimaculatus Moderate + Wolfson and Winter, 1946
A. quadrimaculatus High + Huff and Coulston, 1948
A. quadrimaculatus High + Hawking et al, 1948
A. quadrimaculatus High + Coulston, 1949
A. quadrimaculatus High + Eyles, 1960a
A. quadrimaculatus High + Beye et al,1961
A. quadrimaculatus High + Collins et al, 1965
A. quadrimaculatus High + Collins, 1969

A. riparis High – Warren et al, 1963

A. riparis Moderate – Bennett et al, 1966

A. roperi Low – Bennett et al, 1966

A. sacharovi High – Omar,1968

A. separatus Low – Warren et al, 1963

A. separatus Moderate + Bennett et al, 1966

A. sinensis Low – Warren et al, 1963

A. sinensis Low + Bennett et al, 1966

A. splendidus High – Mulligan, 1935

A. stephensi Moderate – Garnham and Lainson, 1957

A. stephensi High – Garnham,1959
A. stephensi Moderate + Ramakrishnan and Mohan, 1962
A. stephensi High – Choudhury et al, 1963a
A. stephensi High + Collins et al, 1965
A. stephensi High + Omar,1968
A. stephensi High – Omar, 1968a
A. stephensi High + Collins, 1969
A. stephensi High – Hawking et al, 1966, 1968
A. stephensi High – Dissanaike et al, 1965

A. subpictus Moderate – Ind. Res. Fund. As., 1947

A. subpictus Refractory – Warren et al, 1963
A. subpictus Refractory – Bennett et al, 1966

A. sundaicus High + Warren et al, 1963

A. sundaicus High + Bennett et al, 1966

A. tessellatus Refractory – Warren et al, 1963

A. tessellatus High + Choudhury et al, 1963

A. umbrosus Low – Warren et al, 1963

A. vagus Moderate – Warren et al, 1963

A. vagus Low – Green, 1932
A. vagus Moderate + Bennett et al, 1966
94 PRIMATE MALARIAS

TABLE 6.--Comparative infectivity of Plasmodium cynomolgi to 23 species of anophelines.

Mosq. species Number Number of mosquitoes Percent infection GII**

Comparison* tests Standard Other Standard Other ratios
Mac 100
Mac : Bal 30 225 190 81.8 81.1 148.7
Mac : F-1 58 405 365 80.5 83.6 116.6
Mac : St-1 28 316 269 49.4 68.8 102.8
Mac : Sun 11 102 89 92.2 75.3 100.1
Mac : Q-1 56 489 515 65.6 58.8 78.2
Mac : Koc 15 257 92 84.4 79.3 76.2
Mac : Bar 14 137 111 91.2 22.5 35.3
Mac : Les 6 59 62 84.7 54.8 27.8
Mac : Phi 10 123 70 74.0 75.7 21.5
Mac : Vag 22 263 168 83.3 46.4 17.0
Mac : Hod 3 49 9 65.3 11.0 9.9
Mac : Let 14 150 208 79.3 47.6 8.7
Mac : Sep 3 34 14 50.0 14.3 5.0
Mac : Sin 10 107 56 95.3 32.1 4.6
Mac : Atr 7 90 29 81.1 44.8 4.4
Mac : Cam 3 22 10 100.0 30.0 1.7
Mac : Ped 12 158 166 72.8 4.2 0.8
Mac : Umb 3 11 79 100.0 29.1 0.8
Mac : Arg 7 75 126 100.0 11.5 0.6
Mac : Alb 18 204 313 71.6 3.2 0.3
Mac : Don 10 107 71 83.2 12.7 0.2
Mac : Rop 2 15 12 100.0 8.3 0.1

* Mac = A. maculatus, Bal = A. b. balabacensis, F-1 = A. freeborni, St-1 = A. stephensi, Sun = A. sundaicus, Q-1 = A.
quadrimaculatus, Koc = A. kochi, Bar = A. barbirostris, Les = A. lesteri, Phi = A. philippinensis, Vag = A. vagus, Hod = A.
hodgkini, Let = A. letifer, Sep = A. separatus, Sin = A. sinensis, Atr = A. atroparvus, Cam = A. campestris, Ped = A.peditaeniatus,
Umb = A. umbrosus, Arg = A. argyropus, Alb = A. albimanus, Don = A. donaldi, Rop = A. roperi.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A.
maculatus to another species where the GII of A. maculatus = 100.

uniformis has been experimentally infected with

several strains of P. cynomolgi (Warren et al,
Immunity and Antigenic
1962). Both oocyst and sporozoite infections Relationships
were demonstrated but no transmissions were
obtained (Warren and Wharton, 1963; Bennett et Mulligan and Sinton (1933) demonstrated
al, 1966). Culex vishnui (Mulligan, 1935), C. that immunity produced by a given strain of the
tritaeniorhynchus (Warren and Wharton, 1963) parasite in monkeys appears to be specific
and Aedes butleri (Warren and Wharton, 1963; mainly for the same strain. There was some
Bennett et al, 1966) have been reported evidence, however, to suggest a slight degree of
susceptible to infection as far as the oocyst stage common immunity. Chronic infections with P.
only. cynomolgi conferred effective immunity against
In our own studies, 23 species of the clinical effects of superinfection with the
anophelines have been compared for same strain of parasite. They were unable,
susceptibility to infection with P. cynomolgi however, to demonstrate any cross-immunity
(Table 6). The most readily infected was A. b. between infections due to P. knowlesi and P.
balabacensis and the least susceptible was A. cynomolgi. Singh and Singh (1940) found that
roperi.
 PLASMODIUM CYNOMOLGI 95

chronic infections due to P. cynomolgi and P. (1962). They found that when antisera to P.
inui failed to prevent, or modify, the course of cynomolgi from human volunteers was allowed
infection with heterologous parasites. It was also to react with the homologous and heterologous
shown that P. cynomolgi produces an immunity parasites, considerable cross reaction was
to homologous superinfection which remains obtained. They noted, however, that the
effective for at least 18 months. maximum antibody titers were obtained with the
In 1966, Voller et al reported on cross- homologous parasite. Voller (1962)
immunity studies with a number of species of demonstrated a strong IFA cross reaction
monkey malaria. They demonstrated that between P. bastianellii (= P. cynomolgi
infections with P. cynomolgi bastianellii and P. bastianellii) and P. vivax, P. gonderi, and P.
cynomolgi ceylonensis produced no cross- osmaniae (= P. inui shortti). Subsequent studies
immunity either to each other or to P. knowlesi, indicated that antisera to P. falciparum, P.
P. coatneyi, P. fragile, P. inui, P. inui shortti, malariae, and P. ovale would also cross react
and P. gonderi. In a later extensive with the P. cynomolgi antigen (Kuvin and
immunological study, Voller and Rossan (1969, Voller, 1963; Collins et al, 1966a; Meuwissen,
1969a, 1969b) demonstrated that M. mulatta 1968).
monkeys with chronic infections due to P. In our studies (Collins et al, 1966), antisera
knowlesi were still susceptible to infection with to P. cynomolgi gave a fluorescent antibody
P. cynomolgi bastianellii. Animals with chronic cross reaction at a high level to P. fieldi antigen
infections with P. cynomolgi bastianellii were (mean reciprocal titer ratio of 100:76) and lesser
immune to challenge with the homologous reactions to P. knowlesi, P. gonderi, and P.
parasite and to a high degree to P. cynomolgi brasilianum (mean reciprocal titer ratios of
ceylonensis. In contrast, monkeys with chronic 100:54, 100:36, and 100:31, respectively). In the
infections of P. cynomolgi bastianellii produced reverse procedure, P. cynomolgi antigen gave
severe infections when challenged with P. the highest cross reaction to P. inui, P. knowlesi,
cynomolgi ceylonensis. Their studies also P. fragile, and P. fieldi (mean reciprocal titer
indicated that parasite populations isolated from ratios of 100:46, 100:41, 100:33, and 100:31,
a late relapse of P. cynomolgi were respectively).
immunologically different from those isolated El-Nahal (1967), using the exoerythrocytic
from the primary infection and from an early stages of P. cynomolgi as antigen in a
relapse. They concluded that these variants arose fluorescent antibody test, showed that whereas
from different antigenic variants released from the homologous antisera responded well, the
the liver. heterologous antisera to P. inui and P. malariae
The first observations on the serologic cross failed to respond.
reactions between P. cynomolgi and P. vivax
were made, using the fluorescent antibody test,
by Tobie and Coatney (1961) and Tobie et al

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DISSANAIKE, A. S., 1963. Plasmodium infection in Ceylon GARNHAM, P. C. C., 1966. Malaria parasites and other
monkeys. Trans. Roy. Soc. Trop. Med. & Hyg. 57 : 488- haemosporidia. Blackwell Scientific Publications. Oxford.
489. pp.1114.
DlSSANAlKE, A. S. NELSON, P., and GARNHAM, P. C. C., GREEN, R., 1932. A malaria parasite of Malayan monkeys and its
1965. Two new malaria parasites, Plasmodium cynomolgi development in anopheline mosquitoes. Trans. Roy. Soc.
ceylonensis subsp. nov. and Plasmodium fragile sp. nov., Trop. Med. & Hyg. 25 : 455-477.
from monkeys in Ceylon. Ceylon J. Med. Sci. (D) 14 : 1-9.
 PLASMODIUM CYNOMOLGI 97

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HALBERSTAEDTER, L. and PROWZEK, S., 1907. and Plasmodium inui (Halberstadter and Prowazek, 1907)
Untersuchungen uber die malariaparasiten der affen. from naturally occurring mixed infections in Macaca
Arb. K. Gesundh. -Amte (Berl.) 26 : 37-43. radiata radiata monkeys of the Nilgiris, Madras state,
HAWKING, F., PERRY, W. L. M., and THURSTON, J. P., 1948. India. Ind.J. Malariol. 16 :303-311.
Tissue forms of a malaria parasite, Plasmodium RAMAKRISHNAN, S. P. and MOHAN, B. N., 1962. An enzootic
cynomolgi. Lancet 1 : 783-789. focus of simian malaria in Macaca radiata radiata
Geoffroy of Nilgiri;, Madras state, India. Ind. J. Malariol.
HAWKING, F., WORMS, M. J., GAMMAGE, K., and
16 : 87-94.
GODDARD, P. A., 1966. The biological purpose of the
blood-cycle of the malaria parasite Plasmodium RODHAIN, J. and VAN HOFF, T., 1940. Contribution a l'etude
cynomolgi. Lancet 2 : 422-424. des Plasmodium des singes africians. Le comporte- ment
HAWKING, F., WORMS, M. J., and GAMMAGE, K., 1968. 24- different des PI. gonderi et PI. kochi chez les moustiques.
and 48-hour cycles of malaria parasites in the blood; Bull. Soc. Path. Exot. 33 : 107-113.
their purpose, production and control. Trans. Roy. Soc. ROSSAN, R. N., FISHER, K. F., GREENLAND, R. D.,
Trop. Med. & Hyg. 62 : 731-760. GENTHER, C. S., and SCHMIDT, L. H., 1964. The
HSIEH, H. C., 1960. Malaria parasites of the Taiwan monkey. localization of infective pre-erythrocytic forms of
Formosan Science 14 : 477-487. Plasmodium cynomolgi. Trans. Roy. Soc. Trop. Med. &
HUFF, C. G. and COULSTON, F., 1944. The development of P. Hyg. 58 : 159-163.
gallinaceum from sporozoite to erythrocytic trophozoite. J. SCHMIDT, L. H., FRADKIN, R., SQUIRES, W., and GENTHER,
Infect. Dis. 75: 231-249. C. S., 1948. Malaria chemotherapy: II. The response of
HUFF, C. G., 1947. Life cycle of malarial parasites. Ann. Rev. sporozoite-induced infections with Plasmodium cynomolgi
Microbiol. 1 : 43-60. to various antimalarial drugs. Fed. Proc. 7 : 253-254.
HUFF, C. G. and COULSTON, F., 1948. A search for pre- SCHMIDT, L. H., GREENLAND, R., and GENTHER, C. S.,
erythrocytic stages of Plasmodium vivax and P. 1961. The transmission of Plasmodium cynomolgi to man.
cynomolgi. J. Parasit. 34 : 264-274. Am. J . Trop. Med. & Hyg. 10 : 679-688.
Indian Research Fund Assoc., 1947. Report of the scientific SCHMIDT, L. H., GREENLAND, R., ROSSAN, R., and
advisory board for 1st January to the 31st December, 1946. GENTHER, C., 1961a. Natural occurrence of malaria in
9-14. rhesus monkeys. Science 133 : 753.
INOKI, S., TAKEMURA, S., MAKIURA, Y., and HOTTA, F., SCHMIDT, L. H., ROSSAN, R. N., and FISHER, K. F., 1963. The
1942. A malaria parasite, Plasmodium inui var. cyclopis activity of a repository form of 4,6-Diamino-1-(p-
Inoki, Takemura, Makiura, and Hotta 1941 in Macaca chlorophenyl)-1 ,2-dihydro-2,2-dimethyl-s-triazine against
cyclopis Swinhoe. (Japanese text). Osaka Igakkai Zassi 41 infections with Plasmodium cynomolgi. Am. J. Trop. Med.
: 1327-1343. (NS). & Hyg. 12 : 494-503.
INOKI, S., 1951. Studies on the exoerythrocytic schizogony of the SCHMIDT, L. H., ROSSAN, R. N., FRADKIN, R., WOODS, J.,
malaria parasite. Exoerythrocytic forms of the Formosan SCHULEMANN, W., and KRATZ, L., 1966. Studies on
monkey malaria parasite, Plasmodium inui var. cyclopis. the antimalarial activity of 1,2-Dimethoxy-4-(bis-
Med. J. Osaka Univ. 2 : 45-53. diethylami- noethyl)-amino-5-bromobenzene. Bull. WId.
INOKI, S., OKUNO, Y., and AOYAMA, A., 1951. On the length Hlth. Org. 34 : 783-788.
of the asexual life cycle of Plasmodium inui var. cyclopis. SCHMIDT, L. H., HARRISON, J., ELLISON, R., and
Med. J. Osaka Univ. 2 : 37-43. WORCESTER, P., 1970. The activities of chlorinated
KUVIN, S. F. and VOLLER, A., 1963. Malarial antibody titres in lincomycin derivatives against infections with
West Africans in Britain. Brit. Med. Jour. 2 : 477-479. Plasmodium cynomolgi in Macaca mulatta. Am. J. Trop.
MAYER, M. 1907. Ueber malaria beim affen. Med. Klin, Berl. 3 : Med. & Hyg. 19 :1-11.
579-580. SCHNEIDER, J., 1961. P. cynomolgi bastianelli: Hematozoaire du
MAYER, M., 1908. Uber malariaparasiten bei affen. Arch. f. singe transmissible a l'homme. Essai d'impaludation
Protist. 12 : 314-321. therapeutique. Bull. Soc. Path. Exot. 54 : 7-11.
MEUWISSEN, J. H. E. TH., 1968. Antibody responses of patients SHORTT, H. E., 1948. The pre-erythrocytic cycle of Plasmodium
with natural malaria to human and simian Plasmodium cynomolgi. 4th Int. Cong. Trop. Med. & Malaria 1 : 607-
antigens measured by the fluorescent antibody test. Trop. 617.
georg. Med. 20 : 137-140. SHORTT, H. E. and GARNHAM, P. C. C., 1948. Pre- erythrocytic
stage in mammalian malaria parasites. Nature 161 : 126.
MULLIGAN, H. W. and SINTON, J. A., 1933. Studies in
immunity in malaria. II. Superinfection with various SHORTT, H. E. and GARNHAM, P. C. C., 1948a. The pre-
strains of monkey malarial parasites. Rec. Mal. Surv. India erythrocytic development of Plasmodium cynomolgi and
3 : 529-568. Plasmodium vivax. Trans. Roy. Soc. Trop. Med. & Hyg.
MULLIGAN, H. W., 1935. Descriptions of two species of monkey 41 : 785-795.
plasmodium isolated from Silenus irus. Arch. f. Protist. 84 SHORTT, H. E. and GARNHAM, P. C. C., 1948b. Demonstration
: 285-314. of a persisting exo-erythrocytic cycle in Plasmodium
OMAR, M. S., 1968. Die empfiinglichkeit verschiedener cynomolgi and its bearing on the production of relapses.
Anopheles-Arten fur den erreger der affenmalaria Brit. Med. Jour. 1 : 1225-1228.
Plasmodium cynomolgi bastianellii. Zschr. Tropenmed. SHORTT, H. E., GARNHAM, P. C. C., and MALAMOS, B.,
Parasit. 19 : 152-170. 1948. The pre-erythrocytic stage of mammalian malaria.
OMAR, M. S., 1968a. Vergleichende beobachtungen uber die Brit. Med. Jour. 1 : 192-194.
entwicklung von Plasmodium cynomolgi bastianellii im SHORTT, H. E., BRAY, R. S., and COOPER, W., 1954. Further
Anopheles stephensi und Anopheles albimanus. Zschr. notes on the tissue stages of Plasmodium cynomolgi.
Tropen- med. Parasit. 19 : 370-389. Trans. Roy. Soc. Trop. Med. & Hyg. 48 : 122-131.
PRAKASH, S. and CHAKRABARTI, S. C., 1962. The isolation
and description of Plasmodium cynomolgi (Mayer, 1907)
98 PRIMATE MALARIAS

REFERENCES—Continued
SINGH, J. and SINGH, H., 1940. Observations on immunity in VOLLER, A. and ROSSAN, R. N., 1969a. Immunological studies
monkey malaria as evidenced by the results of super- with simian malarias. II. Heterologous immunity in the
infections. J. Malar. Inst. Ind. 3 : 99-114. "cynomolgi" group. Trans. Roy. Soc. Trop. Med. & Hyg.
SODEMAN, T., SCHNITZER, B., CONTACOS, P. G., and 63 : 57-63.
DURKEE, T., 1970. The fine structure of the VOLLER, A. and ROSSAN, R. N., 1969b. Immunological studies
exoerythrocytic stage of Plasmodium cynomolgi. Science. on simian malaria parasites. IV. Heterologous
170 : 340-341. superinfection of monkeys with chronic Plasmodium
THAYER, W. S., 1897. Lectures on the malarial fevers. D. knowlesi infections. Trans. Roy. Soc. Trop. Med. & Hyg.
Appleton & Co., New York, pp. 326. 63 : 837-845.
TOBIE, J. E. and COATNEY, G. R., 1961. Fluorescent antibody WARREN, McW., EYLES, D. E., and WHARTON, R. H., 1962.
staining of human malaria parasites. Exp. Parasit. 11 : Primate malaria infections in Mansonia uniformis. Mosq.
128-132. News 22: 303-304.
TOBIE, J. E., KUVIN, S. F., CONTACOS, P. G., COATNEY, G. WARREN, McW., EYLES, D. E., WHARTON, R. H., and OW
R., and EVANS, C. B., 1962. Fluorescent antibody studies YANG CHEE KONG, 1963. The susceptibility of
on cross reactions between human and simian malaria in Malayan anophelines to Plasmodium cynomolgi
normal volunteers. Am. J. Trop. Med. & Hyg. 11 : 589- bastianellii. Ind. J. Malariol. 17 : 85-105.
596. WARREN, McW. and WHARTON, R. H., 1963. The vectors of
VOLLER, A., 1962. Fluorescent antibody studies on malaria simian malaria: identity, biology, and geographical
parasites. Bull. WId. Hlth. Org. 27 : 283-287. distribution. J. Parasit. 49 : 892-904.
VOLLER, A., GARNHAM, P. C. C., and TARGETT, G. A. T., WEYER, F., 1937. Versuche zur Übertragung der affen-malaria
1966. Cross immunity in monkey malaria. J. Trop. Med. durch stechmucken. Arch. f. Schiffs- u. Trop. -Hyg. 41 :
Hyg. 69 : 121-123. 167-172.
VOLLER, A. and ROSSAN, R. N., 1969. Immunological studies WOLFSON, F. and WINTER, M. W., 1946. Studies of
with simian malarias. I. Antigenic variants of Plasmodium Plasmodium cynomolgi in the rhesus monkey, Macaca
cynomolgi bastianellii Trans. Roy. Soc. Trop. Med. & mulatta. Am. J. Hyg. 44 : 273-300.
Hyg. 63 : 45-56.
(NS) = Not seen.
 7
Plasmodium eylesi Warren, Bennett, Sandosham,
and Coatney, 1965
There was no doubt in the minds of the authors
THIS was the second species of as to its being a new species. They gave it the
malaria described from Malayan gibbons and the name Plasmodium eylesi in honor of the late Dr.
third described for the world. A mature male Don E. Eyles who contributed so much to our
Hylobates lar was taken in the Kedah-Perlis area knowledge of malaria in general and to simian
of the country and made available to the authors malaria in particular.
for study. It was found infected with a malaria
parasite (Warren et al, 1965, 1965a) which was
studied subsequently in five other gibbons.

99
 PLASMODIUM EYLESI 101

Cycle in the Blood cell (Fig. 29). They stain a grayish-blue and
exhibit coarse, granular pigment which is
PLATE VIII scattered rather evenly throughout the parasite.
Immediately after the young parasite enters The deep staining, generally oval, nucleus may
an erythrocyte, the cell becomes enlarged. have a vacuole adjacent to it (Fig. 29). The
Schüffner’s stippling becomes evident even with mature microgametocytes are in an enlarged,
the youngest forms but is not pronounced (Figs. circular to oval, host cell which takes a deep
2-10). Amoeboid forms may occur, but ring brilliant reddish-purple stain with a slightly
forms are by far the most common aspect of the deeper staining nucleus. The pigment is
early part of the asexual cycle. Not only are somewhat prominent and scattered throughout
multiple infections present but they are common the cytoplasm. Once this parasite is seen, the
with as many as six rings in a single cell (Fig. 8). trained eye will "not forget it" (Fig. 30).
The red-staining nucleus is generally single. The One of the most striking features of this
older trophozoites do not fill the cell; they may parasite is its tendency for multiple invasion of
be slightly amoeboid (Fig. 13) and may surround the host red blood cells, a phenomenon probably
a vacuole (Fig. 16). Pigment is scarce, granular, associated with the parasites' predilection for
and yellowish-brown; Schüffner’s stippling reticulocytes. This multiple development in a
continues. The mature trophozoites exhibit a single host cell is responsible for a second
more dense blue-staining cytoplasm (Fig. 19); characteristic feature of P. eylesi, namely the
they are not amoeboid; pigment is granular, and consistent appearance of parasitized cells with as
is a yellow-brown to gray. Stippling is scattered many as 33 merozoites (Fig. 27).
and coarse. The host cell is enlarged and some- The parasite has a highly synchronous
times distorted (Fig. 17). tertian periodicity.
Young schizonts almost fill the enlarged
host cell except for small areas where Sporogonic Cycle
Schüffner’s inclusion bodies may be prominent
(Fig. 24). The cytoplasm remains dense grayish- The natural vector of this parasite is
blue as schizogony progresses. Pigment remains unknown. However, the sporogonic cycle has
granular and difficult to distinguish; oval-shaped been studied in three laboratory reared species of
forms are not uncommon (Fig. 23). Older mosquitoes indigenous to peninsular Malaysia
schizonts are frequently oval (Figs. 25, 27) and (Anopheles kochi, A. maculatus, and A.
the cytoplasm stains a deep bluish-red. The sundaicus). Observations began 36 hours after
chromatin bodies are randomly distributed (Figs. the mosquitoes fed and continued through 14.5
26, 27); pigment is yellowish-brown and not days. Extrinsic incubation took place in an
clumped. The number of chromatin bodies insectary maintained at approximately 27° C.
ranges from 20 to 34 with an average of 25. The oocyst measurements are presented in Table
Fully differentiated mature schizonts have not 7.
been observed. In A. kochi, at day 1.5, the mean oocyst
The gametocytes are distinctive, especially diameter was 7.5 µ with a range of 7 to 9 µ.
the microgametocytes. The fully mature
macrogametocytes fill the much enlarged host

PLATE VIII.—Plasmodium eylesi.

Fig.1 Normal red cell. Figs. 26-27. Mature or nearly mature schizonts.
Fig. 2-8. Young trophozoites. Fig. 28. Young macrogametocyte.
Figs. 9-15. Growing trophozoites. Fig. 29. Mature macrogametocyte.
Figs. 16-20. Mature trophozoites. Fig. 30. Mature microgametocyte.
Figs. 21-25. Developing schizonts.
102 PRIMATE MALARIAS

The oocysts continued to grow so that on day patent infection the parasitemia increased
9.5, they had an average size of 53 µ with a rapidly, so that by the end of the first week the
range of 27 to 69 µ. Sporozoites were first seen count was 20,000 per mm3 where it remained,
in the salivary glands on day 9.5. more or less, until day 20. From then until day
The examination of oocysts in A. maculatus 40, the count averaged about 10,000 parasites
and A. sundaicus mosquitoes indicated that the per mm3 of blood, and thereafter declined
growth rate of the parasite was similar in all slowly. The details of the actual infection in
three species. Sporozoites were present in the each animal were different; i.e., gibbon G 19,
salivary glands of each species by day 9.5. whose parasitemia reached 90,000 per mm3, was
A comparison of the growth rate of P. treated and then survived a second episode of
eylesi with that of P. cynomolgi in A. maculatus above 100,000 per mm3. It was finally cleared of
mosquitoes (Fig. 24) shows the mean oocyst the infection by a curative dose of chloroquine
diameters were quite similar and that sporozoites on the 128th day of the patent infection, whereas
of each of the parasites were present in the the other animals exhibited more moderate
salivary glands at 9.5 days. parasitemias and were able to handle their
The sporozoites in A. kochi were infective infections without treatment.
as shown by their ability to initiate an infection The original investigators were able to
in a gibbon. The prepatent period was 12 days. study a single infection induced by the
inoculation of sporozoites from A. kochi fed on
G 19. This was accomplished in gibbon G 8 on
Cycle in the Tissue 30 May 1964; young parasites appeared in the
blood 12 days later. The parasite count increased
This part of the cycle is unknown. from a second day count of 401 per mm3 of
blood to a count of 40,900 on day 8 which was
Course of Infection the highest count encountered during an
observation period of 32 days.
Infections, induced by the inoculation of Because of the possible zoonotic nature of
parasitized blood, have been studied in four these malarias in the higher apes one is always
gibbons (Fig. 25). Beginning on the first day of tempted to inquire of Nature whether she will

FIGURE 24.—Mean oocyst growth curve and ranges in oocyst diameters of Plasmodium eylesi and P. cynomolgi in Anopheles
maculates mosquitoes. Incubation temperature 27° C. (D = oocyst differentiation; SP = sporozoites present in the
salivary glands). (Data courtest [sic] of Dr. Gordon Bennett).
 PLASMODIUM EYLESI 103

accept such a parasite in man. With A. kochi eylesi because blood passaged from him to a
mosquitoes harboring sporozoites of P. eylesi in parasite-free gibbon failed to produce an
their salivary glands and lacking certified human infection in the animal and, also, because the P.
volunteers the only hope of a trial in man was eylesi infection, if that is what it was, was too
for one of the investigators to act as a volunteer. low to allow positive identification from
This Dr. Gordon F. Bennett (1968) did. He was parasites encountered in the blood smears.
bitten by A. kochi mosquitoes, known to be
infected. On the 15th post exposure day, he Host Specificity
exhibited pronounced clinical symptoms
comparable to those he had previously
The natural host of P. eylesi is the white-
experienced when infected with P. cynomolgi.
handed gibbon, Hylobates lar. The parasite is
The symptoms persisted for about two weeks.
not transferable to the rhesus monkey (Macaca
During this time, parasites were evident at a very
mulatta) by the inoculation of infected blood.
low level for about one week. There is
The natural invertebrate host of P. eylesi is
reasonable doubt that Bennett's symptoms,
unknown. On an experimental basis, 12 species
although real, were due to infection with P.
of anopheline mosquitoes indigenous to its area

FIGURE 25.—Median parasitemia curve for infections of Plasmodium eylesi (4 blood-induced and one sporozoite-induced) in
five gibbons, Hylobates lar.

TABLE 7.—Oocyst diameters of Plasmodium eylesi in Anopheles kochi, A. maculatus, and A. sundaicus.

Days after A. kochi A. maculatus A. sundaicus

infection
No. Range Mean* No. Range Mean No. Range Mean

1.5 6 7-9 7.5

2.5 48 7-12 9 17 6-12 9 52 6-14 10
3.5 76 8-17 12 62 8-15 10 60 10-15 12
4.5 204 11-24 18 112 10-21 15 96 12-24 18
5.5 188 12-35 24 215 12-32 26 113 15-39 25
6.5 279 15-50 33 65 14-42 26 98 21-57 36
7.5 268 23-63 42† 81 22-53 38 111 24-56 45
8.5 334 21-68 48† 151 19-63 45† 50 41-68 52†
9.5 210 27-69 53†** 100 27-60 46†** 211 29-75 54†**
10.5 128 26-71 54†** 12 43-58 44†** 138 30-71 54†**
11.5 110 38-65 52†**

TotaIs 1741 7-71 815 6-63 1039 6-75

* Measurements expressed in microns.

† Oocyst differentiation.
** Sporozoites present in the gland.
104 PRIMATE MALARIAS

of the world became infected when allowed to and intensity of infection of the salivary glands
feed on a gibbon parasitized with P. eylesi. is greater in A. kochi than in A. maculatus.
These were A. kochi, A. maculatus, A. It is possible that a single human infection
sundaicus, A. leucosphyrus, A. umbrosus, A. was obtained through the agency of mosquito
roperi, A. letifer, A. b. introlatus, A. riparis bites (A. kochi) but further work is needed
macarthuri, A. vagus, A. sinensis, and A. lesteri. before this observation can be inserted into the
All but the latter three species delivered realm of fact.
sporozoites to the salivary glands. The intensity
of the infections varied from one species to
another (Table 8). Anopheles vagus was the Antigenic Relationships
most susceptible followed by A. kochi, A. and Immunity
maculatus, A. sundaicus, A. umbrosus, and A.
lesteri. Comparative feedings were not made
Not much is known about antigenic
with the other species.
relationships and immunity as applied to this
Infections were obtained in A. kochi, A. species, although it is well to point out that the
maculatus and A. sundaicus mosquitoes when animal in which P. eylesi was induced by
they were allowed to feed on two different sporozoite inoculation was already carrying a
gibbons (G 11 and G 19) between the 6th and low grade infection with P. youngi. This may
the 18th days of patent parasitemia (Fig. 26). show that infection with P. youngi did not
Maximum infection (100 percent) was obtained, exclude infection with P. eylesi, although it must
from feedings on both animals, on day 10. be recognized that the previous and current P.
A comparison of the salivary gland youngi infection may have modified the course
infection rates (Table 9) shows that when the gut of the superimposed P. eylesi infection.
infection levels are comparable, the percentage

TABLE 8.—Comparative infectivity of Plasmodium eylesi in Anopheles kochi, A. vagus, A. maculatus, A. sundaicus, A. umbrosus, and A. lesteri.

Number of Percent
Mosq. species Number mosquitoes infection GII**
Comparison* tests ratios
Standard Other Standard Other

Kochi 100
Kochi : Vagus 1 10 10 60.0 30.0 113.3
Kochi : Mac 3 156 179 59.0 55.3 47.0
Kochi : Sund 1 10 21 100 100 36.0
Kochi : Umb 1 35 7 77.1 28.6 3.6
Kochi : Les 2 18 12 100 50.0 1.4

* Kochi = Anopheles kochi, Vagus = A. vagus, Mac = A. maculatus, Sund = A. sundaicus, Umb = A. umbrosus, Les = A lesteri.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. kochi to another
species where the GII of A. kochi = 100.
 PLASMODIUM EYLESI 105

TABLE 9.—Comparison of salivary gland infection rates of Plasmodium eylesi in Anopheles kochi and A. maculatus (gut infection rates were 27
oocysts per gut for each species).

A. kochi A. maculatus
Days after
infection Positive/ Positive/
PGI* PGI
Dissected Dissected
8.5 0/24 0/10
9.5 16/34 3.4 7/45 2.1
10.5 24/36 3.3 7/18 2.1
11.5 22/37 3.2 21/32 2.4
12.5 15/20 2.7 18/35 2.9
13.5 7/17 3.7 8/23 3.0
14.5 6/14 2.5 2/10 1.5

Totals 90/182 3.2 63/173 2.5

• PGI = Positive Gland Index = Average gland rating of salivary glands found to be positive.

FIGURE 26.—Relationship of parasitemia to mosquito infection in two different gibbons, Hylobates lar, infected with
Plasmodium eylesi.

REFERENCES
BENNETT, G. F., 1968. Personal communication.
WARREN, McW., BENNETT, G. F., and SANDOSHAM, A. A., WARREN, McW., BENNETT, G. F., SANDOSHAM, A. A., and
1965. A new malaria parasite from the white-handed COATNEY, G. R., 1965a. Plasmodium eylesi sp. nov.,
gibbon, Hylobates lar lar in Malaya. Singapore Med. J. a tertian malaria parasite from the white-handed gibbon,
6 : 50. Hylobates lar. Ann. Trop. Med. Parasit. 59 : 500-508.
This page intentionally left blank.
 8
Plasmodium gonderi Sinton and Mulligan, 1933

THIS parasite was first seen by Gonder proceeded to study it. They, as Gonder and
and von Berenberg-Gossler (1908) in the blood Rodenwalt before them, found its periodicity to
of a mangabey, Cercocebus fuliginosus (= C. be tertian. This being true, it could not be
atys), housed in the Hamburg Zoo. These considered as belonging to the inui group of
malaria parasites and, therefore, they raised it to
authors identified the organism as Plasmodium
specific rank under the name Plasmodium
kochi. Berenberg-Gossler saw the parasite again
gonderi.
in 1909 in the same host, and, in two long-tailed
According to Garnham et al (1958), Duke,
green monkeys which Sinton and Mulligan
in 1956, found a malaria parasite in a drill
(1933) identified as Cercopithecus (C. sabaeus).
Each of the Berenberg-Gossler papers concerned (Mandrillus leucophaeus), taken in the
with this parasite was illus trated with beautiful Cameroons, and succeeded in infecting other
drills with it. An infected animal was eventually
colored plates comprising some 72 figures. In
sent to Garnham who, after careful study,
1910, Gonder and Rodenwalt again studied the
concluded that the parasite was P. gonderi. The
parasite in the natural host and pointed out its
significance of this find was that it expanded the
morphological resemblance to P. vivax and its
range of the parasite some one thousand miles
tertian periodicity.
northwest of its then known habitat. The
Sinton and Mulligan in 1932-33 did a
following year (1959) Bray encountered the
complete review of all the known malarias from
same parasite in mangabeys (Cercocebus
the lower monkeys (Cercopithicidae and
fuligin osus) in Liberia which further extended its
Colobidae) and concluded that the parasite
range to include the west coast of Africa from
described by Gonder and von Berenberg-Gossler
the mouth of the Congo river to Liberia.
was a true Plasmodium allied to the P. inui
group. They proposed the name P. inui gonderi.
At that time, it was not recognized that P. inui
had a 72-hour cycle. Rodhain and van den
Berghe (1936) isolated the parasite from a
Cercocebus galeritus agilus from the Congo and

107
 PLASMODIUM GONDERI 109

Cycle in the Blood a suggestion of a vacuole. The pigment is

clumped into a yellow-gold to black central
PLATE IX mass. The cytoplasm of the host cell is
The young merozoites invade the blood hypochromic almost to the point of being
stream and prefer to enter reticulocytes, inapparent so that the schizont may appear free
according to Rodhain and Lassman (1939) (Figs. 22, 23).
where they are seen as small bodies which The young gametocytes appear as heavy
quickly grow into the "signet ring" stage. These rings with large deep-staining nuclei. As the
young forms, sometimes two in a cell (Fig. 3), or parasite grows, the cytoplasm becomes dense
as many as four, show a pale blue cytoplasm and and the presence of prominent dark pigment sets
a deep red nucleus with no enlargement of the it off from the asexual parasites. The
host cell (Figs. 2-4). With further growth of the macrogametocyte stains a deep blue with
parasite, Schüffner’s stippling appears in the scattered pigment. The nucleus, generally
cytoplasm. The host cell is increased in size and eccentric, stains a deep red generally with a
there may be some distortion (Figs. 5, 6, 8). The lighter portion toward the center. The parasite
parasite displays a large vacuole, except in the may not entirely fill the enlarged host cell (Fig.
amoeboid forms (Fig. 10) and fine to granular 24). The mature microgametocyte stains a light
greenish-brown scattered pigment which may be purplish-pink with dark pigment granules
located along the periphery. In the older forms, scattered in the cytoplasm with some tendency
the cytoplasm stains a deeper blue; the nucleus to collect toward the periphery. The nucleus is
is larger, irregular to bar-shaped, and takes a somewhat diffuse and occupies a large part of
deep red stain, often with a lighter stained area. the parasite; it takes a slightly purplish-pink
The pigment is now in small aggregates (Figs. stain and encloses a deep red oval body. This
14, 15) which Rudzinska et al (1960) showed by parasite, too, may fill the host cell which is
electron microscopy were actually in individual somewhat enlarged (Fig. 25).
food vacuoles due to intracellular phagotrophy. The parasite has a 48-hour cycle.
Host cell stippling is prominent (Figs. 11-14).
Further growth produces the young
schizont. In the early nuclear divisions, the Sporogonic Cycle
chromatin appears as flat-oval to irregular red PLATE X
masses located on the periphery of the parasite
(Figs. 16, 17). The pigment is more condensed According to Garnham (1966), each gamete
but scattered. Stippling is prominent. The older produced by the exflagellating gametocyte
schizont may almost fill the host cell and shows measures about 16 µ in length with a dot at one
a purplish cytoplasm with deeper reddish-purple end, and, what is taken to be, two nuclei near the
nuclei. The nuclei still appear as if on the center. The ookinetes appear in the gut of the
periphery of the parasite and some look as if mosquito at about 21 hours after feeding. The
they were about to escape from it (Fig. 20). The parasite is about 12 µ long and, when fully
mature schizont may not fill the host cell. There mature, pigment collects at the broad end as a
may be 12 to 20 merozoites, generally about 16, brown mass in a yellowish sac. A clear circular
and each one appears to have a purple-staining
broad end which occupies 1/3 to 1/2 of the body
with a lighter trailing area sometimes with only

PLATE IX.—Plasmodium gonderi.

Fig. 1. Normal red cell. Figs. 16-20. Developing schizonts.
Figs. 2-4. Young trophozoites. Figs. 21-23. Mature schizonts.
Figs. 5-11. Growing trophozoites. Fig. 24. Mature macrogametocyte.
Figs. 12-15. Mature trophozoites. Fig. 25. Mature microgametocyte.
110 PRIMATE MALARIAS

PLATE X.—Developing oocysts of Plasmodium gonderi in Anopheles freeborni mosquitoes. X 580.

Fig. 1. 5-day oocyst. Fig. 6 10-day oocyst.
Fig. 2. 6-day oocyst showing scattered pigment. Fig. 7 10-day differentiating oocyst.
Fig. 3 7-day oocyst. Fig. 8. 11-day oocyst showing atypical form of
Fig. 4. 8-day oocyst showing small vacuoles. differentiation.
Fig. 5. 9-day oocyst showing a large number of small Fig. 9. Prematurely ruptured 11-day oocyst showing
vacuoles. sporozoites attached to sporoblastoid body.
 PLASMODIUM GONDERI 111

vacuole is generally present in the cytoplasm; oocyst development appeared to be normal in

the nucleus is not too well defined. the A. b. balabacensis, sporozoites did not
Bano (1959) studied the early sporogony of appear in the salivary glands until day 16, and in
P. gonderi in Anopheles aztecus and reported some lots, not until day 20.
that at 50 hours the oocysts measured about 12 µ A comparison of the oocyst growth curve
in diameter. At 58 hours, as a result of mitosis, of P. gonderi with that of P. cynomolgi (Fig. 27)
the haploid number of chromosomes was shown indicates that these species in A. freeborni are
to be three (two large and one small). Growth similar in size through 11 days of incubation.
proceeds rapidly at 28° C. At 6 days, the oocysts However, sporozoites were present in the
measured 25 µ, at 7 days 40 µ and when mature, salivary glands of mosquitoes infected with P.
up to 60 µ. Nine days after the blood meal, cynomolgi two days earlier than with P. gonderi.
sporozoites, measuring about 10 µ in dried The sporozoites were shown to be infective
films, were in the salivary glands. because the infection was transmitted to rhesus
We have observed the oocyst growth in six monkeys by the bites of A. freeborni (3 times),
species of anophelines when incubated at 25° C A. maculatus (once), A. stephensi (3 times), and
(Table 10). In A. freeborni, the mean oocyst A. b. balabacensis (twice). The prepatent periods
diameter at day 4 was 13 µ with a range of 9 to of these nine transmissions ranged from 9 to 17
19 µ. The oocysts continued to grow so that by days with a mean of 12.7 days. Garnham et al
day 12, the mean diameter was 65 µ, with a (1958) reported prepatent periods of 8 days in
range of 41 to 94 µ, and sporozoites were each of two M. mulatta monkeys infected by the
present in the salivary glands. intravenous inoculation of infected salivary
In the other mosquitoes, A. stephensi, A. glands from A. aztecus mosquitoes.
maculatus, A. b. balabacensis, A.
quadrimaculatus, and A. atroparvus, the oocyst Cycle in the Tissue
diameters were within the limits found in A.
freeborni. Sporozoites were present in the
We have tried to demonstrate the EE cycle
salivary glands of the A. quadrimaculatus and A.
of P. gonderi on one occasion but met with
atroparvus on day 12; in A. maculatus on day
13; and in A. stephensi on day 15. Although the

TABLE 10.--Oocyst diameters of Plasmodium gonderi in Anopheles freeborni, A. stephensi, A. maculatus, A. b. balabacensis, A.
quadrimaculatus, and A. atroparvus.

Days after
A. freeborni A. stephensi A. maculatus A. b. balabacensis A. quadrimaculatus A. atroparvus
Infection
No. Range* Mean No. Range Mean No. Range Mean No. Range Mean No. Range Mean No. Range Mean
4 100 8-19 13 100 9-25 14 100 8-18 13 100 8-19 13 100 9-26 16
5 100 11-25 19 100 12-31 22 100 12-27 20 100 12-26 20 100 12-26 19 8 14-18 16
6 204 12-34 23 100 18-32 25 222 12-32 23 216 12-33 23 102 18-33 25 49 18-31 24
7 100 13-30 22 88 15-27 21 111 18-35 28 100 14-28 22
8 100 18-51 37 100 21-54 38 121 18-59 39 101 18-50 32 99 20-55 40
9 100 30-64 50 41 22-41 33 100 24-61 44 100 24-57 42 101 24-53 39 158 24-63 44
10 100 24-72 58 100 30-77 53 100 24-72 45 100 24-77 50 100 24-72 50 8 40-59 48
11 100 30-85 56† 100 41-89 62† 100 30-65 50 100 41-85 61† 100 35-83 60† 100 33-70 49†
12 100 41-94 65†** 100 30-77 55† 100 35-89 61† 100 41-94 63† 100 35-85 63†** 100 30-80 57†**
13 100 41-89 68†** 100 22-89 59† 100 41-107 70†** 90 35-104 71† 100 18-100 61†**
14 100 35-100 71†** 100 30-94 65† 100 33-94 63†** 100 34-94 66† 100 51-98 71†**
15 100 44-81 67†** **
Totals 1304 8-100 841 9-94 1210 8-107 1238 8-104 1104 9-100 431 14-80

* Measurements expressed in microns.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
112 PRIMATE MALARIAS

FIGURE 27.—Mean oocyst growth curve and ranges in oocyst diameters of Plasmodium cynomolgi and P. gonderi in Anopheles
freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

failure even though the test animal developed an contained in an oval outline. The size of these
infection after a suitable prepatent period. bodies was variable, the largest was 27 by 32 µ,
Garnham et al (1958) were able to demonstrate and estimated to carry about two thousand
the EE cycle in the rhesus monkey on days 5, 7, merozoites.
and 8 and reported that the 5-day schizonts were It is not known if there are secondary EE
within a definite limiting membrane surrounding bodies in the life-cycle of P. gonderi.
a granular cytoplasm with about 20 nuclei. The
largest schizont measured 13 by 19 µ. Seven-day
forms were generally oval in shape with a Course of Infection
regular outline. The largest ones measured 18 to
27 µ. These forms showed numerous large dense According to Garnham et al (1958), blood-
spherical to rod-shaped nuclei; the cytoplasm induced infections in the rhesus monkey are
was granular and without vacuoles. characterized by initial high parasitemias which
The eight-day forms represented nearly decline slowly during the following weeks and
mature, mature, and post-mature parasites. They then persist; parasites were found easily, in thin
expressed no decided increase in size over the 7- films, after twelve months. The peak of
day forms but there were many more nuclei schizogony was found to occur about mid-day.
packed closely together. The nuclei were small Infected animals showed no signs of illness.
and appeared as densely stained granules in a Zuckerman (1960), on the other hand, reported
paler matrix. Some of the schizonts showed that P. gonderi produced an excessive degree of
lobations. The cytoplasm was granular and anemia in these animals.
 PLASMODIUM GONDERI 113

In our studies, the course of infection was reported it from the drill, Mandrillus
followed in 25 M. mulatta monkeys; 17 were leucophaeus.
infected by the inoculation of parasitized blood Numerous investigators have successfully
and 8 by the inoculation of sporozoites (Fig. 28). transferred P. gonderi to the rhesus monkey, M.
In the former animals, the peak parasitemia mulatta, via blood and by sporozoites. It was
(approximately 140,000/mm3) occurred after passaged by blood to Papio anubis, P. jubilaeus,
about 10 days of parent parasitemia. The and Cercopithecus aethiops by Rodhain and van
parasite count then declined slowly to a more or den Berghe (1936). We have also infected M.
less persistent level. After 60 days of patent radiata by blood inoculation.
parasitemia, the median count was about Rodhain and van den Berghe (1936) made
5,000/mm3. one attempt to transfer P. gonderi to man by
In the 8 monkeys infected by sporozoite- blood inoculation but without success. We
inoculation, the peak parasite count obtained on attempted to transmit this species to 8 volunteers
the 10th day of patent parasitemia by mosquito bite; all attempts failed with
(approximately 190,000/mm3). The parasite observation continued for 180 days.
count then declined but to a lower level than The natural vector of P. gonderi is
found in the blood-induced infections. The unknown, but being an African-based parasite, it
median parasite count at 60 days was about would be expected that A. gambiae would
350/mm3. None of the animals required transmit the infection readily. However, Bray
chemotherapy for survival. (1959), given favorable conditions was unable to
obtain infections. Rodhain and van Hoof (1940)
Host Specificity successfully transmitted the infection using A.
atroparvus. Garnham et al (1958) obtained
The natural hosts of P. gonderi are the transmission using A. aztecus. We have been
mangabeys and drills found on the west coast of able to infect A. freeborni, A. maculatus, A. b.
Africa and in the Cameroons. It was reported balabacensis, A. stephensi, A. quadrimaculatus,
from mangabeys, Cercocebus fuliginosus (= A. atroparvus, A. sundaicus, and A. albimanus.
atys) by Gonder and von Berenberg-Gossler The level of susceptibility varied (Table 11),
(1908), and from C. galeritus, C. aterrimus, and with A. freeborni, A. b. balabacensis, A.
C. atys by Bray (1963). Garnham et al (1958) maculatus, A. stephensi, and A. quadrimaculatus
being readily susceptible whereas the other
species were not.

FIGURE 28.—Median curves of the parasitemia of blood-induced (17 animals) and sporozoite-induced (8 animals) infections
of Plasmodium gonderi in Macaca mulatta monkeys.
114 PRIMATE MALARIAS

Immunity and Antigenic gonderi give a fluorescent antibody cross-

reaction at only a low level to P. fieldi antigen
Relationships (mean reciprocal titer ratio of 100:41) and much
lower levels of reactivity to other primate
Garnham and Bray (1955) reported no malaria antigens (Collins et al, 1966). In the
cross-immunity between P. gonderi and P. reverse procedure, P. gonderi antigen produces
cynomolgi in that a monkey cured of one low level responses to P. cynomolgi, P. fragile,
infection was completely susceptible to infection and P. inui antisera (mean reciprocal titer ratios
with the other species. Voller et al (1966) of 100:36, 100:35, and 100:32, respectively).
reported that monkeys originally infected with Although antisera to P. falciparum and P.
P. cynomolgi or with P. knowlesi could be malariae responded to the P. gonderi antigen,
infected with P. gonderi and that a normal the cross reactions were much lower than the
infection developed. homologous responses (Collins et al, 1966a).
Data from serology show that antisera to P.

TABLE 11.—Comparative infectivity of Plasmodium gonderi to eight species of Anopheles.

Number Percent
Mosq. species Number mosquitoes infection GII**
Comparison* tests ratios
Standard Other Standard Other
F-1 100
F-1 : Bal 11 121 106 50.4 44.3 87.0
F-1 : Mac 22 245 236 58.8 64.4 84.7
F-1 : St-1 9 69 82 73.9 75.6 55.4
F-1 : Q-1 28 563 569 59.0 43.8 50.6
F-1 : Atro 7 210 186 58.6 8.6 6.5
F-1 : Sund 7 178 143 64.0 9.8 2.6
F-1 : Alb 9 124 117 25.8 2.6 0.3

* F-1 = Anopheles freeborni, Bal = A. b. balabacensis, Mac = A. maculatus, St-1 = A. stephensi, Q-1 = A. quadrimaculatus, Atro = A.
atroparvus, Sund = A. sundaicus, Alb = A. albimanus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. freeborni to another
species where the GII of A. freeborni = 100.

REFERENCES
BANO, L., 1959. A cytological study of the early oocysts of seven including its sporogony in Anopheles aztecus and its
species of Plasmodium and the occurrence of post- pre-erythrocytic schizogony in the rhesus monkey.
zygotic meiosis. Parasitol. 49 : 559-585. Trans. Roy. Soc. Trop. Med. & Hyg. 52 : 509-517.
BERENBERG-GOSSLER, VON H. V., 1909. Beitrage zur GARNHAM, P. C. C., 1966. Malaria parasites and other
naturgeschichte der malariaplasmodien. Arch. f. Protist haemosporidia. Blackwell Scientific Publications.
(Jena) 16 : 245-280. Oxford.
BRAY, R. S., 1959. Range of Plasmodium gonderi. Trans. Roy. GONDER, R. and BERENBERG-GOSSLER, VON H. V., 1908.
Soc. Trop. Med. & Hyg. 53 : 300. Untersuchungen über malaria-plasmodien der affen.
Malaria-Intem. Arch., Leipzig 1 : 47-56.
BRAY, R. S., 1963. Malaria infections in primates and their
importance to man. Ergeb. Mikrob. Immunit. Experim. GONDER, R. and RODENWALDT, E., 1910. Experimentelle
Therap. 36 : 168-213. untersuchungen über affenmalaria. aentralbl. f. Bakt. I.
Abt. Orig. 54 : 236-240.
COLLINS, W. E., SKINNER, J. C., and GUINN, E. G., 1966.
Antigenic variations in the plasmodia of certain RODHAIN, J ., and VAN DEN BERGHE, L., 1936. Contribution
primates as detected by immuno-fluorescence. Am. J. a l'etude des plasmodiums des singes africains. Ann.
Trop. Med. & Hyg. 15 : 438-485. Soc. Belg. Med. Trop. 16 : 521-531.
COLLINS, W. E., JEFFERY, G. M., GUINN, E., and SKINNER, RODHAIN, J. and LASSMAN, P., 1939. Le comportement
J. C., 1966a. Fluorescent antibody studies in human different de Plasmodium cynomolgi Mayer et de
malaria. IV. Cross-reactions between human and simian Plasmodium gonderi Rodhain et van den Berghe vis-a-
malaria. Am. J. Trop. Med. & Hyg. 15 : 11-15. vis des reticulocytes. C. R. Sac. BioI. 132 : 71-75.
RODHAIN, J. and VAN HOOF, T., 1940. Contribution a l'etude
GARNHAM, P. C. C. and BRAY, R. S., 1955. Absence of cross-
des Plasmodium des singes africains. Le comportement
immunity between Plasmodium cynomolgi and
different des PI. gonderi et PI. kochi chez les
plasmodium gonderi. Ind. J. Malariol. 9 : 255-260.
moustiques. Bull. Sac. Path. Exot. 33 : 107-113.
GARNHAM, P. C. C., LAINSON, R. and COOPER, W., 1958. RUDZINSKA, M. A., BRAY, R. S. and TRAGER, W., 1960.
The complete life cycle of a new strain of Plasmodium Intracellular phagotrophy in Plasmodium falciparum
gonderi from the drill (Mandrillus leucophaeus), and Plasmodium gonderi. J. Protozoal. 7 : 24-25.
 PLASMODIUM GONDERI 115

REFERENCES—Continued
SINTON, J. A., and MULLIGAN, H. W., 1932 and 1933. A
critical review of the literature relating to the
identification of the malarial parasites recorded from
monkeys of the families Cercopithecidae and
Colobidae. Rec. Malar. Surv. India III: 357-380; 381-
444.
VOLLER, A., GARNHAM, P. C. C., and TARGETT, G. A. T.,
1966. Cross immunity in monkey malaria. J. Trop.
Med. & Hyg. 69 : 121-123.
ZUCKERMAN, A., 1960. Blood loss and replacement in
plasmodial infections. III. Plasmodium cynomolgi,
Plasmodium gonderi, and Plasmodium knowlesi in
Macaca mulatta mulatta, the rhesus monkey. J. Infec.
Dis. 106 : 123-140
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 9
Plasmodium hylobati Rodhain, 1941

ON the 2nd of June 1939, Dr. so that further studies could be carried out.
Professor Garnham also believed the parasite to
Jerome Rodhain examined the blood of a
be the long-sought P. hylobati and, anxious to
gibbon, Hylobates lensciscus Geoff. from Java
have a demonstration of the exoerythrocytic
and in the blood, he found a few parasites of a
stages, he sent the animal to us in Chamblee,
malaria. He made a second examination five
Georgia, because we maintained a colony of A.
days later and saw that the parasites were more
b. balabacensis believed to be a potential vector.
numerous. They then disappeared and he never
The gibbon was received at Chamblee in good
saw them again. From the material collected on
health and carrying a low-grade infection of a
those two occasions, Rodhain described the
malaria. Later, the animal was splenectomized;
parasite and named it Plasmodium hylobati in
and with increased parasitemia, it was evident
1941. His description lacked completeness, in
that the parasite was P. hylobati. Under those
that he did not see microgamecocytes; but, it
favorable conditions, mosquitoes were allowed
does allow for the recognition of the parasite. He
to feed on the animal. At the same time, there
predicted that the asexual cycle was quartan.
was ample material for studying the periodicity
According to Garnham (1966), Wenyon saw a
of the asexual cycle and for transfer of
similar parasite in other species of gibbons
parasitized blood to monkeys. Five species of
which had died in the London Zoo in 1946 and
mosquitoes were allowed to feed on the gibbon
he, Garnham, saw it in films sent to him from
whereupon, its malaria was eliminated by
Sarawak. This extra material was evidently used
treatment with chloroquine. When the test
by Garnham in writing his description of the
mosquitoes were found infected, they were
parasite.
dissected and their sporozoites inoculated
The parasite has not been found in
intravenously into the original gibbon and,
peninsular Malaysia or in Thailand and none of
intrahepatically at laparotomy, into an owl
us had ever seen it. In fact, we despaired of
monkey (Aotus trivirgatus). Following exposure
locating material for a colored plate, and other
to infection, liver biopsies were done on day 7
studies, when a fortunate happening occurred. A
and 14 in the gibbon and on day 7 in the owl
mature male Hylobates moloch from North
monkey. Exoerythrocytic parasites were found
Borneo was received at the University of
in each of the animals. Human volunteers were
Singapore and found to be infected with a
exposed to infection through bites of infected
malaria tentatively identified as P. hylobati. Dr.
mosquitoes.
Zaman, knowing the rarity of the parasite, sent
the infected animal to Professor Garnham at the
London School of Hygiene & Tropical Medicine

117
 PLASMODIUM HYLOBATI 119

Cycle in the Blood of moderately coarse, yellowish-black granules

(Figs. 21, 23, 24). The mature schizont has from
PLATE XI 12 to 20 merozoites, which may be arranged to
form what Rodhain called a rosette; 14 to 16 is
The youngest erythrocytic parasites consist the most common number. When the merozoites
of a prominent nucleus and a small fragment of are completely formed, the pigment assumes a
cytoplasm (Fig. 2). As the young parasite grows, single dense yellowish-black mass (Figs. 25,
the nuclear mass remains essentially unchanged 26).
while the cytoplasm increases both in volume Young gametocytes are difficult to
and in the area of the host cell it occupies (Figs. distinguish from young and mature trophozoites.
3-6). Multiple chromatin masses are not However, the young microgametocyte does
uncommon (Figs. 5, 6). Dual invasion of a show some of the rare staining qualities of the
single host cell has been observed (Fig. 4) but cytoplasm fairly early (Fig. 29).
the condition is not considered characteristic of The mature macrogametocyte fills the host
the parasite. The nucleus begins to change as the cell and displays smooth, uniformly staining
trophozoite continues to develop, stretching blue cytoplasm. The pigment is in moderately
around the periphery of the vacuole or branching coarse, randomly distributed granules. The
as it grows (Figs. 7-10). Pigment first appears as nucleus is circular to oval, dense, and usually
one or two accumulations of very fine, grayish- located at the periphery of the cell (Fig. 28).
black granules causing areas of the cytoplasm to The fully adult microgametocyte fills the
appear more gray than blue in the Romanowsky- host cell and the cytoplasm as well as the
stained young trophozoite (Fig. 10). The nucleus stains a bright rose-pink. The nucleus is
pigment becomes identifiable though not large and diffuse with a dense central mass.
prominent in the young adult forms (Fig. 11). As Frequently, the nucleus and the cytoplasm stain
the trophozoite approaches maturity, the a uniform pink. Separation is achieved because
cytoplasm thickens, takes a deeper stain, and the pigment granules are randomly distributed
nucleus becomes more dense (Figs. 10-12). The through the cytoplasm but they are absent from
pigment remains scarce and frequently hard to the nucleus (Fig. 30).
identify. There is no obvious host cell The asexual cycle in the blood occupies 48
enlargement. Schüffner’s stippling or other types hours.
of inclusions in the host cell have not been
observed. Schizogony is unremarkable except
that the host cell cytoplasm sometimes appears Sporogonic Cycle
greatly depleted, leaving the parasite enmeshed PLATE XII
in a network of fine cytoplasmic threads (Figs.
16-18, 20, 23). In some cases, the nuclear
The natural vector of this parasite is
material is dominated by thick, tortuous threads
unknown. However, we were able to study the
or globs (Figs. 20, 24). As the schizont
sporogonic cycle in five laboratory reared
approaches maturity the pigment becomes more
species of mosquitoes: A. b. balabacensis from
abundant and begins to accumulate into groups

PLATE XI.—Plasmodium hylobati.

Fig. 1. Normal red cell. Figs. 14-24. Schizogonic stages
Figs. 2-13. Developmental stages of the trophozoite. Fig. 25. Submature schizont.
Figs. 2-4. Rings stages. Fig. 26. Mature schizont.
Figs. 5, 6. Young trophozoites. Figs. 27-30. Gametocytes.
Figs. 7-9. Adolescent trophozoites. Fig. 27. Immature macrogametocyte.
Fig. 10. Young adult trophozoite. Fig. 28. Mature macrogametocyte.
Figs. 11, 12. Mature or adult trophozoites. Fig. 29. Immature microgametocyte.
Fig. 13. Mature trophozoite with dividing nucleus. Fig. 30. Mature microgametocyte.
120 PRIMATE MALARIAS

PLATE XII.—Developing oocysts and sporozoites of Plasmodium hylobati in Anopheles b. balabacensis mosquitoes. X 580.
Fig. 1. 8-day oocysts. Fig. 5. 12-day differentiated oocyst. Sporozoites free in
Fig. 2. 9-day oocysts. fluid near gut.
Fig. 3. 10-day oocysts. Fig. 6. Sporozoites present near salivary gland tissue 12
Fig. 4. 11-day oocyst showing differentiation. days after feeding.
 PLASMODIUM HYLOBATI 121

Thailand, A. stephensi from India, A. maculatus made at a different extrinsic incubation

from Malaysia, A. freeborni and A. temperature.
quadrimaculatus from the United States. The sporozoites of P. hylobati in A. b.
Observations began 6 days after feeding and balabacensis were infective because they
continued through day 13. The extrinsic initiated infection in a gibbon. The prepatent
incubation temperature was 25° C. period was nine days.
The results of the oocyst measurements are
presented in Table 12. In A. b. balabacensis, on Cycle in the Tissue
day 6, the mean oocyst diameter was 15 µ with a
range of 11 to 20 µ. The oocysts continued to PLATE XIII
grow so that by day 12, the average size was 53
µ with a range of 30 to 70 µ. Sporozoites were Sodeman et al (1971) ably described the
first seen in the salivary glands on day 12. tissue stages of Plasmodium hylobati found on
The examination of the oocyst diameters in day 7 and 14 in the gibbon and those seen on
the other test mosquitoes indicated that the day 7 in the owl monkey.
growth rate of the parasite was similar in each of The EE bodies were round to elliptical in
the five species. Sporozoites were present in the shape with smooth edges. Some were retracted
salivary glands of A. stephensi on day 12 and in from their surrounding liver cell, probably as a
A. freeborni and A. maculatus on day 13. No result of fixation. The nuclei were round,
sporozoites were found in the salivary glands of although, infrequent bar-shapes were seen. The
the A. quadrimaculatus through day 16. nuclei stained magenta, measured 0.5-1.5 µ in
In comparing the extrinsic development of diameter, and were evenly distributed through
P. hylobati in A. b. balabacensis with P. the cytoplasm. The latter was granular in texture
cynomolgi, from the rhesus monkey (Fig. 29), and stained pale blue. In many of the parasites
and P. jefferyi, from the gibbon (Fig. 30) one irregular shaped, dark blue aggregates
finds that the P. cynomolgi oocysts are much ("flocculi") were scattered diffusely through the
larger and that its sporozoites appear in the cytoplasm but this feature was not universal.
salivary glands two days earlier. Plasmodium The flocculated material contained small holes.
jefferyi oocysts are considerably smaller than Parasitized liver cells were enlarged and their
those of P. hylobati and its sporozoites appear in nuclei displaced peripherally. There was no
the glands one day later. nuclear enlargement of the parasitized liver
Comparison studies with P. eylesi could not cells; and, vacuolation of the cytoplasm was
be made because the growth studies with it were seen only occasionally. No mononuclear cell or

TABLE 12.--Oocyst diameters of Plasmodium hylobati in Anopheles b. balabacensis, A. stephensi, A. freeborni,

A. maculatus, and A. quadrimaculatus.

Days after A. b. balabacensis A. stephensi A. freeborni A. maculatus A. quadrimaculatus

Infection
No. Range* Mean No. Range Mean No. Range Mean No. Range Mean No. Range Mean

6 150 11-20 15
7 100 12-30 21 90 13-25 19 107 12-25 17 105 12-26 16 117 12-27 20
8 100 15-33 26 111 17-35 24 105 13-35 25 113 14-35 24 128 18-40 29
9 100 18-47 35 124 19-45 33 135 13-42 30 111 14-37 24 109 18-45 34
10 100 25-55 41† 100 27-55 42† 100 19-47 35 114 20-52 34 150 21-55 37
11 100 28-59 45† 100 24-65 39† 110 20-47 34 111 19-63 46† 100 21-61 41†
12 100 30-70 53†** 108 22-65 45†** 100 26-68 51† 114 20-64 45† 100 32-72 51†
†** †** †

Totals 750 11-70 †** 633 13-25 †** 657 12-68 †** 668 12-64 †** 704 12-72 †

* Measurements expressed in microns; incubation temperature 25° C.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
122 PRIMATE MALARIAS

FIGURE 29.—Range in oocyst diameters and the mean oocyst diameter curve of Plasmodium hylobati and P. cynomolgi in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

acute inflammatory cell infiltrate was present. vacuoles were present and vague clefts were
The seven day forms (Figs. 1 & 2) observed in only one body.
sectioned at 3 µ extended through 5 sections; Only two exoerythrocytic bodies were seen
those sectioned at 6 µ, through 2.6 sections. The in the 7-day material from the owl monkey
average size was 15.2 µ in length and 11.3 µ in (Figs. 5 & 6). In the 6 µ sections one body was
width. The space occupied by the EE bodies complete. It extended through 4 sections and
averaged 15.8 µ in length and 11.7 µ in width. measured 25.2 µ by 14.4 µ. Sections of the EE
The limiting membrane was thin but distinct. body measured up to 22.8 µ by 19.2 µ. There
Only two vacuoles were seen in fifty specimens was a distinct limiting membrane. Flocculi were
examined; they measured 5 µ in diameter and large and more frequent than in the gibbon
were clear. material. The parasites exhibited small, clear
Only four EE bodies were seen in the 14- vacuoles. The nuclei were round and measured
day material (Figs. 3 & 4). In the 6 µ sections 1-1.5 µ. Clefts were present in the cytoplasm.
they extended through 4 sections. Their average It seems safe to say that the morphology of
size was 26.1 µ in length and 18.6 µ in width. P. hylobati is not unlike that of the other primate
The space occupied by the parasites in the liver malarias (Held et al, 1967). If one compares the
cell averaged 26.4 µ in length and 19.5 µ in 6-day parasite of P. jefferyi, the only other EE
width. A limiting membrane was distinct and parasite of a gibbon so far described (Sodeman
flocculi were present, however, they were small
and less frequent than in the 7-day forms. No
 PLASMODIUM HYLOBATI 123

PLATE XIII.—Exoerythrocytic bodies of Plasmodium hylobati. X 580.

Figs. 1, 2. 7-day EE body in liver tissue of gibbon, Hylo- Figs 5, 6. 7-day EE bodies in liver tissue of monkey, Aotus
bates moloch. trivirgatus.
Figs. 3, 4. 14-day EE bodies in gibbon.
124 PRIMATE MALARIAS

et al, 1969), with the 7-day forms of P. hylobati transient and is eliminated in a few weeks.
one sees considerable difference in the average Infections in Macaca nemestrina and M.
size which suggests, if there were sufficient fascicularis monkeys have also been obtained by
comparative measurements, that they might be the inoculation of parasitized blood. The
separated at day 7 on the basis of size. The host parasitemias were of a low level. Gametocytes
animal exhibited a patent infection on day 9 yet were produced, and numerous mosquito
EE bodies were found on day 14. We are not feedings were carried out; none of the
able to say, because of their large size, whether mosquitoes became infected.
these were 'left-over' EE bodies of the initial
generation, or, possibly, second generation EE Host Specificity
bodies because some were of the same size as 7-
day forms; maybe both. This will not be
The natural host of P. hylobati is the
resolved until we have additional information on
gibbon; infections have been reported in H.
the tissue stages of this and other ape malarias.
moloch from Java and North Borneo.
Experimentally, M. mulatta, M. fascicularis, and
Course of Infection M. nemestrina have been infected by the
inoculation of parasitized blood.
We know very little about the course of the Two human volunteers were exposed to
infection in the normal host except that it does infection through the bites of A. b. balabacensis
become latent and that it is provoked to mosquitoes heavily infected with this parasite.
exacerbation following splenectomy. No patent infection was produced.
The parasite will infect splenectomized and The natural invertebrate host of P. hylobati
intact rhesus monkeys. Infection induced by is unknown. On an experimental basis, five
inoculation of parasitized blood in a species of anopheline mosquitoes have been
splenectomized animal is marked by a very high infected. Anopheles b. balabacensis was the
parasitemia (up to 28/100 RBC) which persists most susceptible followed by A. stephensi, A.
at a detectable level for up to four months. freeborni, A. maculatus and finally A.
Reinoculation has produced infections as high as quadrimaculatus. The intensity of the infections
8/100 RBC. In intact rhesus, the infection is varied from one mosquito species to another
(Table 13).
TABLE 13.--Comparative infectivity of Plasmodium hylobati in Anopheles b. balabacensis, A. stephensi, A. freeborni,
A. maculatus, and A. quadrimaculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

Bal 100
Bal : St-1 2 20 15 100 100 49
Bal : F-1 2 20 20 100 90 43
Bal : Mac 2 20 29 100 100 22
Bal : Q-1 2 20 47 100 68 7

* Bal = Anopheles b. balabacensis, St-1 = A. stephensi, F-1 = A. freeborni, Mac = A. maculatus, Q-1 = A. quadrimaculatus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII
of A. balabacensis to another species where the GII of A. balabacensis = 100.

REFERENCES
GARNHAM, P. C. C., 1966. Malaria parasites and other SODEMAN, T. M., CONTACOS, P. G., COATNEY, G. R., and
haemosporidia. Blackwell Scientific Publications, JUMPER, J. R., 1969. Studies of the exoerythrocytic
Oxford. stages of simian malaria. V. Plasmodium jefferyi. J.
HELD, J. R., CONTACOS, P. G., and COATNEY, G. R., 1967. Parasit. 55 : 1247-1252.
Studies of the exoerythrocytic stages of simian malaria. SODEMAN, T. M., CONTACOS, P. G., GARNHAM, P. C. C.,
I. Plasmodium fieldi. J. Parasit. 53 : 225-232. JUMPER, J. R., and SMITH, C. S., 1971. Studies of the
RODHAIN, J., 1941. Sur un Plasmodium du gibbon Hylobates exoerythrocytic stages of simian malaria VI.
lensciscus Geoff. Acta. Bioi. Beige 1 : 118-123. Plasmodium hylobati. J. Parasit. (in press)
 10
Plasmodium jefferyi Warren, Coatney, and Skinner, 1966

a new species and depicted what they saw in a

IN the case of this malaria, as is well executed colored plate.
true of the early history of many others, there is Shortly before the closing of the LPC, NIH
an interesting side-light. In 1964, Dr. A. A. laboratory in Kuala Lumpur in 1964, the dual
Sandosham, the Director of the Institute of infection, P. youngi and the then undescribed
Medical Research in Kuala Lumpur, Malaysia, species, was passed by inoculation of parasitized
and the senior author (GRC) were visiting an blood from G 31 to another gibbon (G 8).
area in north Malaysia when we chanced to meet Shortly thereafter, both animals were shipped to
a trapper who had, on numerous occasions, the Laboratory of Parasite Chemotherapy,
obtained animals for us. When queried as to why Section on Primate Malaria at Chamblee,
he had not sent in animals lately, he replied that Georgia. There, it was discovered that G 8 had a
his traps were in disrepair and that he had no high parasite count so blood was withdrawn and
money to pay for wire to fix them. He was given deep frozen. Each of the animals died a short
money immediately so his traps could be time later. The blood was left in the deep freeze
repaired; and, upon taking our leave, he was told until 1968 when a power failure early in that
to notify us immediately if he managed to catch year necessitated immediate action if the
a gibbon. The fates were kind. A young female specimens were to be saved. The blood was sent
gibbon, G 31, Hylobates lar, was brought to the to the senior author in New Orleans, Louisiana,
laboratory in July, and when her blood was and inoculated forthwith into a parasite-free
examined it was found to harbor Plasmodium gibbon, H. lar, (G 420), at the Delta Regional
youngi and a low-grade population of another Primate Research Center, Covington, Louisiana.
parasite which was morphologically different The animal (G 420) developed a patent
from any other gibbon malaria. In the hope of infection 14 days later. However, its infection
obtaining a heavier infection of the then was quite different from the one in the donor
undescribed species, the infection was animal (G 8) where P. youngi accounted for
transferred to a malaria-free gibbon, G 32, by more than 80 percent of the parasite population
the inoculation of parasitized blood. The original at the time blood was drawn for freezing. In G
description of the parasite to which Warren, 420, only one species of parasite was present
Coatney, and Skinner (1966) gave the name and, although it resembled P. jefferyi in many
Plasmodium jefferyi, in honor of their colleague, respects, other of its characteristics were quite
Dr. G. M. Jeffery, was based largely on material different. For example, fully mature schizonts,
from that animal. not seen in previous P. jefferyi infections, were
In reporting on this parasite, the authors abundant in the peripheral blood as were
were careful to point out the scarcity of young distinctive microgametocytes. In addition, the
schizonts and the complete lack of mature infection appeared to be of a fulminating type.
schizonts in the peripheral blood. They were Our first thought was that maybe the
convinced, however, that they were dealing with parasite resulted from a latent infection in G 420
triggered by the splenectomy and subsequent

125
126 PRIMATE MALARIAS

manipulations. The splenectomy had been understandable when one remembers that the
performed some 40 days prior to the transfer of original natural infection exhibited only a low-
parasitized blood and the prepatent period was grade parasitemia with the undescribed species
within reasonable limits, that is, 14 days. The (now carrying the designation P. jefferyi) and
idea of a latent infection in G 420 therefore that that state of affairs was not greatly
seemed remote; the question was unde venit, improved when the infection was transferred to
where did it come from? There was only one G 32.
possible answer. The parasite had come from the The presence of mature schizonts in the
blood of G 8. peripheral blood of G 420 and a
That animal, as mentioned earlier, had had microgametocyte of arresting characteristics had
a dual infection in which the predominant led us away from considering the parasite P.
parasite was P. youngi and a low-grade infection jefferyi. Later, the gaps and the inaccuracies in
with the then undescribed new species (= P. the original description were recognized.
jefferyi). The infection in G 420 was not P. Whereupon, Coatney, Orihel, and Warren
youngi--that parasite apparently failed to survive redescribed the parasite (1969) and included a
the freezing episode. Therefore, the parasite in G colored plate to show the complete asexual
420 was P. jefferyi but with characteristics not cycle, the true macrogametocyte, and the
observed and, consequently, not mentioned in distinctive microgametocyte.
the original description. The situation is
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 PLASMODIUM JEFFERYI 129

Cycle in the Blood parasite, and golden brown in color (Figs. 23,
24).
PLATE XIV The mature schizonts have a body size less
than that of the host cell (Fig. 25) and exhibit 10
The earliest ring forms display a deep red to 18 blue-stained nuclei. The gold-black
chromatin dot, measuring 1µ in diameter, pigment is clumped. The parasite at this stage
sometimes an accessory chromatin dot, and a often assumes bizarre shapes with the
delicate circle of blue-staining cytoplasm, or merozoites piled on each other.
there may be two chromatin masses of unequal The young macrogametocytes, 3 to 4 µ in
size. Marginal forms are rare, as are multiple diameter, display a deeply stained red nucleus,
infections in early infections. The host cell is not with blue cytoplasm and with, or without, a
enlarged (Figs. 2-6). Growth forms may occupy vacuole. Older forms may have one, sometimes
up to one-half or more of the host cell. The two, large vacuoles and fill or almost fill the
nucleus stains a reddish-purple and may lie host cell. The nucleus is bright pink with a deep
within a vacuole. The cytoplasm stains a pale reddish-purple bar or skein. The cytoplasm is
blue (Figs. 7-9). The older trophozoites are grayish-blue with evenly distributed dust-like
frequently paisley-shaped with the nucleus, pigment (Fig. 27). The adult forms virtually fill
sometimes double, at the broad end and, the host cell. Their cytoplasm is without
occasionally, an accessory chromatin dot. The vacuoles and stains a light blue; pigment is in
pigment is fine to seed-like and may be arranged greenish-gold granules scattered evenly. The
along the periphery of the parasite (Figs. 10, 16, nucleus is bright red with a deep red bar or
17). Multiple infections of the host cell are strand (Fig. 28).
common in developed infections (Figs. 5, 12, 14, The young microgametocytes, 3 to 4 µ in
15). diameter, have a deeply stained red nucleus,
Stippling is absent and there is no increase sometimes two nuclear masses, compact
in the size of the host cell. The cytoplasm of the cytoplasm, and only a suggestion of a vacuole
young schizont stains a pale blue and nearly fills next to the nucleus; the cytoplasm appears a
the host cell. The pigment is in dust-like very light brown. Older growth forms are
granules scattered throughout the cytoplasm. roughly ellipsoidal, jug-shaped with one side
The nuclei stain a deep red (Figs. 18, 19). The depressed. The cytoplasm is generally without
older schizonts are more compact and do not fill color and with fine dust-like pigment. The
the host cell. Their nuclei stain a deep red to nucleus stains a light pink with a red to purple
reddish-purple and number from 4 to 18. The bar or skein (Fig. 29). Adult forms are
youngest of these forms exhibit a jagged predominantly oval, sometimes circular, with a
periphery with pale blue cytoplasm confined to slightly ragged appearance. The nucleus is
the center of the parasite. Light brown pigment located at the small end of the parasite and stains
granules are distributed unevenly in the dark rose with a reddish-purple bar, band, or
cytoplasm (Figs. 20-22). Many of the 6-nucleate skein. The cytoplasm stains reddish-pink and is
forms display an eosinophilic ring reminiscent overlaid by a golden-brown bead-like pigment
of P. fieldi (Fig. 21). In the older forms, the sometimes arranged to present a stocking-cap
periphery is smoother, pigment granules are effect to the more bulbous portion of the
coalesced, massed toward the center of the parasite.

PLATE XIV.—Plasmodium jefferyi from the gibbon.

Fig. 1. Normal red blood cell. Figs. 25-26. Mature schizonts.
Figs. 2-6. Young trophozoites. Figs. 27, 28. Immature and mature macrogametocytes.
Figs. 7-17. Older trophozoites. Figs. 29, 30. Immature and mature microgametocytes.
Figs. 18, 19. Young schizonts. (Plate reprinted, courtesy of the Journal of Parasitology.)
Figs. 20-24. Older schizonts.
130 PRIMATE MALARIAS
 PLASMODIUM JEFFERYI 131

The parasite has a 48-hour periodicity. µ, versus 57 µ for A. b. balabacensis, and some
of the oocysts had differentiated. Sporozoites
Sporogonic Cycle were not seen in the salivary glands until day 17
and then at a low level and, in only one
PLATE XV mosquito.
In A. quadrimaculatus, the growth rate was
During the course of the infection of P. difficult to determine because of the limited
jefferyi in gibbon 420, mosquitoes were shipped number of oocysts. Oocyst differentiation was
by air from our laboratory in Chamblee, present by day 13 but salivary gland infections
Georgia, to New Orleans, Louisiana, and carried were not found through 17 days of observation.
to the Delta Regional Primate Research Center It thus appears that of the four test mosquitoes
in Covington, Louisiana, where they were only A. b. balabacensis was a favorable host for
allowed to feed on the animal; after feeding they P. jefferyi.
were returned by air. The total travel time did A comparison of the growth curve of P.
not exceed 30 hours. Upon return, the jefferyi with that of P. cynomolgi in A. b.
mosquitoes were held at 25° C for the remainder balabacensis mosquitoes (Fig. 30) shows that P.
of the extrinsic incubation period. Beginning on cynomolgi is considerably larger and completes
day 5 and continuing through day 14, sample its development approximately 3 days sooner
mosquitoes were examined for the presence of than does P. jefferyi. A comparison with another
oocysts (Collins and Orihel, 1969). gibbon malaria parasite, P. hylobati indicates
The oocyst diameters of P. jefferyi in four that P. jefferyi is much smaller but takes only
species of Anopheles are presented in Table 14. one day longer for sporozoites to appear in the
In A. b. balabacensis, on day five, the oocysts salivary glands.
had a mean diameter of 9 µ , with a range of 7 to As discussed later, it was not possible to
11 µ . They continued to grow so that on day 13, determine if the sporozoites were infective. Even
the mean diameter was 57 µ , with a range of 33 though EE bodies were produced after
to 77 µ . Sporozoites were present in the salivary inoculation of these sporozoites into a clean,
glands on day 13. splenectomized gibbon, no erythrocytic infection
In A. freeborni, the oocysts grew from a resulted.
mean diameter of 8 µ, on day 5 to a mean of 45
µ, on day 13. Although differentiation was
apparent by day 12, sporozoites were not found Cycle in the Tissue
in the salivary glands until day 15 and then at a PLATE XVI
very low level.
In A. maculatus, the oocysts grew at a Although four species of gibbon malarias
slower pace than in A. b. balabacensis and in A. are known, exoerythrocytic parasites of
freeborni. The mean diameter on day 13 was 30

Plate XV.—Developing oocysts and sporozoites of Plasmodium jefferyi in Anopheles b. balabacensis mosquitoes. X 580 (except
Figs. 1 and 2).
Fig. 1 4-day oocyst. X 1300. Fig. 9. 12-day oocysts showing two with normal
Fig. 2. 6-day oocyst showing peripheral pigment. development and one which has failed to develop.
X1300. Fig. 10. 12-day oocysts, one of which is showing early
Fig. 3. 7-day oocyst showing prominent pigment. differentiation.
Fig. 4. 8-day oocyst with pigment becoming less distinct. Fig. 11. 13-day oocyst showing more advanced
Fig. 5. 9-day oocyst. differentiation.
Fig. 6. 10-day oocyst. Fig. 12. 13-day oocyst showing full differentiation.
Fig. 7. 10-day oocysts. Fig. 13. Sporozoites from salivary gland tissue 14 days
Fig. 8. 11-day oocyst showing vacuole containing after feeding.
pigment.
132 PRIMATE MALARIAS

TABLE 14.—Oocyst diameters of Plasmodium jefferyi in Anopheles b. balabacensis, A. freeborni, A. maculatus, and
A. quadrimaculatus.

A. b. balabacensis A. freeborni A. maculatus A. quadrimaculatus

Days after
Infection
No. Range* Mean No. Range Mean No. Range Mean No. Range Mean

5 70 7-11 9 101 5-10 8 20 8-11 9 5 7-11 10

6 57 7-17 12 62 6-13 9 31 7-13 10 4 8-14 8
7 95 8-24 14 100 7-18 12 24 9-19 13 8 8-18 12
8 100 9-26 16 54 8-25 14 27 9-21 12 5 15-26 20
9 38 14-27 19 61 8-20 14 35 9-31 17 38 12-37 18
10 100 9-46 24 100 12-40 19 16 12-50 17 18 14-51 28
11 24 13-36 25 100 15-50 30 34 8-36 21 29 11-40 24
12 100 19-73 40† 29 14-59 31† 23 14-44 27 16 17-54 40
13 41 33-77 57†** 76 14-73 45† 56 19-64 30† 4 28-59 46†
14 80 12-61 35† 2 53-55 54†

* Measurements expressed in microns; incubation temperature 25° C.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.

FIGURE 30.—Mean oocyst growth curve and ranges in oocyst diameters of Plasmodium cynomolgi and P. jefferyi in Anopheles b.
balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
 PLASMODIUM JEFFERYI 133

PLATE XVI.—Exoerythrocytic bodies of Plasmodium jefferyi in Hylobates lar.

Fig. 1. 6-day exoerythrocytic body surrounded by Fig. 2. 6-day exoerythrocytic body containing two
mononuclear cell infiltration. X 580 prominent vacuoles. X 930

Plasmodium jefferyi were the first to be The EE bodies caused enlargement of the
described (Sodeman et al, 1969). An liver cell and pushed the normal sized nucleus to
experimental infection of P. jefferyi in a gibbon one side. Vacuoles were seldom found in
(H. lar pileatus) was induced by inoculating unparasitized liver cells. Focal mononuclear cell
sporozoites directly into the liver at laparotomy, infiltrates were scattered through the sections;
and, by sporozoites introduced intravenously. some were associated with portal regions.
On day 6, following the inoculations, a biopsy at Infrequently, EE bodies were found within these
the site of infection of the liver was taken and infiltrates. Mononuclear cell infiltration was not
the tissue sectioned at 1, 2, 3, 4, and 6µ. infrequent in areas surrounding the EE bodies,
Numerous EE bodies of P. jefferyi were found in which suggests that the parasite provoked an
the sections. unfavorable host response (Fig. 1).
The tissue parasites were round to elliptical. The general morphological features of P.
The average dimensions were 16.8 by 19.4 µ jefferyi EE bodies are similar to those of other
with a range of 10.8 to 21.6 by 14.4 to 24.0 µ. primate malarias as discussed by Held et al,
The edge of the parasite was smooth and usually (1967). At that time it appeared that the most
enclosed by a distinct thin limiting membrane. consistant feature of a given species was the
The nuclei were usually round but frequently average size of its EE bodies in relation to the
appeared diploid; some had bar and triangular day of infection. However, comparison shows
shapes. The nuclei stained magenta, were about that this is hazardous because their
1.0 to 1.5 µ in diameter, and did not display a measurements present overlapping ranges. If,
pattern of distribution. with the present data, that criterion is not
The cytoplasm was granular in texture and reliable, and there appear to be no other
stained a pale blue with irregular-shaped, dark distinguishing features for recognizing the EE
blue, aggregates ("flocculi") scattered through stages of P. jefferyi, such as nuclear clefts or
the cytoplasm. The aggregates stained cytoplasmic patterns different from those in
homogeneously but in thin sections small holes many other simian malaria species; then, species
were evident. A prominent feature of P. jefferyi cannot be separated, at the present time, on the
EE bodies was the presence of 0 to 5 large, basis of the morphology of the fixed tissue
round to oval, vacuoles in the body, (Fig. 1, 2). schizonts.
These structures, 4.1 by 4.8 µ , often had pink or
deep-purple stained material in them.
134 PRIMATE MALARIAS

Sodeman et al in discussing the presence of (G 453) but because it was housed some
the small holes, seen in the 1 to 2 µ sections of distance away from the laboratory and not under
the flocculi, mentioned that they might be there our immediate control--it could have happened.
to increase surface area. These structures are not
mentioned as occurring in the flocculi of other
primate malarias, but Sodeman et al were able to
Course of Infection
demonstrate them in thin sections of the EE
bodies of P. cynomolgi B strain showing that This species of malaria has been observed
they are not limited to P. jefferyi and, therefore, in only 4 gibbons. In three of them, it was mixed
not a character for separating the species. The with P. youngi. The infections were of a low
presence of vacuoles in the P. jefferyi material level with the P. youngi parasite predominating
has been reported repeatedly in other species. (Fig. 31). In splenectomized gibbon 420,
Held et al (1967) felt that they might be fixation however, the infection with P. jefferyi reached a
artifacts, or that they had a host relationship high level, (285,000 per mm3 on the 8th day of
(Eyles, 1960) or that they are present in the patency); the animal became weak and lethargic.
living form and have a respiratory function Treatment with antimalarial drug was necessary
(Shortt and Garnham, 1948). and the animal recovered. Upon recrudescence
It is known that certain drugs may cause of the infection, the animal again became ill and
vacuoles (Eyles and Coatney, 1962) and, also, very weak, requiring additional antimalarial
vacuoles may occur in degenerating EE bodies treatment. It thus appears that P. jefferyi in the
(Sodeman et al, 1969a). When all aspects of splenectomized gibbon in the early and/or acute
vacuolization in the P. jefferyi material were stages may be fulminating in character and may
considered it seemed likely that they were due to be fatal.
the process of degeneration, mainly because of
the presence of more mononuclear cell infiltrates Host Specificity
than usually seen, and, to the fact that the host
animal failed to develop a recognizable patent
The only known natural host is the gibbon,
parasitemia; a fact that was discussed earlier.
Hylobates lar from West Malaysia. Attempts to
We can say that no drugs were authorized and,
to our knowledge, none was given to the animal

FIGURE 31.—Mean parasitemia curve of Plasmodium jefferyi infections in two Hylobates lar gibbons infected by the inoculation
of parasitized blood.
 PLASMODIUM JEFFERYI 135

infect two volunteers by the bites of heavily

infected A. b. balabacensis mosquitoes were Immunity and Antigenic
unsuccessful.
The susceptibility of different mosquito Relationships
species to P. jefferyi is based on our single
feeding of four species. The comparative It would appear from the observations on
susceptibility of these mosquitoes to infection is three gibbons that in dual infections with P.
shown in Table 15. As indicated earlier, youngi and P. jefferyi, the former is
however, only A. b. balabacensis produced predominant. No additional information is
heavily infected salivary glands. The A. available.
freeborni and A. maculatus mosquitoes had only
rare salivary gland infections.
TABLE 15.--Infectivity of Plasmodium jefferyi to Anopheles b.
balabacensis, A. freeborni, A. maculatus, and A. quadrimaculatus.

No. positive/ Oocysts/pos.

Species Oocysts/gut
no. dissected gut
A. b. balabacensis 16/16 53.9 53.9
A. freeborni 21/22 38.1 39.9
A. maculatus 60/79 4.4 5.8
A. quadrimaculatus 36/86 1.5 3.5

REFERENCES
COATNEY, G. R., ORIHEL, T. C., and WARREN, McW., 1969. SHORTT, H. E., and GARNHAM, P. C. C., 1948. The pre-
Plasmodium jefferyi: A redescription. J. Parasit. 55 : erythrocytic development of Plasmodium cynomolgi
1235-1239. and Plasmodium vivax. Trans. Roy Soc. Trop. Med. &
COLLINS, W. E., and ORIHEL, T. C., 1969. The sporogonic Hyg. 41 : 785-795.
cycle of Plasmodium jefferyi J. Parasit. 55 : 1240-1246. SODEMAN, T. M., CONTACOS, P. G., COATNEY, G. R., and
EYLES, D. E., 1960. The exoerythrocytic cycle of Plasmodium JUMPER, J. R., 1969. Studies of the exoerythrocytic
cynomolgi and Plasmodium cynomolgi bastianellii in stages of simian malaria V. Plasmodium jefferyi. J.
the rhesus monkey. Am. J. Trop. Med. & Hyg. 9 : 543- Parasit. 55 : 1247-1252.
555. SODEMAN, T. M., CONTACOS, P. G., SMITH, C. S., JUMPER,
EYLES, D. E., and COATNEY, G. R., 1962. Effect of certain J. R., and COLLINS, W. E., 1969a. The
drugs on exoerythrocytic parasites of Plasmodium exoerythrocytic stages of Plasmodium falciparum in
cynomolgi. Am. J. Trop. Med. & Hyg. 11 : 175-185. Aotus trivirgatus. J. Parasit. 55 : 682-683.
HELD, J. R., CONTACOS, P. G., and COATNEY, G. R., 1967. WARREN, McW., COATNEY, G. R., and SKINNER, J. C., 1966.
Studies of the exoerythrocytic stages of simian malaria. Plasmodium jefferyi sp. n. from Hylobates lar in
I. Plasmodium fieldi. J. Parasit. 53 : 225-232. Malaya. J. Parasit. 52 : 9-13.
This page intentionally left blank.
 11
Plasmodium pitheci Halberstaedter and
von Prowazek, 1907

the parasite and the color plate is based on that

THIS was probably the first material.
true simian plasmodium to be seen and As in any endeavor, success or failure may
described. Laveran, in 1905, mentions a blood hang by a slender thread. Such might have been
parasite seen in smears taken from an orangutan the case in the above instance. In 1966, McWW
in Paris. In 1907, Halberstaedter and von was given a short-term assignment to study the
Prowazek, working in Borneo, described the zoonotic aspects of simian malaria in West
parasite they found in the orangutan and named Malaysia. During the planning of that work, the
it Plasmodium pitheci. Shibayama (1910) saw desirability of his making a side-trip to Borneo
the parasite in the blood of an orangutan in the vain hope of retrieving the malaria
imported to Japan and remarked that Schüffner’s parasite of the orangutan, last seen in 1913, was
dots were not present but, as Wenyon (1926) discussed. It was realized that the Malaysia
pointed out, his figures indicate that he used a assignment would be difficult because of the
weak stain which could account for the lack of limitation in time and funds, but it was agreed
Schüffner’s dots. Dodd (1913) reported the that the trip would be made if at all possible. He
death of an orangutan in the Zoological Gardens went to Malaysia and near the end of his tour he
of New South Wales as probably due to let one of us (GRC) know that it would be
Haemoproteus (= Plasmodium ) pitheci. In difficult for him to complete the original work
1920, Reichenow put forward the idea, but on schedule and therefore he would probably
without conviction, that maybe P. pitheci was have to abandon the trip to Borneo. It seemed
actually a human parasite although Koch (1900) that to miss this chance of finding P. pitheci
had failed in an attempt to infect the orangutan again would be catastrophic--the effort must be
and the gibbon with P. vivax or with P. made. A cable was sent to him to the effect: "Go
falciparum. Donovan (1921) carried out a study to Borneo or don't come back--letter follows".
of blood parasites in simians at the foot of the The letter merely reiterated what we both knew
Nilgiri hills in southern India and reported as to the importance of the project and urged
finding P. pitheci in the orangutan. This finding him to go to Borneo if at all possible. Upon
is open to question because, as all authorities receipt of the cable, he left immediately for
agree, these animals occur on Sumatra and Borneo with excellent results as mentioned
Borneo only, although, according to Darlington earlier. It goes without saying that he finished
(1957), they were in India during the Lower the original assignment in a highly creditable
Pleistocene era. manner and on schedule. One wonders would he
The parasite was not seen again until one of have gone to Borneo sans cable? Who can say--
us (McWW) visited Borneo in 1966 and while the point is: he did!
there succeeded in obtaining blood smears from
18 orangutans; ten of the animals harbored P.
pitheci. The description of the blood stages of

137
 PLASMODIUM PITHECI 139

Cycle in the Blood The macrogametocyte fills the host cell

completely and without enlargement (Fig. 19).
PLATE XVII The nucleus is large, usually oval, dark-staining
The youngest parasites consist of a small and generally found near the periphery of the
amount of bluish-gray cytoplasm associated with parasite. The cytoplasm is uniform and stains
a deep purple-staining nucleus. The cytoplasm grayish-blue. The pigment is abundant, coarsely
may appear as a ring or only as an elongate granular, and evenly distributed. The
smudge (Fig. 4). Double invasion of the cell is microgametocyte also fills the host cell without
rare but it may occur (Fig. 3). As growth increasing its size (Fig. 20). The nucleus is
proceeds, there is an increase in the amount of larger than its counterpart in the
cytoplasm with the nucleus remaining macrogametocyte and it takes a lighter stain. It is
essentially unchanged. The cytoplasm becomes oval to circular and stains wine-red. The bluish-
amoeboid. Very light stippling appears in the red gray staining cytoplasm is frequently vacuolated
cell as the trophozoite approaches maturity. and lacks the uniform consistency seen in the
Pigment first appears as a coarse, greenish- macrogametocyte. The pigment is coarse,
brown granule. As growth continues, the granular, and generally less abundant than in the
increase in pigment is by the addition of more macrogametocyte.
greenish-brown granules. The nucleus stains
wine-red as it increases in size. There is little or
no host cell enlargement. Sporogonic Cycle
The cytoplasm of the mature trophozoite is There are no data on the sporogonic cycle.
amoeboid and frequently assumes a web
formation which almost fills the cell. The Cycle in the Tissue
nucleus may be distorted. The stippling is sparse No exoerythrocytic stages are known.
and granular with little or no host cell
enlargement. The pigment is greenish-black and
appears as uniform coarse granules.
Course of Infection
The nuclear divisions are not out of the The course of the infection, as described by
ordinary. The color of the nuclei changes from the original investigators, runs a chronic course
wine-red in the early stages, to deep purple in with little if any pathology. The temperature in
the mature forms. The cytoplasm gradually infected animals was within the normal range. In
changes from light blue to a reddish-purple and contrast, Dodd (1913) was of the opinion that
virtually disappears in the mature forms. the death of an orangutan kept for some 13
Pigment granules tend to gather in one part of months in the Zoological Gardens in Sydney,
the cell and then the granular bodies coalesce NSW, was probably due to an overwhelming
into one or more greenish-yellow masses (Fig. infection with P. pitheci. It is unfortunate that a
17). Stippling remains sparse and is sometimes thorough post mortem was not done on the
inapparent since the developing schizont usually animal, or, if it was, that the findings should
fills the entire host cell with little or no host cell have been limited to a single note stating that the
enlargement or distortion. The mature schizont bone marrow of the femur, humerus, and ribs
normally has 12 to 14 merozoites (Fig. 18). was decidedly congested. None of the infected

PLATE XVII.—Plasmodium pitheci.

Fig. 1. Normal red cell. Figs. 12-16. Developing schizonts.
Figs. 2-4. Young trophozoites. Figs. 17-18. Mature schizonts.
Figs. 5-9. Growing trophozoites. Fig. 19. Mature macrogametocyte.
Figs. 10-11. Mature trophozoites. Fig. 20. Mature microgametocyte.
140 PRIMATE MALARIAS

animals examined by McWW showed any trivirgatus), a pig-tailed (Macaca nemestrina),

clinical evidence of infection. and a rhesus (Macaca mulatta). None of these
animals became infected. A similar inoculation
Host Specificity into a gibbon was not successful.
Also, during the fall of 1967, parasitized
The normal host of P. pitheci is the
blood was given to a second young
orangutan, Pongo pygmaeus. According to
splenectomized chimpanzee (#1394), which
Halberstaedter and Prowazek (1907) the parasite
exhibited a patent infection after 18 days. The
could be transferred successfully by blood
parasitemia remained low for ten days
inoculation to other orangutans but not to
whereupon it terminated spontaneously. These
gibbons or to monkeys. In an attempt to augment
limited data tend to show that the parasite does
the above findings regarding host specificity,
not infect other apes well and does not grow in
blood obtained from three infected orangutans in
monkeys. It is hoped that opportunities will be
Borneo was shipped to the United States and
found whereby this parasite, whose host is one
given intravenously to a young splenectomized
of our near relatives evolutionarily, can be given
chimpanzee (#31) on 11 July 1967. The
the opportunity to infect man.
infection became patent on the 21st of
The natural invertebrate host is not known
September and remained so, always with a low
and as far as we can learn there have been no
parasite count, until 17 October 1967. On 2
attempts to find a mosquito capable of accepting
October 1967 parasitized blood was transferred
infection with the parasite.
to three different monkeys: an owl (Aotus

REFERENCES
DARLINGTON, P. J., JR., 1957. Zoogeography: the geographical LAVERAN, M. A., 1905. Haemocytozoa. Essai de classification.
distribution of animals. John Wiley & Son, Inc., New Bull. Inst. Pasteur 3 : 809-817.
York. REICHENOW, E., 1920. Ueber das vorkommen der
DODD, S., 1913. Anaplasms or jolly bodies? J. Comp. Path. & malariaparasiten des menschen bei den afrikanischen
Therap. 26 : 97-110. menschenaffen. Centralbl. f. Bakt. I. Abt. Orig. 85 :
DONOVAN, C., 1921. Malaria of Monkeys. Ind. J. Med. Res. 7 : 207-216.
717-720. SHIBAYAMA, G., 1910. On malaria parasites of the orangoutan.
HALBERSTAEDTER, L. and VON PROWAZEK, S., 1907. Philippine J. Sci. 5 : 189-191.
Untersuchungen über die malariaparasiten der affen. WENYON, C. M., 1926. Protozoology. II. William Wood & Co.,
Arb. K. Gesundh. -Amte (Berl.) 26 : 37-43. New York.
KOCH, R., 1900. Zweiter bericht über die thatigkeit der malaria
expedition. Deutsche Med. Wochenschr. 26 : 88-90.
 12
Plasmodium schwetzi Brumpt, 1939
resemblance of these parasites to the human
THE credit for being the first ones, investigators were prompted to attempt
to see parasites of malaria in chimpanzees must cross-infection experiments. The initial results
go to Ziemann but we are not able to determine were not altogether convincing which prompted
with certainty just which species he saw. In 1920 Brumpt (1939), to propose the name
Reichenow, while working in the Cameroons, Plasmodium schwetzi for the ovale-vivax
examined the blood of sixteen apes among parasite of the chimpanzee under the firm belief
which he found human-like tertian, quartan, and that it was enough different, morphologically
falciparum parasites of malaria. The parasite he and biologically, from P. vivax or P. ovale, to
considered the counterpart of P. vivax was found justify the name.
in three of the chimpanzees and in two of the As work continued on these forms the
gorillas. He described and illustrated that concensus appeared to be that the schwetzi
parasite along with the others. In 1922 parasite was more like P. vivax than it was like
Blacklock and Adler, working in Freetown, P. ovale and hence Bray (1958) felt justified in
Sierra Leone, saw each of the three human-like making it a subspecies of P. vivax. We are not
parasites of malaria in the chimpanzee as had sympathetic toward subspecies designations,
Reichenow earlier. Probably because the except under very special conditions, and,
population of the ovale (vivax)-like parasites because our studies have convinced us that P.
was so scanty, they elected not to give it a name; schwetzi is an entity, more closely allied to P.
however, they did include it in their plate. ovale than to P. vivax, we have elected to
For a little over a decade these interesting consider the parasite Plasmodium schwetzi
parasites appear to have been virtually ignored Brumpt.
but in the early thirties, Schwetz began his work Plasmodium schwetzi is an African-based
in the Belgian Congo (Schwetz, 1933, 1933a). In parasite with the apes in Sierra Leone and
the blood of two adult gorillas and three young Liberia having the heaviest incidence of
chimpanzees he found Plasmodium vivax-forms infection. The infection continues east and
along with malariae- and falciparum-forms. In south, almost running out in the eastern Congo.
the 1933a paper, in discussing the vivax-forms, It has been reported as absent in the Lake Kivu
he mentioned their ovale-like characteristics and area of the Democratic Republic of the Congo
his beautifully executed colored plate makes this by Schwetz and by van den Berghe and, so far, it
point doubly clear. In 1934 Schwetz, in has not been reported in Uganda. However, we
describing a double infection in a young isolated the parasite recently from a chimpanzee
chimpanzee, again mentioned the close taken in the vicinity of Lake Edward, north of
resemblance of the vivax-like parasites to P. Lake Kivu, which places its distribution east to
ovale. And, here again, in a beautifully painted about 29°. Plasmodium schwetzi can therefore
plate, which emphasizes the heavy stippling so be said to occur in an area from the Cameroons
characteristic of P. ovale, he figured fourteen of to 29° E in the lower Congo and thence west
the peripheral blood stages. Because of the close into Liberia and Sierra Leone.

141
 PLASMODIUM SCHWETZI 143

Cycle in the Blood growing trophozoites (Figs. 12-15). The

cytoplasmic vacuole, which is quite common in
PLATE XVIII the younger stages, gradually disappears as the
The earliest parasites are relatively compact
parasite matures. Pigment increases in amount
rings with a round to oval nucleus which stains
and the size of the individual granules seems to
dark reddish-black with Giemsa. There is
become larger and more prominent. There is an
practically no cell enlargement, no stippling, and
increase in the size or the nucleus which is
no visible pigment in these early developmental
irregular in shape and now displays a lighter
stages (Figs. 2, 3). The first evidence of the
staining reaction than that seen in the younger
parasite's growth is seen in the older ring forms
stages (Fig. 15).
with the expansion of the cytoplasm; the nucleus
During the early stages of nuclear division
remains circular to oval, compact, and deep
there is little or no change in the parasite or the
staining. Some enlargement of the invaded red
host cell except that with continued nuclear
blood cell is seen by the time the parasite
division there may be some host cell distortion,
occupies one quarter of the host cell (Figs. 4, 5).
reminiscent of P. ovale, (Figs. 22, 23) which
Light, granular stippling also appears at this
proceeds as the schizont continues to grow
time. Multiple infections are not uncommon
(Figs. 23-25). Following the 6 to 8 nucleate
(Fig. 5). The trophozoite grows with some
stage, the cytoplasm appears more purple than
increase in amoeboidity; the cytoplasm takes a
blue. It is frequently fragmented and irregular in
more intense stain indicating that the
shape although, in some instances, a large
cytoplasmic density increases as the parasite
segment of the cytoplasm is free of nuclei and
matures (Figs. 7-10). The host cell is definitely
these segments retain their initial blue color
enlarged, the size is stabilized and does not
(Fig. 25). The pigment organizes into one or
change markedly with the development of the
more distinct masses and these masses take on a
schizonts so long as only one parasite is
yellowish cast (Fig. 25). The stippling becomes
involved in a single blood cell. The stippling is
difficult to differentiate as the parasite
abundant, evenly distributed, and uniformly
frequently fills most of the red blood cell leaving
coarse. The amount of pigment increases as the
only a pale eosinophilic web around the border
parasite matures and appears as greenish-black
of the parasite (Figs. 25, 27).
moderately coarse granules scattered through the
The mature schizont usually has from 12 to
cytoplasm. At times this pigment seems to
16 distinct nuclear masses not infrequently
appear in clusters (Fig. 10). The nucleus
discretely organized around a combination of a
increases in size as the parasite grows and
blue staining cytoplasmic residual and clusters
generally maintains its oval to circular outline
of pigment (Fig. 27). Mature schizonts are
although the specific border may be somewhat
frequently in distinctly oval red blood cells. As
irregular. With the increase in size the staining
the number of nuclei increases, the individual
of the nucleus lightens considerably from very
nuclei decrease in size, lose their wine-red color,
deep purple to a wine-red with darker inclusions.
and assume the dark purple color seen in the
In the mature trophozoite, the cytoplasm in-
young ring stages.
creases in amount but the intensity of the
The macrogametocyte is regular in shape.
staining is much the same as that found in the

PLATE XVIII.—Plasmodium schwetzi.

Fig. 1. Normal red cell. Figs. 19-24. Developing schizonts.
Figs. 2, 3. Young trophozoites. Figs. 25-27. Nearly mature and mature schizonts.
Figs. 4-14. Growing trophozoites, showing double Figs. 28, 29. Half-grown and mature macrogametocytes.
and triple host cell infections. Fig. 30. Mature microgametocyte.
Figs. 15-18. Older and mature trophozoites.
144 PRIMATE MALARIAS
 PLASMODIUM SCHWETZI 145

PLATE XIX.—Developing oocysts of Plasmodium schwetzi in Anopheles b. balabacensis mosquitoes. X 580 (except where
indicated).
Fig. 1. 4.5-day oocyst showing scattered pigment. Fig. 7. 8.5-day oocysts.
Fig. 2. 5.5-day oocyst. X 928 Fig. 8. 9.5-day oocysts showing less prominent
Fig. 3. 6.5-day oocysts showing small vacuoles and vacuolation.
pigment. Fig. 9. 10.5-day oocyst.
Fig. 4. 7.5-day oocysts showing numerous small Fig. 10. 11.5-day oocyst.
vacuoles. Fig. 11. 12.5-day oocysts. X145
Fig. 5. 8.5-day oocysts showing prominent vacuolation. Fig. 12. 12.5-day oocyst showing first signs of
X 145. differentiation.
Fig. 6. 8.5-day oocysts.

PLATE XX.—Developing oocysts and sporozoites of Plasmodium schwetzi in Anopheles b. balabacensis mosquitoes. X 580
(except where indicated).
Fig. 13. 13.5-day oocysts. X 145. Fig. 15. Sporozoites present near salivary gland tissue
Fig. 14. 13.5-day oocyst showing more advanced stage 15.5 days after feeding.
of differentiation.

and stains uniformly blue. The pigment is Sporogonic Cycle

coarse, black to greenish-black and evenly
distributed. The nucleus, usually oval and
PLATE XIX, XX
The natural vector of this parasite is
peripheral, stains a deep wine-red. The mature
unknown. The earliest attempt to find a vector
parasite almost fills the enlarged erythrocyte and
was that of Blacklock and Adler (1922) who
is surrounded generally by the eosinophilic web-
obtained negative results after feeding 40 A.
like border of the host cell (Figs. 28, 29).
costalis (= A. gambiae) on a chimpanzee
The microgametocyte is usually brightly
infected with P. reichenowi and P. schwetzi.
colored with reddish-purple cytoplasm and a
Rodhain and Lassman (1940) successfully
large, diffuse wine-red nucleus. The cytoplasmic
infected Anopheles maculipennis var. atroparvus
edge of the parasite is frequently crenated or
with P. schwetzi. The oocysts were large,
lace-like and tends to merge with the
measuring up to 88.8 µ; P. vivax, in their
eosinophilic web of the enlarged host cell. The
experience, measured up to 66 µ. Rodhain
pigment is coarse, black to greenish-black and
(1955) obtained a 66 percent infection rate in A.
evenly distributed (Fig. 30).
m. atroparvus. The oocysts matured in 13 to 14
The parasite has a 48-hour cycle in the
days; the dimensions were 70 to 74 µ. The
chimpanzee. According to Bray (1963) the cycle
sporozoites were in the glands by day 14 to day
may lengthen to a 50- to a 52-hour cycle as the
15; their chromatin was centrally located.
infection progresses; we did not observe this
Bray (1958) was able to study the sexual
phenomenon.
146 PRIMATE MALARIAS

development in A. gambiae. In that host, the 14.5, the mean diameter was 81 µ, with a range
cycle required 10 days at 75-80° F. He did not of 47 to 103 µ. The most obvious morphological
feel that A. gambiae constituted a good vector feature of the oocysts was the presence of
because the salivary glands were scantily numerous small, spherical inclusions which
infected. The mature oocysts had an average appeared to be vacuoles (Plate XIX). Although
diameter of 60.6 µ on the 10th day which is such inclusions are found in the oocysts of most
larger than P. vivax (45 to 55 µ). At the 4- and 5- of the plasmodia, they are more abundant in P.
day level Bray found the P. schwetzi oocysts schwetzi. Inclusions were also found abundant in
resembled exactly those of P. vivax. P. gonderi and P. simium but to a lesser extent.
Garnham (1966) reported that P. schwetzi The oocysts are larger than those of most
developed readily in A. aztecus at a temperature species; their size was comparable to those
of 22° C. The oocysts had grown to 18 µ at day measured by Rodhain in A. m. atroparvus.
6 and to 68 µ after day 13. In the young oocysts Sporozoites were present in the salivary glands
the pigment was found in straight or curved on day 14.5 and were viable in that P. schwetzi
lines. We have been able to infect the Asian infection was transmitted to human volunteers as
anophelines, A. b. balabacensis and A. discussed below.
maculatus, as well as the California-based A comparison of the growth rate of P.
anopheline, A. freeborni (Collins et al, 1969). schwetzi with that of P. cynomolgi in A. b.
The mosquitoes were incubated at 25° C balabacensis mosquitoes (Fig. 32) shows that
beginning 30 hours after exposure. The oocyst they are approximately of the same size through
diameters are presented in Table 16. In A. b. 10.5 days. However, the oocysts of P. cynomolgi
balabacensis, at day 3.5, the mean oocyst have differentiated by that time and sporozoites
diameter was 16 µ with a range of 12 to 21 µ. are present in the salivary glands. In contrast, the
The oocysts continued to grow so that by day oocysts of P. schwetzi continue to grow and do

FIGURE 32.—Range in oocyst diameters and the mean oocyst diameter curves of Plasmodium schwetzi and P. cynomolgi in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
 PLASMODIUM SCHWETZI 147

not differentiate until day 13.5 with sporozoites Plasmodium schwetzi generally occurs as a
in the glands on day 14.5. In other words, P. mixed infection with P. reichenowi and P.
schwetzi requires approximately 4.5 days longer rodhaini and, prior to our studies, begun in
to complete the sporogonic cycle than does P. 1967, it was assumed that initially one or both of
cynomolgi. the latter parasites dominated or suppressed the
P. schwetzi infection. This may be true for intact
Cycle in the Tissue animals in nature; however, when P. reichenowi
and P. schwetzi are introduced together into a
We regret to say there are no data on the
splenectomized and an intact chimpanzee the
exoerythrocytic cycle of P. schwetzi. We were
schwetzi malaria assumes almost complete
fortunate in obtaining good infections in mos-
dominance over P. reichenowi.
quitoes, discussed above, but unfortunately no
So far all attempts to infect any of the mon-
chimpanzees were available to us at that time
keys with this parasite have failed.
and therefore the opportunity for finding these
The parasite will grow and produce disease
forms was lost.
in man as will be detailed below. The early
attempts to infect man with P. schwetzi resulted
Course of Infection in failure. The first was that of Blacklock and
The natural hosts of Plasmodium schwetzi Adler (1922) who transferred blood from a
are the chimpanzee and the gorilla. It was from chimpanzee infected with P. schwetzi, P.
these hosts that Reichenow described the reichenowi, and P. rodhaini to two people. One
parasite originally, having found it in three of person received the blood both intravenously
eight chimpanzees and in two of eight gorillas. and intramuscularly and the other received it
One of the young chimpanzees showed a subcutaneously. Neither one exhibited evidence
moderate infection but in the other animals the of a malarial infection after an observation
parasitemia was low. It does not appear to evoke period of 28 and 17 days, respectively. Rodhain
clinical symptoms even in young chimpanzees and Muylle (1938) tried to infect three patients
and we observed no clinical evidence of requiring malaria therapy. The first patient
infection in splenectomized older chimpanzees received the parasitized chimpanzee blood by
even though they exhibited high parasitemias. intramuscular injection, the second and third by
the intravenous route. None of the patients

TABLE 16.—Oocyst diameters of Plasmodium schwetzi in Anopheles b. balabacensis, A. maculatus, and A. freeborni.

A. b. balabacensis A. maculatus A. freeborni

Days after
Infection
No. Range Mean* No. Range Mean No. Range Mean

3.5 100 12-21 16 16 13-15 14 41 9-14 12

4.5 81 12-27 23 12 15-22 19 45 15-21 18
5.5 20 20-32 27 34 13-22 19 75 9-25 20
6.5 65 20-44 35 26 17-26 21 64 19-35 27
7.5 75 21-59 45 64 20-45 32 40 27-53 41
8.5 56 21-74 58 52 26-60 42 60 32-71 55
9.5 60 35-74 53 52 28-59 43 9 51-64 56
10.5 60 41-79 62 60 31-84 55
11.5 60 47-96 77 15 39-80 63‡
12.5 15 59-94 79 63 35-83 57‡
13.5 54 35-90 74† 13 39-76 51†‡
14.5 41 37-103 81†** 40 28-83 58†‡

* Measurements expressed in microns; incubation temperature 25° C.

† Oocyst differentiation.
‡ Oocyst degeneration.
** Sporozoites present in the salivary glands.
148 PRIMATE MALARIAS

contracted the infection. In the same year (1938) obtained from a chimpanzee at the National
Rodhain, van Hoof and Muylle reported their Communicable Disease Center, Atlanta-
failures in trying to infect man with the vivax Chamblee, Georgia, was inoculated into a
parasite (= P. schwetzi) and in 1939 Rodhain chimpanzee in Covington, Louisiana. A patent
again tried to infect man with P. schwetzi by the infection of the two malaria parasites developed
inoculation of infected blood; it failed. He drew in this chimpanzee; but, in a rather short period
the conclusion that P. schwetzi was a true of time, the P. schwetzi parasite became the
parasite of the chimpanzee and not infective to predominant one. When gametocytes of this
man. species were numerous, mosquitoes (Anopheles
In 1940 Rodhain, concerned with the freeborni, A. maculatus, and A. b. balabacensis)
possible transfer of P. rodhaini to man, passaged from our laboratories in Chamblee were shipped
blood from a chimpanzee infected with P. by air to New Orleans and carried to Covington,
rodhaini and with P. schwetzi to two people. Louisiana, for feeding on the chimpanzee. These
The quartan infection appeared in each of the were returned to the insectary in Chamblee,
inoculated individuals and with it there appeared Georgia, within 30 hours.
a vivax-like parasite too; i.e., P. schwetzi. When it was observed that there were
Rodhain expressed some doubt about the latter sporozoite positive glands in the A. b.
being P. schwetzi, probably because of his balabacensis mosquitoes on day 15.5, it was
previous failures to infect man, and, because the decided to expose three volunteers (2
chimpanzee had been inoculated with known P. Caucasians and 1 Negro) to infection. Of the
vivax some two years before; that infection had three volunteers exposed to infection by bites of
persisted for some weeks. The infection then 7 to 9 heavily infected mosquitoes, two (both
disappeared and was not seen subsequently Caucasians) developed patent infections. One
during the following two years. In the light of volunteer developed a patent infection at 24 days
what happened later it is more than probable that (day zero being the day of exposure). The other
the vivax-like parasite in the recipients was volunteer experienced generalized malaise and
actually P. schwetzi. For the next fifteen years headache at irregular intervals beginning on day
the question of whether P. schwetzi would infect 14, and on two occasions, day 15 and 17,
man was allowed to lie fallow. exhibited temperatures of 100.4 and 99.8° F,
In 1955 and 1955a Rodhain and Dellaert respectively. However, this volunteer did not
reported that they had been able to infect man develop a patent infection until day 104. The
with P. schwetzi. They detailed the successful third volunteer, a Negro, exposed to infection by
infection of a man and from him to other mosquito bite, did not develop a patent infection
humans, then back to the chimpanzee, and, through 200 days of observation. Ten other
again, back to man. In each instance the volunteers (9 Caucasians and 1 Negro) were
infection was initiated by the inoculation of exposed to infection by the inoculation of
parasitized blood. In their first paper, in parasitized human blood. Only the 9 Caucasians
commenting on the parasite in man they developed patent infections,
mentioned its close resemblance to P. ovale, a Patency of infection persisted for up to 145
fact mentioned as early as 1934 by Schwetz and days. Figure 33 shows the pattern of parasitemia
later by Bray (1958). One wonders, in view of for the above-mentioned infections of P.
their unqualified success in 1955, why they schwetzi in man through 75 days. It can be seen
made no mention of Rodhain's transfer of P. that there is an initial peaking of parasitemia
schwetzi to man in 1940. during the first three weeks of patent parasitemia
Our own studies of this parasite in man (maximum count of 2,750 parasites per mm3 of
came about through a series of fortuitous blood). However, it should be noted that parasite
circumstances (Contacos et al, 1970). Several counts of 1,000 per mm3 or higher were
chimpanzees at the Delta Regional Primate frequently observed through the first 65 days of
Research Center in Covington, Louisiana parasitemia.
became available for studies in malaria. Blood The fever patterns for infections of P.
parasitized with P. reichenowi and P. schwetzi, schwetzi in man were variable with a tertian
 PLASMODIUM SCHWETZI 149

FIGURE 33.—Median parasitemia curve and individual parasite counts in 11 Plasmodium schwetzi infections in man (2
sporozoite-induced and 9 blood-induced).

pattern only occasionally evident in most of the observed. There was, however, one interesting
volunteers. Paroxysms often occurred daily difference. Rodhain and Dellaert made a point of
indicating a two-brood infection. The maximum the absence of splenic enlargement in their
temperature observed in any volunteer was infections. In our group, 5 of the 11 volunteers
105.6° F. Several volunteers had fever free had splenic enlargement ranging from tip to 5
intervals in spite of the presence of patent centimeters below the left costal margin, with
parasitemia. tenderness in 6 of the volunteers.
Major complaints consisted of headache, One of the more interesting observations of
generalized malaise, anorexia, and nausea. the schwetzi infections in man was its close
Vomiting and frank chills were frequently resemblance to human ovale malaria as com-
observed. Even though antimalarial therapy was pared to its appearance in the chimpanzee. The
given to several of the volunteers for various schwetzi parasites as seen in human blood films
reasons, it was not necessary for clinical and/or are illustrated in Plate XXI. On superficial
parasitologic reasons. observation the P. ovale parasites (Plate XXV)
An intriguing facet of our human trials was do not resemble the P. schwetzi parasites as they
that the two Negro volunteers failed to develop appear in man. However when one examines the
patent infections. This was totally unexpected, compact nature of the parasite, the ovaling
especially, in view of the fact that P. schwetzi is tendency of many of the parasitized red blood
an African-based parasite. We would not have cells and the coarse red stippling, the similarities
been too surprised if the sporozoite-induced case become apparent.
had failed to come down but failure in the blood- In addition to what has been discussed
induced trial left us entirely "at sea". above, it is recognized that during the initial
If one compares the characteristics of our phase of an ovale infection the mature schizonts
infections with the blood induced infections generally show eight merozoites, the half
reported by Rodhain and Dellaert (1955), one number for P. schwetzi, but during relapse
finds little difference in maximum parasite (Garnham et al, 1955), or following continuous
counts, maximum temperatures, and in numbers passage (Hauer, 1937), the merozoite number is
of paroxysms. They observed parasitemias doubled--the schwetzi number, and the host cell
ranging up to 2,060 whereas in our volunteers is appreciably enlarged (Garnham loc. cit.), also
the range was up to 2,750 parasites per mm3. a schwetzi characteristic. It is also recognized
They reported maximum temperatures of from that Plasmodium ovale generally exhibits a low
104 to 105.8° F and up to 14 paroxysms. In our parasitemia. The infections in our volunteers and
volunteers, maximum temperatures ranged from in those reported by Rodhain and Dellaert
100.2 to 105.8° F and up to 19 paroxysms were followed the same pattern.
 PLASMODIUM SCHWETZI 151

The distribution of P. ovale in Africa was Host Specificity

mentioned earlier and if a map showing the
distribution of the chimpanzee (and gorilla) is
Plasmodium schwetzi naturally infects
superimposed over one of P. ovale one finds
chimpanzees and gorillas (Reichenow, 1920;
very close agreement. Apropos of that situation,
Schwetz, 1933a). Experimentally, infections
Languillon (1957) working in a forest area of the
have been induced in man by the inoculation of
Cameroons which supported a large chimpanzee
parasitized blood (Rodhain and Dellaert, 1955)
population, encountered in a small native village
and by the bites of infected mosquitoes
five infants infected with malaria. He
(Contacos et al, 1970). The natural vector is
determined four of these to be P. ovale and one
unknown and, in fact, very little information is
to be P. schwetzi. In his comments he suggested
available on the susceptibility of African
that P. ovale in man may be an adaptation of P.
anophelines to infection with this parasite. Bray
schwetzi. He and Rodhain had alluded to the
(1958) reported the infection of Anopheles
same relationship two years earlier.
gambiae but considered it to be an unsuitable
Rodhain had a continuing interest in the
host. Other mosquitoes which have been
parasite specificity of the ape malarias beginning
reported as susceptible to infection are A.
in 1940 and in a paper published shortly before
atroparvus (Rodhain, 1955) and A. aztecus
his death (1956) he included a title, with the
(Garnham, 1966). In our own studies (Collins et
notation "in preparation", on “The Paradox of
al, 1969), we have obtained infection of A. b.
Plasmodium schwetzi in Humans”. As far as we
balabacensis, A. freeborni, and A. maculatus
know the manuscript was never completed and
mosquitoes; the average number of oocysts per
we are left to speculate as to what he might have
mosquito gut was 82.8, 52.7, and 31.7,
written. In view of the close resemblance of P.
respectively. The A. b. balabacensis readily
schwetzi in man to P. ovale, one may well
transmitted the infection. The latter species is
wonder how much of the malaria being
not coindigenous with the parasite and,
diagnosed as ovale malaria is truly schwetzi
therefore, cannot serve as its natural vector.
malaria, especially in areas where man and the
chimpanzee co-exist.

REFERENCES
BLACKLOCK, B., and ADLER, S., 1922. A parasite resembling CONTACOS, P. G., COATNEY, G. R., ORIHEL, T. C.,
Plasmodium falciparum in a chimpanzee. Ann. Trop. COLLINS, W. E., CHIN, W., and JETER, M. H., 1970.
Med. Parasit. 16 : 99-106. Transmission of Plasmodium schwetzi from the
BRAY, R. S., 1958. Studies on malaria in chimpanzees. V. The chimpanzee to man by mosquito bite. Am. J. Trop.
sporogonous cycle and mosquito transmission of Med. & Hyg. 19 : 190-196.
Plasmodium vivax schwetzi. J. Parasit. 44 : 46-51. GARNHAM, P. C. C., BRAY, R. S., COOPER, W., LAINSON,
BRAY, R. S., 1963. The malaria parasites of anthropoid apes. J. R., AWAD, F. I., and WILLIAMSON, J., 1955. The
Parasit. 49 : 888-891. pre-erythrocytic stage of Plasmodium ovale. Trans.
BRUMPT, E., 1939. Les parasites due paludisme des chimpanzes. Roy. Soc. Trop. Med. & Hyg. 49 : 158-167.
C. R. Soc. Bioi. 130 : 837-840. GARNHAM, P. C. C., 1966. Malaria parasites and other
COLLINS, W. E., ORIHEL, T. C., CONTACOS, P. G., JETER, haemosporidia. Blackwell Scientific Publications.
M. H., and GELL, L. S., 1969. Some observations on Oxford.
the sporogonic cycle of Plasmodium schwetzi, P. vivax HAUER, A., 1937. Ueber neue beobachtungen an einem
and P. ovale in five species of anopheles. J. Protozool. Plasmodium ovale-Stamm. Arch. f. Schiffs. u. Trop.-
16 : 589-596. Hyg. 41 : 153-157.

Plate XXI.—Plasmodium schwetzi in man.

Fig.1. Normal red cell. Figs. 17-26. Developing schizonts.
Figs. 2, 3. Young trophozoites. Fig. 27. Mature schizont.
Figs. 4-13. Growing trophozoites. Figs. 28, 29. Developing and mature macrogametocytes.
Figs. 14-16. Nearly mature and mature trophozoites. Fig. 30. Mature microgametocyte.
152 PRIMATE MALARIAS

REFERENCES—Continued
LANGUILLON, J., 1957. Carte epidemiologigue du paludisme au RODHAIN, J., and MUYLLE, G., 1938. Sur la specificite des
Cameroun. Bull. Soc. Path. Exot. 50 : 585-600. plasmodium des anthropoides de I'Mrique Centrale. C.
REICHENOW, E., 1920. Ueber das vorkommen der R. Soc. Bioi. 127 : 1467-1468.
malariaparasiten des menschen bei den afrikanischen RODHAIN, J., and LASSMAN, P., 1940. Le cycle schizogonique
menschenaffen. Centralbl. f. Bakt. I. Abt. Orig. 85 : de Plasmodium schwetzi et l'evolution de ce parasite
207-216. chez Anopheles maculipennis var atroparvous. Ann.
RODHAIN, J., 1939. La receptivite du chimpanze Pan satyrus au Soc. Beige Med. Trop. 20 : 179-186.
Plasmodium vivax humain. C. R. Soc. BioI. 132 : 69- RODHAIN, J. and DELLAERT, R., 1955. Contribution a l'etude
70. de Plasmodium schwetzi E. Brumpt (2me note).
RODHAIN, J.,1940. Les plasmodiums des anthropoides de I' Transmission du Plasmodium schwetzi a l'homme. Ann.
Afrique Central et leurs relations avec les plasmodiums Soc. BeIge Med. Trop. 35 : 73-76.
humains. Ann. Soc. BeIge Med. Trop. 20 : 489-505. RODHAIN, J. and DELLAERT, R., 1955a. Contribution a l'etude
RODHAIN, J ., 1955. Contribution a I'Ctude de Plasmodium du Plasmodium schwetzi E. Brumpt. (3me note).
schwetzi, E. Brumpt. Ann. Soc. BeIge Med. Trop. 35 : L'infection a Plasmodium schwetzi chez l'homme. Ann.
69-73. Soc. Beige Med. Trop. 35 : 757-777.
RODHAIN, J ., 1956. Les formes preerythrocytaires du SCHWETZ, J.,1933. Sur les parasites malariens (Plasmodium) des
Plasmodium vivax chez le chimpanze. Ann. Soc. BeIge singes superieurs (Anthropoides) Africains. C. R. Sac.
de Med. Trap. 36 : 99-103. Bioi. 112 : 710-711.
RODHAIN, J. VAN HOOF, L., and MUYLLE, G., 1938. SCHWETZ, J., 1933a. Sur une infection malarienne triple d'un
Contribution a I'etude des plasmodium des singes chimpAnze. Zentralb. f. Bakt. I. Abt. Orig. 130 : 105-
africains. Les plasmodium des chimpanzes du Congo 111.
BeIge. Ann. Sac. BeIge Med. Trop. 18 : 237-253. SCHWETZ, J., 1934. Contribution a l'etude des parasites
malariens des singes superieurs africains. Riv. Malariol.
13 : 143-147.
 13
Plasmodium simium Fonseca, 1951
mentions that he and Fonseca studied the
THERE is an interesting parasite again in 1955 in another young howler
story connected with the discovery of P. simium from the Itapecerica Forest.
as related by Dr. Deane (Deane et al, 1969) who, In recent years Deane and his coworkers
by his action, actually contributed to its (1969) have made extensive studies of the
discovery. In 1939 Fonseca was engaged in the malaria parasites in Brazilian monkeys,
study of the yellow fever virus in monkeys from concentrating mainly on the southeast and north-
the Itapecerica Forest near São Paulo, Brazil. An west sections of the country. The studies show
unusual temperature curve in the infected howler that P. simium occurs only in howlers (A. fusca)
monkey (Alouatta fusca) prompted him to make in the states of Rio Grande Do Sul, Santa
smears of its blood. When he examined the Catarina, and São Paulo, but in the state of
preparations he came across a plasmodium Esperito Santo it occurs not only in howlers but
which he assumed to be P. brasilianum. He also in woolly spider monkeys (Brachyteles
made a series of smears over the next several arachnoides). Adult howlers show infection
days and saved them. Ten years later Deane, more frequently than immature or very old ones;
anxious to obtain a strain of P. brasilianum, very young specimens are generally negative.
asked Fonseca where the howler carrying the Infections are present throughout the year but
brasilianum parasite had been caught. This query there is an increase in incidence during the
prompted Fonseca to reexamine the original summer. Why P. simium is limited to one small
smears whereupon, to his surprise, he did not area of Brazil is an interesting question. The
find P. brasilianum, as he had recorded in 1939, answer probably lies with a vector which
but a new parasite which he described and occupies a very special ecological niche.
named P. simium in 1951. Garnham (1966)

153
 PLASMODIUM SIMIUM 155

Cycle in the Blood Sporogonic Cycle

PLATE XXII PLATE XXIII
The young parasites are rings, or modified Although Deane et al (1966), on purely
forms, which may occupy about a fifth of the epidemiological grounds, forecast that P. simium
host cell, with a compact dark red nucleus and a was transmitted by Anopheles cruzi, the only
rim of blue cytoplasm; these forms sometimes studies reported on the sporogonic cycle have
exhibit a double chromatin dot (Fig. 4). The been with exogenous mosquito species. In our
developing parasites are amoeboid and retain the laboratory, (Collins et al, 1969 and later) we
vacuole. Double infections are quite common have allowed six different species of
(Fig. 8). Schüffner's stippling is prominent in all anophelines (A. freeborni, A. maculatus, A.
but the youngest rings. Some of these forms stephensi, A. quadrimaculatus, A. atroparvus,
virtually fill the host erythrocyte (Figs. 9, 10). and A. b. balabacensis) to feed on squirrel
The young schizonts are marked by the monkeys (Saimiri sciureus) infected with P.
disappearance of the vacuole; the nucleus simium. Beginning as early as 3 days after
elongates and divides; and small, barely visible infection, dissected midguts of the mosquitoes
pigment granules appear in the cytoplasm. In the were examined to determine the presence and
older schizonts the nuclei take a deep reddish- the diameters of the oocysts.
purple stain, are large, oval or crescent-shaped. The results of the oocyst measurements are
The mature schizonts display 12 to 18 presented in Table 17. In A. freeborni at day 3,
merozoites. The schizonts are ragged in outline; the mean oocyst diameter was 9 µ with a range
medium to dark brown pigment is clumped into of 7 to 11 µ. The oocysts continued to grow so
a mass, and sometimes these forms appear that on day 12 they had an average size of 46 µ
smaller than slightly younger forms (Fig. 21). with a range of 22 to 63 µ. Sporozoites were
The parasitized host cell increases in size about first seen in the salivary glands on day 12.
the time Schüffner’s dots appear and later may In A. stephensi the 5-day oocyst measured
reach a diameter of 11 µ. 13 µ with a range of 11 to 17 µ. On day 13 they
The gametocytes have some resemblance to averaged 53 µ, with a range of 24 to 71 µ.
those of P. vivax. The young forms are compact Sporozoites were present in the salivary glands
with dense blue cytoplasm and a red-staining on day 12.
nucleus (Fig. 28). The mature gametocytes fill The examination of the A. maculatus
and distend the host cell. The macrogametocytes revealed a lower intensity of infection. On the
have a small dense-staining red nucleus; rather 5th post-feeding day the oocysts had an average
deep blue cytoplasm, and scattered, small diameter of 12 µ with a range of 11 to 13 µ. On
granules of pigment (Fig. 29). The day 13, sporozoites appeared in the salivary
microgametocytes exhibit a diffuse pink glands and on that day oocysts had an average
nucleus, pale blue cytoplasm and small diffuse measurement of 47 µ with a range of 27 to 67 µ.
pigment granules (Fig. 30). The number of oocysts available for
The asexual cycle in the blood is about 48 measurement in A. quadrimaculatus, A.
hours. atroparvus and A. b. balabacensis was limited
but the oocyst diameters in each of these species

PLATE XXII.--Plasmodium simian.

Fig. 1. Normal red cell. Figs. 19-21. Older schizonts.
Figs. 2-8. Young trophozoites. Figs. 22-26. Nearly mature schizonts.
Figs. 9-12. Growing trophozoites. Figs. 27. Mature schizont.
Figs. 13-15. Mature trophozoites. Figs. 28-29. Young adult and mature macrogametocytes.
Figs. 16-18. Young schizonts. Fig. 30. Mature microgametocyte.
156 PRIMATE MALARIAS
 PLASMODIUM SIMIUM 157

were within the range for the other test Course of Infection
mosquitoes. Sporozoites were found in the
salivary glands of A. b. balabacensis on day 12.
Until recently it was thought that the only
Plasmodium simium is a smaller parasite
natural host of P. simium was the black howler
than P. cynomolgi and it takes about two days
monkey (Alouatta fusca); however, Deane et al
longer to complete its sporogonic cycle. This is
(1968, 1968a) record finding this parasite as a
shown graphically in Figure 34 where its growth
natural infection in the woolly spider monkey
curve in A. freeborni, the most acceptable test
(Brachyteles arachnoides), also. Deane and his
vector, is compared with that of P. cynomolgi.
coworkers (1969) were able to observe natural
Heavy salivary gland infections were present in
infections in several A. fusca for varying periods
each of the test species by day 12.
of time before splenectomy. All of the animals
The sporozoites in A. freeborni and in A.
exhibited mild symptoms and moderate to light
maculatus were fully viable and infective
parasitemias. However, following splenectomy
because bites by each of these species
the parasitemia generally increased rapidly up to
transferred sporozoites which initiated infection
as high as 225,000 per mm3 in about 3 weeks
in splenectomized squirrel monkeys.
(Deane, 1964). He also reported anemia, loss of
Parasites in 9 splenectomized squirrel mon-
hair, diarrhea, and fever as high as 41.5° C. It
keys were infective to A. freeborni mosquitoes
must be kept in mind that howlers are difficult to
when they were allowed to feed as early as 6 and
keep in captivity and consequently some of these
as late as 41 days after onset of patent
manifestations might have been due to their
parasitemia. Oocyst densities of 5 or more per
failure to accept food. In this connection
gut occurred in mosquitoes fed 6 to 21 days after
Crandall (1964) reported that three howlers lived
onset of parasitemia. Indications are that the best
in captivity between 3 and 5 years. More
infections result when feedings are carried out
recently, Malinow et al (1968) reported keeping
during the first 6 to 15 days of patent
two male howlers in good health, at the Oregon
parasitemia.
Regional Primate Center, for a period of two
years. The latter investigators have since
Cycle in the Tissue discontinued observations on this species
because of the extreme mortality rate in
Fonseca reexamined tissue smears of the captivity.
spleen, liver, brain, and kidneys of the original So far no one has reported the infection of
monkey whose parasites were seen in 1939 but the rhesus monkey with this parasite.
was unable to find "elements of the In the splenectomized squirrel monkey
exoerythrocytic cycle." We have sought these (Saimiri sciureus) Deane and Okumura (1965)
stages repeatedly, following heavy sporozoite were able to obtain heavy infections in three out
inoculations in owl and squirrel monkeys. Only of four animals infected by the inoculation of
recently we have found 7-day forms in each of parasitized blood; in the fourth animal, the
these animals; these forms are similar to P. vivax parasitemia was low and all evidence of
in the owl monkey. infection had disappeared on the fourth day.

PLATE XXIII.—Developing ooccysts of Plasmodium simium in Anopheles freeborni mosquitoes. X 580 (Except Fig. 2).
Fig. 1. 4-day oocyst. Fig. 6. 9-day oocyst and mosquito gut membrane.
Fig. 2. 5-day oocysts showing scattered pigment. X 740. Fig. 7. 9-day oocyst.
Fig. 3. 6-day oocyst showing pigment and presence of Fig. 8. 10-day oocyst showing numerous small vacuoles.
small vacuoles. Fig. 9. 10-day differentiating oocyst.
Fig. 4. 7-day oocyst. Fig. 10. 11-day fully differentiated oocyst.
Fig. 5. 8-day oocyst showing prominent vacuoles.
 TABLE 17.—Oocyst diameters of Plasmodium simium in Anopheles freeborni, A. stephensi, A. b. balabacensis,
A. quadrimaculatus, A. atroparvus, and A. maculatus.

Days after
A. freeborni A. stephensi A. b. balabacensis A. quadrimaculatus A. atroparvus A. maculatus
Infection
No. Range* Mean No. Range Mean No. Range Mean No. Range Mean No. Range Mean No. Range Mean

3 8 7-11 9
4 97 9-20 14
5 200 8-22 14 100 11-17 13 2 14-17 15 6 13-19 17 16 11-13 12
6 201 12-32 19 111 12-26 17 27 13-27 19 25 13-24 18 17 11-18 15
7 136 13-35 23 126 18-39 25 3 24-26 25 12 20-38 28 20 12-35 20
8 132 13-49 26 111 14-53 27 18 20-44 34 28 26-47 35 85 25-53 39 48 15-51 28
9 120 17-54 31 103 19-44 33 8 26-46 36 58 26-59 41 30 20-55 39† 92 14-53 32
10 196 18-55 42† 201 20-61 47† 33 26-48 38 33 31-59 46† 32 24-57 39† 105 18-60 41†
11 110 21-60 47† 200 30-63 49† 20 31-54 43† 34 32-73 47† 13 26-63 49† 66 27-63 49†
12 100 22-63 46†** 194 18-64 46†** 2 40-59 50†** 6 51-63 57† 86 24-71 48†
13 106 24-71 53†** 5 30-65 47† 48 27-67 47†**

Totals 1300 7-63 1252 11-71 110 13-59 198 13-65 172 20-63 498 11-71

* Measurements expressed in microns; incubation temperature 25° C.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
 PLASMODIUM SIMIUM 159

FIGURE 34.—Mean growth curves and ranges in oocyst diameters of Plasmodium simium and P. cynomolgi in Anopheles
freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

Less severe infections were obtained in A. We were able to transmit the parasite to two
paniscus and in young A. fusca (Deane et al, splenectomized S. sciureus through the bites of
1966a) and mild infections in Lagothrix A. maculatus in one and A. freeborni in the
lagotricha (Deane et al, 1965a). The marmoset, other; the prepatent periods were 24 and 38
Catticebus iacchus, was found susceptible but days, respectively. The maximum parasite
the infection was of low order (Deane and counts in these animals ranged from 63,000 to
Okumura, 1965b). All the animals, as reported 160,000 per mm3 of blood. The animal with the
by the Brazilian workers, recovered from their highest parasite count died; in the other,
infections spontaneously except a single very parasitemia persisted for 100 days.
young specimen of S. sciureus. In 1966b Deane et al reported that one of
In our own studies, infection was obtained the members of his research crew, working in
in 12 splenectomized squirrel monkeys by the the government forest reserve outside São Paulo,
inoculation of parasitized blood (Fig. 35). The had developed fever with a tertian pattern (up to
parasitemia rose rapidly to a median count 39.5° C) which lasted about a week. Smears of
greater than 10,000 per mm3 by day 8 and the man's blood showed scanty parasites of
remained at that level or higher for malaria. Some of his blood was given to a
approximately 25 days. The highest parasitemia, splenectomized squirrel monkey which
approximately 440,000 per mm3 was obtained in developed a high parasitemia. The authors
one animal 22 days after inoculation. In another, attributed the human infection to P. simium
the parasite count dropped to zero on day 26; 9 because the parasite was P. vivax-like, and,
of the 12 test animals were negative by day 60. because it grew well in S. sciureus. In our
Patent infections persisted in the other three for opinion it could just as well have been P. vivax
as long as 112 days. because in the same year (1966b) Deane et al
160 PRIMATE MALARIAS

FIGURE 35.—Parasite counts with the median curve of parasitemia for Plasmodium simium in 12 splenectomized squirrel
monkeys, Saimiri sciureus, inoculated with parasitized blood.

showed that P. vivax would grow in vector and the proof of its ability may come
splenectomized squirrel monkeys. Furthermore, shortly. However, when sporozoites from A.
in the following year, using a strain of P. simium cruzi were transferred to three different squirrel
received from Dr. Deane, we failed in five monkeys, patent infection did not occur. As
attempts to infect human volunteers, via mentioned earlier, we were able to infect six
mosquito bite, employing A. stephensi and A. species of anophelines with this parasite. The
freeborni. In three of these attempts, infectivity intensity of infections in the mosquitoes varied
of the sporozoites was confirmed by their ability from one species to another (Table 18).
to initiate infection in splenectomized squirrel Anopheles freeborni was the most acceptable
monkeys. Since these trials, we have learned that host followed by A. stephensi, A. maculatus, A.
by the use of splenectomized Saimiri (squirrel) b. balabacensis, A. quadrimaculatus, and finally
monkeys sporozoites of P. simium, produced by A. atroparvus. Average oocyst densities greater
these mosquitoes, are routinely infective. In than one per gut in A. freeborni may be expected
view of these considerations the acceptance of a to yield salivary gland infections of 50 percent
P. simium infection occurring naturally in man or greater after an incubation period of 14 days
should be held in abeyance until there is more and a temperature of 25° C.
evidence in support of its zoonotic nature.
Antigenic Relationships
Host Specificity and Immunity
The natural vector of P. simium is
Two splenectomized S. sciureus monkeys
unknown. In 1966a, Deane et al incriminated A.
were inoculated with sporozoites of P. simium
cruzi as the probable natural vector because it
but failed to develop a patent infection with that
occurs in the area where there are infected
species. One of the animals was splenectomized
howler monkeys and because it feeds in the
two days prior to inoculation and the other 18
forest canopy as well as at ground level. In a
days after inoculation. Subsequent to the
more recent paper summarizing all his studies
splenectomy, each developed a patent infection
through July, 1968 (Deane et al, 1969) he
with P. brasilianum rather than the expected P.
contends "one can hardly incriminate another
mosquito. ..". It may well be that A. cruzi is the
 PLASMODIUM SIMIUM 161

TABLE 18.—Comparative infectivity of Plasmodium simium to Anopheles freeborni, A. stephensi, A. b. balabacensis,

A. quadrimaculatus, A. atroparvus, and A. maculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

F-1 100
F-1 : St-1 20 269 332 38.3 25.3 46.2
F-1 : Bal 9 81 173 60.5 20.2 10.3
F-1 : Q-1 12 89 248 78.7 20.2 10.1
F-1 : Atro 6 43 151 83.7 19.9 8.6
F-1 : Mac 26 286 670 51.7 19.7 8.1

* F-1 = Anopheles freeborni, St-1 = A. stephensi, Bal = A. b. balabacensis, Q-1 = A. quadrimaculatus, Atro = A. atroparvus,
Mac = A. maculatus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A.freeborni to another
species where the GII of A.freeborni = 100.

simium. It may well be that a latent P. simium infections in intact squirrel monkeys by
brasilianum will prevent the development of a the inoculation of sporozoites indicating either
patent P. simium infection. Also, in this the necessity for splenectomy or the presence of
connection, we have failed to obtain patent P. a latent infection with P. brasilianum.

REFERENCES
COLLINS, W. E., CONTACOS, P. G., and GUINN, E., arredores de São Paulo. VI. Infeccao
1969. Observations on the sporogonic cycle and experimental de Macaco coata Ateles paniscus
transmission of Plasmodium simium da Fonseca. pelo Plasmodium simium. Rev. Paul. Med. 68 :
J. Parasit. 55 : 814-816. 181-182.
CRANDALL, L. S., 1964. The management of wild DEANE, L. M., DEANE, M. P., and FERREIRA NETO,
animals in captivity. Uni. Chicago Press, J., 1966b. Studies on transmission of simian
Chicago. malaria and on a natural infection of man with
DEANE, L. M., 1964. Studies on simian malaria in Brazil. Plasmodium simium in Brazil. Bull. WId. Hlth.
Bull. WId. Hlth. Org. 31 : 752-753. Org. 35 : 805-808.
DEANE, L. M. and OKUMURA, M., 1965; Malaria de DEANE, L. M., FERREIRA NETO, J., and SITONIO, J.
macacos dos arredores de São Paulo. I. G., 1968. Novo hospedeiro natural do
Susceptibilidade do macaco-de-cheiro Saimiri Plasmodium simium e do Plasmodium
sciureus ao Plasmodium simium do bugio brasilianum: o mono, Brachyteles arachnoides.
Alouatta fusca. Rev. Paul. Med. 66 : 171-172. Rev. Inst. Med. Trop. sao Paulo, 10 : 287-288.
DEANE, L. M., DEANE, M. P., and OKUMURA, M., DEANE, L. M., FERREIRA NETO, J. A. and SITONIO, J.
1965a. Malaria de macacos dos arredores de Sao G., 1968a. Estudos sobre malaria de macacos no
Paulo. III. Susceptibilidade do macaco-barrigudo Estado de Espirito Santo. Rev. Brasil. BioI. 28 :
Lagothrix lagotricha, a infeccao pelo 531-538.
Plasmodium simium. Rev. Paul. Med. 66 : 363. DEANE, L. M., FERREIRA NETO, J. A., OKUMURA,
DEANE, L. M., and OKUMURA, M., 1965b. Malaria de M., and FERREIRA, M. O., 1969. Malaria
IIlacacos dos arredores de São Paulo. II. parasites of Brazilian monkeys. Rev. Inst. Med.
Susceptibilidade do sagui Callithrix Jacchus a Trop. São Paulo, 11 : 71-86.
infeccao pelo Plasmodium simium. Rev. Paul. FONSECA, F. DA, 1951. Plasmodio de primata do Brasil.
Med. 66 : 174. Mem. Inst. Osw. Cruz. 49: 543-551.
DEANE, L. M., DEANE, M. P., and FERREIRA NETO, J GARNHAM, P. C. C., 1966. Malaria parasites and other
., 1966. A naturally acquired human infection by haemosporidia. Blackwell Scientific Publications.
Plasmodium simium of howler monkeys. Trans. Oxford. pp.1114.
Roy. Soc. Trop. Med. & Hyg. 60 : 563-564. MALINOW, M. R., POPE, B. L., DEPAOLI, J. R., and
DEANE, L. M., OKUMURA, M., HERTHA, B., and DE KATZ, S.,1968. Biology of the howler monkey
SOUZA, W. T., 1966a. Malaria de macacos dos (Alouatta caroya). Biblio. Primat. #7 : 224-230.
This page intentionally left blank.
 14
Plasmodium youngi Eyles, Fong, Dunn, Guinn, Warren,
and Sandosham, 1964
et al, 1965, 1966) and in Thailand (Ward and
THE first report of a true Cadigan, 1966; Cadigan et al, 1969).
malaria from the gibbon was that of Rodhain In May of 1962, personnel of the Far East
who described Plasmodium hylobati in 1941. Research Unit (NIH) located in Kuala Lumpur,
Earlier, Fraser (1909) mentions finding a Malaysia, obtained a young gibbon (Hylobates
"malaria-like parasite" Plasmodium kochi in lar) which had been captured in the state of
Hylobates symphalangus (= Symphalangus Kalantan in peninsular Malaysia. The animal
syndactylus) in Malaya. Plasmodium kochi is harbored a true Plasmodium which did not fit the
actually a hepatocystis considered generally to description of P. hylobati. Eyles and his
be limited to the monkeys. However, Shiroishi et coworkers transferred the parasite to other
al (1968) reported Hepatocystis sp. in Hylobates gibbons and after careful study concluded it was
concolor from northern Thailand and therefore a new species. They named the parasite
the Fraser parasite could have been a Plasmodium youngi in honor of the American
representative of either genus. It probably was a malariologist, Dr. Martin D. Young.
plasmodium since we know now that true
malarias are common in the gibbons of
peninsular Malaysia (Eyles et al, 1964; Warren

163
 PLASMODIUM YOUNGI 165

Cycle in the Blood adult forms fill the host cell (Fig. 28). The
cytoplasm of the microgametocyte stains
PLATE XXIV reddish-purple and exhibits a large deep reddish-
pink nucleus sometimes with a deeper staining
The youngest parasites are frequently ring- bar-like area. The black pigment is prominent
shaped and measure about 2 µ. The nucleus is and Schüffner’s stippling collects toward the
generally single but there may be up to four periphery of the host cell. The parasite may not
nuclei of unequal size (not shown on plate). fill the host cell (Figs. 29, 30).
Sometimes marginal (applique) forms are The asexual cycle follows a tertian
present, a characteristic of P. coatneyi of periodicity.
macaques and of P. falciparum of man.
Stippling of the Schüffner type appears as the
young forms mature and is prominent in cells Sporogonic Cycle
harboring old trophozoites (Fig. 10). Host cells
are not enlarged. The older trophozoites are The natural vector of P. youngi is unknown
amoeboid (Figs. 11, 12). The cytoplasm stains a but is likely to be a member of the leucosphyrus
pale blue; pigment becomes heavier and is seen group of anopheline mosquitoes. Eyles et al
as yellowish-brown granules (Figs. 15, 16). (1964) found A. maculatus partially susceptible
Stippling is fairly prominent and there is no host in that oocysts developed slowly but failed to
cell enlargement. The original describers made a produce sporozoites. Warren et al (1965) also
point of the latter fact because their figures gave found only partial development in A. sundaicus,
the illusion that the host cell was enlarged. A. balabacensis introlatus, and A. maculatus
The cytoplasm of the young schizont takes (see Table 19).
a slightly darker stain than the earlier forms and
may fill the host cell completely. The pigment
collects into larger granules and Schüffner’s Cycle in the Tissue
stippling is prominent (Figs. 17-19). The older
schizonts appear to have depleted the host cell The tissue stages of this parasite are
cytoplasm to the extent that it stains poorly; the unknown.
cytoplasm of the parasite takes a light blue stain.
The merozoites may number from 12 to 20 with
an average of about 14. The pigment is Course of Infection
concentrated in large yellowish-brown granules
and comes together in a single mass during the According to Eyles et al (1964) gibbons (H.
final stages of schizogony (Figs. 23-26). lar) which received their infection via
The cytoplasm of the young gametocytes inoculation of parasitized blood exhibited peak
takes a deep blue stain and displays a prominent parasitemias of 43,000 to 130,000 per mm3 12 to
red nucleus. Schüffner’s stippling may be 16 days following inoculation. Although the
prominent. The cytoplasm of the parasitemia declined after the peak, parasites
macrogametocyte is compact and stains a pale persisted in the circulating blood for up to 192
blue with prominent pigment sometimes located days. Infections in these animals were more
toward the periphery of the parasite. The nucleus severe than usually seen in malaria infections in
is generally eccentric, takes a deep red stain, and lower primates. The maximum temperature
encloses a deeper staining, bar-like area. The

PLATE XXIV.—Plasmodium youngi.

Fig. 1. Normal red cell. Figs. 17-22. Developing schizonts.
Figs. 2-5. Young trophozoites. Figs. 23-26. Nearly mature and mature schizonts.
Figs. 6-15. Growing trophozoites. Figs. 27-28. Young adult and mature macrogametocytes.
Fig. 16. Mature trophozoites. Figs. 29-30. Young adult and mature microgametocytes.
166 PRIMATE MALARIAS

encountered was 106.5° F. The animals were H. concolor and another gibbon, possibly H.
clinically ill, anemic, and listless. It is not agilis. Neither of these animals developed an
unlikely that this parasite may actually intense parasitemia signifying the generally held
incapacitate some animals in the wild. belief that each of the malarias of gibbons is
Subsequent to the work of Eyles et al more or less host specific.
(1964) blood-inoculated infections have been The natural vector is unknown.
studied in 7 additional gibbons and the data Experimentally, Anopheles maculatus, A.
pooled. The median parasitemia curve for the 12 sundaicus, and A. b. introlatus have been proven
animals, during the first 60 days of patent susceptible to at least partial development of the
parasitemia (Fig. 36), shows that a peak count of parasite (Eyles et al, 1964; Warren et al, 1965).
approximately 30,000 per mm3 occurred on day
13. The parasite level then declined to about 100 Antigenic Relationships
per mm3 by day 50, followed by a secondary
rise. and Immunity
In instances where blood parasitized with
P. youngi has been inoculated into rhesus No antigen-antibody studies have been carried
monkeys no infection resulted. No attempts have out. There appears to be little cross-immunity.
been made to infect man with this parasite. between P. youngi and the other malarias of
gibbons, according to Warren et al (1966).
Those investigators were able to obtain a full-
Host Specificity blown infection with P. jefferyi in a gibbon that
had had prior infections with both P. youngi and
The normal host of P. youngi is the white-
with P. eylesi.
handed gibbon (H. lar). The infection has been
transferred by inoculation of parasitized blood to

FIGURE 36.—Median curve of Plasmodium youngi parasitemia in 12 gibbons, Hylobates lar, infected by the inoculation of
parasitized blood.
 PLASMODIUM YOUNGI 167

TABLE 19.—Oocyst diameters of Plasmodium youngi in Anopheles b. introlatus, A. maculatus, and A. sundaicus.

Days after A. b. introlatus A. maculatus A. sundaicus

infection
No. Range Mean* No. Range Mean No. Range Mean
6.5 13 12-27 18 9 15-22 21
7.5 10 22-30 27 13 15-30 24
8.5 5 20-27 23
9.5 1 30 30
10.5
11.5 8 30-60 44

* Measurements expressed in microns.

REFERENCES
CADIGAN, F. C., JR., WARD, R. A., and CHAICUMPA, V., SHIROISHI, T., DAVIS, J., and WARREN, McW., 1968.
1969. Further studies on the biology of human malarial Hepatocystis in the white-cheeked gibbon, Hylobates
parasites in gibbons from Thailand. Milit. Med. 134 : concolor. J. Parasit. 54 : 168.
757-766. WARD, R. A., and CADIGAN, F. C., JR., 1966. The development
EYLES, D. E., FONG, Y. L., DUNN, F. L., GUINN, E., of erythrocytic stages of Plasmodium falciparum in the
WARREN, McW. and SANDOSHAM, A. A., 1964. gibbon, Hylobates lar. Milit. Med. 131 : 944-951.
Plasmodium youngi n. sp., a malaria parasite of the WARREN, McW., BENNETT, G. F., SANDOSHAM, A. A., and
Malayan gibbon, Hylobates lar lar. Am. J. Trop. Med. COATNEY, G. R., 1965. Plasmodium eylesi sp. nov., a
& Hyg. 13 : 248-255. tertian malaria parasite from the white-handed gibbon,
FRASER, H., 1909. Annual Report, Institute for Medical Hylobates lar. Ann. Trop. Med. Parasit. 59 : 500-508.
Research, Federated Malay States. p. 6. WARREN, McW., COATNEY, G. R., and SKINNER, J. C., 1966.
RODHAIN, J., 1941. Sur un Plasmodium du gibbon Hylobates Plasmodium jefferyi sp. n. from Hylobates lar in
lensciscus Geoff. Acta. Bioi. Belge. 1 : 118-123. Malaya. J. Parasit. 52 : 9-13.
This page intentionally left blank.
 SECTION 3
Ovale-Type Parasites
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 15
Plasmodium ovale Stephens, 1922

Nicol, and Shute (1932, 1933, 1935) plus the

BECAUSE of the close critical comparative study of the sporogonic
resemblance of Plasmodium ovale to P. vivax it cycles by Shute and Maryon (1952), acceptance
is impossible to tell when P. ovale was first was inevitable, although as late as 1935
seen. Macfie and Ingram (1917) described a Giovannola held that P. ovale was a
parasite in a mixed infection which they found modification of P. vivax in chronic infections.
in the blood of a child in the Gold Coast which As mentioned earlier, Craig (loc. cit.) had
may well have been P. ovale. They mentioned described a vivax-like parasite from the
the pronounced Schüffner’s stippling in the Philippines. In 1914 he accepted it to be P. vivax
parasites and at the same time stressed the fact var. minuta Emin. In 1933, he reversed his 1914
that the organism resembled the quartan parasite, opinion after he received stained preparations of
P. malariae, which it does. Earlier, Emin (1914) P. ovale from Professor Yorke in Liverpool,
had found a parasite in pilgrims at Camaran, on stating that the parasite seen by him in 1900
the Red Sea, which he named Plasmodium vivax agreed perfectly with P. ovale. The importance
var. minuta. Ziemann (1915) examined some of of this announcement lies in the fact that, for the
Emin's material and because his observations first time, P. ovale had been found outside of
were at variance with Emin's, he suggested the Africa.
name P. camaranense Emin which, of course, is In 1941, Coatney and Young reviewed all
zoologically unacceptable irrespective of the the papers dealing with the nomenclature of the
worth of his observations. To further complicate ovale parasite and concluded that none of the
the picture, Craig (1900) described a malaria names attributed to it could be placed in
parasite which he found in the blood of synonomy. The present authors hold the same
American soldiers, returned from the opinion, and, that the correct name for the
Philippines, and noted especially its tertian fever parasite is Plasmodium ovale Stephens, 1922.
pattern plus certain peculiar morphological The ancestral home of P. ovale is
characteristics not found in P. vivax. In 1918, undoubtedly tropical Africa although it has been
Stephens had an East African patient with reported from time to time from each of the
malaria at the Liverpool School of Tropical other continents. Plasmodium ovale is a rare
Medicine. In examining blood films from the parasite. In fact, as late as 1949, Brumpt was
patient, he was struck by the fact that among the able to record only 105 cases for the world; only
parasitized red cells some were oval and with 14 were outside of Africa. In 1966, Lysenko and
fimbriated edges. In 1922, he published a full Beljaev made a careful appraisal of the areas of
description of the blood forms and illustrated the distribution of P. ovale, and, after considering
sequence of development in a well executed all the reports, many of which are of doubtful
plate with 22 figures. A fourth species of human validity for reasons readily apparent to
malaria, difficult to separate from P. vivax, was malariologists, their conclusion was that, outside
not readily accepted; but, as evidence of Africa, there are only two areas where P.
accumulated through the work of Stephens and ovale is endemic: the Philippines and New
Owen (1927), Yorke and Owen (1930) James, Guinea.

171
172 PRIMATE MALARIAS

Many hypotheses have been advanced to Brumpt (loc. cit.), in calling attention to the
explain the lack of ovale malaria outside of extreme rarity of P. ovale, mentioned that
Africa among which are: 1) climate and perhaps it was "a plasmodium of a cosmopolitan
vegetation, 2) relation between host and vector, vertebrate wandering in man." Languillon
3) host susceptibility, and 4) relation with simian (1957) was more specific. In commenting on his
malaria. It is generally recognized that he finding P. schwetzi in a child, he suggested that
highest prevalence of P. ovale is in equatorial P. ovale may be an adaptation of the ape
forests and savannah woodland but, as in parasite. In 1967, Contacos et al infected a series
northern Senegal, it may occur almost to the of volunteers with P. schwetzi via mosquito bite
borders of the desert. In the area of West Africa, (Contacos et al, 1970) and the close resemblance
prevalence decreases from south to north in line of that parasite to P. ovale in man lead Coatney
with the decrease in rainfall. Hence, prevalence (1968) to suggest that P. schwetzi was probably
is due, in part at least, to a combination of both a zoonosis and, if that is the case, the infection is
vegetation and climatic conditions. Yet, in other now diagnosed as P. ovale. The present authors
areas of the world where there are almost hold the same view.
identical climatic conditions, there are only If one examines the prevalence figures for
sporadic cases, as in the Western Pacific, or Africa, one finds that it is relatively common in
none, as in South and Central America. the West Coast with, according to Garnham
We will present a full discussion of the (1966), 10 percent of the young children
vectors as they relate to this problem in a later harboring the parasite in Nigeria, Ghana, Sierra
section but suffice it to say here that several Leone, Liberia, and the Gambia. From these
vectors (A. freeborni, A. maculatus, etc.) other areas, it spreads through Central Africa east to
than A. gambiae will transmit P. ovale readily so the coast but with greatly reduced incidence
that sporadic area distribution can hardly be except at the southern tip of its range in
linked to a lack of suitable vectors. In the realm Mozambique. Lacan (1962), in reporting on 105
of host susceptibility, it would appear from the cases of P. ovale from the French-speaking
work of Jeffery et al (1954, 1955) and Chin et al countries of Africa, noted that practically all
(1966) that Negroes and Caucasians are equally were from savannah areas; only 19 were from
susceptible to P. ovale; and, since natural the forest region. Furthermore, all were children
infections appear in New Guinea populated by under 8 years of age with maximum incidence in
Melanesians, and in the Philippines where the children 2 to 4 years old. Onori (1967) carried
people are mainly of Mongoloid stock, it would out an in depth survey in Uganda where, among
appear that all people are susceptible to P. ovale. 251 ovale infections, he found the parasite more
Prior to 1960, when the natural often in infants and adolescents than in older
transmission of a simian malaria to man was people. The highest prevalence rate (2.6) was in
established by Eyles et al, no one had given the 1 to 4 year age group and only 0.7 in those
serious consideration to the possible zoonotic individuals 20 years or older. We think it safe to
nature of the simian malarias although many point out that such figures could be misleading
early observers, namely Reichenow and as far as the general population is concerned
Rodhain, mentioned that the forms they saw in because surveys are more or less limited to
apes were like those in man. The latter author children for obvious reasons. A true prevalence
(1940) after transferring P. rodhaini, and figure for the population in any area probably
incidentally, P. schwetzi, too, from the cannot be had because of the evasive nature of
chimpanzee to man, was convinced that P. the parasite, its easy masking by any one of the
rodhaini was actually P. malariae but he gave other human malarias, and the rapidity with
no indication whether he considered the which chronicity, with very low parasitemias,
infection a zoonosis or an anthroponosis. develops.
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174 PRIMATE MALARIAS
 PLASMODIUM OVALE 175

Cycle in Blood same stage nucleus in P. vivax but only slightly

larger than P. schwetzi.
PLATE XXV Through the early divisions the nuclei
The young ring forms have a prominent
remain large (Figs. 16-19) and at maturity there
circular nucleus eccentrically placed with a wisp
are ordinarily 8 merozoites although during
of cytoplasm surrounding a vacuole (Fig. 2). As
relapse or following continued passage, the
the parasite grows, the erythrocyte becomes
number may be 12 to 16 and located in a much
enlarged, the cytoplasm increases in amount,
enlarged host cell.
and the vacuole disappears (Fig. 10); older
The immature gametocyte is difficult to
trophozoites come to occupy about half the host
separate from the compact late trophozoite; but,
erythrocyte (Figs. 10, 11). The cytoplasm may
as growth proceeds, these forms grow to fill the
appear pulled out and ragged (Figs. 5, 8) or
enlarged host erythrocyte.
assume a band-like form, reminiscent of P.
The cytoplasm of the mature
malariae, (Fig. 6); double infections are not
macrogametocyte (Fig. 24) stains a medium blue
uncommon (Fig. 7). The host cell may appear
with an eccentrically placed red-staining
oval with fimbriated edges (Figs. 8, 13) when
prominent nucleus with darker staining areas.
the smear is dried in an atmosphere of low
The pigment is in granules arranged like a string
humidity; the same feature does not occur in P.
of beads and these short strands lie scattered in
vivax preparations made under the same
the cytoplasm. The stippling is prominent, takes
conditions. The pigment first appears as fine
a red stain, and is arranged in a ring around the
dust-like grains which later come together to
parasite.
form small greenish-brown beads (Fig. 18) and
The mature microgametocyte takes a lighter
later mass together in yellowish-brown patches
and more delicate blue stain. The large nucleus
(Fig. 21). The most distinctive characteristic of
occupies about half the parasite. It has a dark
the circulating ovale parasite is the stippling or
red-staining area from which the color fades to a
Schüffnerization, an apt term probably first
light pink toward the edge. The pigment is in
applied by Hauer (1937). This stippling appears
large to medium sized granules scattered
early in the growth of the parasite (Fig. 4) and
throughout the cytoplasm. The parasite is
becomes intense as the parasite grows (Figs. 15,
completely enclosed by a prominent circle of
17). The host cell cytoplasm becomes pale and
eosinophilic stippling (Fig. 25).
transparent and appears as a mass of stippling
The asexual cycle occupies 48 hours.
(Figs. 17, 19). Schüffnerization in P. ovale is
intense, more pronounced than in P. vivax and
not too different from P. schwetzi. Sporogonic Cycle
The cytoplasm of the parasite takes a PLATE XXVI
decided blue stain which fades somewhat during
early schizogony (Figs. 16-18) only to appear as Within the oocyst of P. ovale, the pigment
purplish-blue during the late stages (Figs. 21- granules are arranged in a highly characteristic
23). The large deep red-staining nuclei of the pattern which differentiates it from the other
young forms become larger with deeper red human malaria parasites (James et al, 1932,
patches as growth proceeds so that just 1933; Shute and Maryon, 1952). The 50 to 60
preceding division it is 2 to 4 times its original
size (Fig. 14) or about double the size of the

PLATE XXV.—Plasmodium ovale.

Fig. Normal red cell. Figs. 16-22. Developing schizonts.
Figs. 2-5. Young trophozoites. Fig. 23. Mature schizont.
Figs. 6-12. Growing trophozoites. Fig. 24. Adult macrogametocyte.
Figs. 13-15. Mature trophozoites. Fig. 25. Adult microgametocyte.
176 PRIMATE MALARIAS
 PLASMODIUM OVALE 177

grains of dark brown pigment granules are stephensi, and A. quadrimaculatus). The results
arranged in several patterns, but the most (Table 20) show a fairly close agreement with
common is two rows or chains which cross each those of previous workers concerning the length
other at right angles in the center of the oocyst. of time required for completion of the cycle (14
This pattern is first seen on the 4th day of to 15 days). However, they found a much greater
growth, but is more plainly visible on the 6th to range in oocyst diameters with maximum sizes
8th days. Jeffery (1954) reported that this pattern of up to 96 µ. There was little difference in the
of the pigment was present in 38.2 percent of the size of the oocysts in the different species until
oocysts of the Donaldson strain. In contrast, day 14, at which time, those in A. maculatus
such a pattern was found in two strains of P. were noticeably smaller. The larger oocysts were
vivax in only 8.0 and 7.2 percent of the oocysts. found in the A. freeborni mosquitoes.
The oocysts, in A. atroparvus mosquitoes, A comparison of the oocyst growth curve
according to Shute and Maryon (loc. cit.), grew of P. ovale with that of P. cynomolgi (Fig. 37),
at a daily rate of about 2 µ from the 3rd to the indicates that the former is a much slower
6th day at an incubation temperature of 25° C. growing parasite. There was approximately a 30
There was a sharp increase in size between the µ difference in oocyst diameters after 10 days of
7th and 8th days of about 5 µ; this daily increase extrinsic incubation. Whereas the P. cynomolgi
of 5 µ continued until the 12th day. Completion parasites in A. freeborni mosquitoes completed
of the cycle required 15 days. Oocysts ranged their development in 11 days, the P. ovale
from a minimum of 9 µ to a maximum of 37 µ parasites required 14 days. A comparison of the
in diameter. growth rate of P. ovale with P. vivax and P.
James et al (1932) found that a strain of P. falciparum (see Chapter 5) indicates that it is the
ovale from the Congo required 16 days to slowest growing of the human tertian malarias
complete its development whereas Sinton et al although it eventually becomes the largest by
(1939), using strains from the Congo and the day 14. However, P. ovale completes its
Gold Coast (Ghana), reported completion of sporogonic cycle 3 days sooner than does P.
development in A. atroparvus in 15 days. Using malariae (see Chapter 18).
the Donaldson strain from the West Pacific and The first experimental transmission of P.
A. quadrimaculatus and A. albimanus ovale was via the bites of A. atroparvus
mosquitoes, Jeffery (1954) found sporozoites in mosquitoes (James et al, 1932, 1933). During
the salivary glands as early as day 14 although the course of their studies, James and his co-
they usually appeared on day 15. workers (1949) obtained 36 transmissions of this
Collins et al (1969), using a strain of P. parasite via A. atroparvus. With the Donaldson
ovale from West Africa, studied the oocyst strain, infections were transmitted to 37 patients
growth rate in 5 species of anophelines (A. using A. quadrimaculatus and A. albimanus
freeborni, A. b. balabacensis, A. maculatus, A.

PLATE XXVI.—Developing oocysts of Plasmodium ovale (West African strain) in Anopheles b. balabacensis and A. freeborni
mosquitoes. X 580.
Fig. 1. 4-day oocyst showing scattered pigment. Fig. 10. 10-day oocyst.
Fig. 2. 5-day oocyst. Fig. 11. 11-day oocyst showing many small vacuoles.
Fig. 3. 6-day oocyst. Fig. 12. 12-day oocyst showing first signs of
Fig. 4. 6-day oocyst showing “Prince of Wales Feathers” differentiation.
arrangement of pigment. Fig. 13. 13-day oocyst showing differentiation.
Fig. 5. 7-day oocyst showing crossing of pigment. Fig. 14. 13-day oocyst.
Fig. 6. 8-day oocyst. Fig. 15. 13-day oocysts.
Fig. 7. 9-day oocyst showing scattered pigment and Fig. 16. 14-day oocyst showing concentration of
small vacuaoles. sporozoites near periphery and small vacuoles in center.
Fig. 8. 10-day oocyst. Fig. 17. 14-day fully differentiated oocyst.
Fig. 9. 10-day oocyst. Fig. 18. 14-day fully differentiated oocyst.
178 PRIMATE MALARIAS

mosquitoes (Jeffery et al, 1954). Later, Jeffery et transmitted a local strain of P. ovale to a
al (1955) reported 4 transmissions of a Liberian Liberian child and to a chimpanzee. Chin et al
strain of P. ovale via the bites of A. (1966) reported transmission of a West African
quadrimaculatus and A. albimanus mosquitoes. strain of this parasite to 5 volunteers using A.
Bray (1957) using A. gambiae mosquitoes, freeborni and A. maculatus mosquitoes.

FIGURE 37.—Range in oocyst diamters and the mean oocyst diameter curves of Plasmodium ovale and P. cynomolgi in
Anopheles freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

TABLE 20.—Oocyst diameters of Plasmodium ovale in Anopheles freeborni, A. b. balabacensis, A. maculatus,

A. stephensi, and A. quadrimaculatus.

Days after
A. freeborni A. b. balabacensis A. maculatus A. stephensi A. quadrimaculatus
Infection
No. Range Mean* No. Range Mean No. Range Mean No. Range Mean No. Range Mean

4 52 9-14 12 38 8-12 11 50 9-14 12 50 8-15 11 53 8-15 12

5 103 11-18 14 100 10-21 14 100 8-19 14 100 10-20 15 100 8-20 15
6 126 9-21 16 100 10-20 16 100 11-24 17 100 10-21 16 100 12-25 19
7 100 11-26 20 98 12-28 20 100 13-34 20 100 13-27 20 100 14-30 24
8 100 15-41 28 100 17-35 26 100 17-35 26 100 15-35 27 100 15-35 30
9 136 15-40 28 109 18-39 29 101 14-35 24 100 15-39 30 100 20-38 35
10 102 15-51 38 100 19-41 32 100 17-43 30 100 14-41 30 100 15-41 29
11 100 20-55 39 55 25-51 36 104 14-50 32 60 25-51 42 108 18-54 39
12 96 24-59 43 89 27-55 40 100 27-53 39 100 12-60 42t 100 19-59 42
13 108 24-65 46† 96 25-65 42 100 20-61 41 100 22-61 45t 30 22-66 44
14 20 41-76 64†** 100 35-71 52† 100 38-71 51†** 108 25-84 54†**
15 50 43-96 68†** 50 35-80 59†** 39 25-55 48†** 8 43-54 48†

Totals 1093 9-96 1035 8-80 1094 8-71 918 8-61 999 8-84

* Measurements expressed in microns; incubation temperature 25° C.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
 PLASMODIUM OVALE 179

Cycle in the Tissue P. ovale tissue stages in the chimpanzee. He

reported 7-day stages measured an average of
36.6 x 30.3 µ (range of 45 to 28 µ. x 39 to 23 µ.
The tissue stages of P. ovale were first
He described 4 different forms of schizonts
described in liver biopsy material from a human
which occurred regularly in the chimpanzee
volunteer by Garnham et al (1955). The
material. Eighteen day schizonts measured from
youngest stages described by these authors were
91 to 40 µ x 50 to 31 µ. One schizont, found in a
considered to be 5-day stages and measured
39-day biopsy, measured 31 x 33 µ.
from 28 to 60 µ in length. The smallest of these
In his paper, Bray (1957) points out that the
forms were oval with regular or smooth edges.
EE bodies of P. ovale display at least 3
The larger of these 5-day forms showed small
characteristics that have not been noted in the
convolutions on the surface. The most striking
tissue stages of P. vivax or P. falciparum;
feature observed in these immature forms was
namely, 1) a definite limiting membrane or
the "relatively" enormous size of the nuclei-
periplast; 2) peripheral nuclear bars tangential
approximately 2 µ. The 9-day tissue stage of
rather than radial; and 3) a minor but distinct
ovale measured from 70 to 80 µ by 50 µ. The
hypertrophy of the host cell nucleus.
surface was lobated and the nuclei were about
Rupture of the mature schizonts of the pre-
half the size found in the 5-day stage, in other
erythrocytic cycle is believed to occur chiefly on
words, roughly 1 µ.
the 9th day after exposure to infection. The
The nuclei of the tissue stages had an
positive biopsies of 18 and 39 days suggest the
uneven margin and the cytoplasm was granular.
apparent existence of second-generation forms.
Sometimes the cytoplasm was clumped around
Bray et al (1963) attempted to demonstrate the
each nucleus so that the tissue stage appeared to
existence of one or more exoerythrocytic cycles
contain clefts or to be vacuolated. A definite
after the initial EE cycle, in the chimpanzee.
limiting membrane was described for many of
Biopsy on the 19th day revealed bursting and
the schizonts.
mature schizonts as had been observed on the
As the EE body of P. ovale approaches
9th day, suggesting the existence of at least a
maturity (on or about the 9th day), it goes
second generation.
through what is described as 3 stages: 1) nuclear
multiplication progresses rapidly with no evident
merozoite formation; 2) the appearance of Course of Infection
merozoites at the edges of the EE bodies, with Sinton et al (1939) alluded to a mean
active nuclear division continuing elsewhere prepatent period for several strains of P. ovale of
within the parasite; and 3) complete maturation about 15 days. James et al (1949) reported
of the schizont with rupture. incubation periods (which may, on occasion, be
The merozoite of the P. ovale tissue the same as the prepatent period) of 11 to 16
schizont is described as a remarkable structure. days with a mean of 13.6 days in sporozoite
It is large (1.8 µ in diameter), spherical, and induced infections with 6 different strains of P.
consists of 2 portions; a larger portion of ovale. They reported one case with a delayed
cytoplasm and a smaller portion which is the incubation period of 85 days. The Donaldson
nucleus situated at one side of the parasite. The strain (Jeffery et al, 1954) exhibited prepatent
number of merozoites in one schizont of P. periods of 12 to 20 days with a mean of 15.3
ovale has been reported to be as high as days. Employing a Liberian strain, Garnham et
15,000±. al (1955), Jeffery et al (1955), and Bray (1957)
In the paper describing the tissue stages of reported prepatent periods of 13.5, 14 to 15
P. ovale, the authors noted that P. ovale tissue (mean 14.5), and 14 days, respectively. Chin et
stages differ markedly from those of P. vivax al (1966) working with a West African strain
and P. inui. They were of the opinion, however, obtained prepatent periods of 14 to 18 days with
that there was a superficial resemblance to those a mean of 16.8 days.
of P. falciparum, at least in regard to size and Generally, P. ovale infections are
form. Bray (1957) described the development of considered to be the least severe of the 4 species
180 PRIMATE MALARIAS

of human malaria and are characterized by a were seen from 14 to 26 days after exposure to
mild clinical course. In the mosquito induced infection, with a mean of 17.7 days or a median
infections reported by James et al (1949) the of 17 days; maximum mean fever was 105.2° F.
onset of the typical primary attack was definitely The number of paroxysms observed in infections
less severe than that observed with vivax (103 cases) with this strain ranged from 1 to 22
malaria. The characteristic rigor of vivax malaria with a mean or median of 8. With the West
was a rarity with ovale malaria; only one or two African strain of ovale malaria, we observed a
characteristic rigors occurring during the early temperature of only 103.6° F when the parasite
stages (10 days or so) of the primary attack. count was as high as 32, 450 per mm3.
Even though the patient may feel chilly, Using the interval between tertian fever
chattering of teeth and the classical shaking with peaks as reference points, the periodicity of
a sensation of being cold from head to foot are Donaldson strain infections was found to range
seldom or never seen. However, they found the from 34 to 61 hours with a median of 49 hours.
duration of the entire paroxysm to be the same Maximum parasitemia is considered to be
for ovale as it is in vivax malaria (6 to 10 hours). generally lower than that observed with vivax
Garnham et al (1955) took exception in that the malaria. Sinton et al (1939) reported variations
paroxysms in their cases were usually in maximum parasitemias ranging from 125 to
accompanied by chills as well as severe and 20,000 per mm3 while James et al (1949)
persistent headache. Temperatures of up to 105° reported maximum parasite densities ranging
F in one volunteer persisted for 3 weeks. The from 500 to 100,000 per mm3. Jeffery et al, in
spleen became palpable after about 10 days. studies with the Donaldson strain, reported
James et al (1949) observed another ranges of maximum counts from 464 to 25,940
obvious difference between ovale and vivax parasites per mm3 with a mean of 8,376. With
malaria; namely, the degree of fever at the peaks the West African strain of P. ovale, we have
of the paroxysms. They reported that only 30 out observed maximum counts ranging up to 32,450
of 197 patients (15 percent) had 10 or more parasites per mm3 in blood-induced infections.
febrile paroxysms with peak temperatures Figure 38 shows the minimum and
exceeding 103° F. With the Donaldson strain maximum parasitemia curves for 24 blood-
(Jeffery et al, 1954), only 10 percent of the induced infections with the Donaldson strain of
subjects had 10 paroxysms with peak P. ovale, (17 Negro and 7 Caucasian) as well as
temperatures exceeding 103° F. The first fevers

FIGURE 38.—Maximum and minimum parasite counts and median parasitemia curves of 24 blood-induced infections of
Plasmodium ovale in man (17 Negro and 7 Caucasian).
 PLASMODIUM OVALE 181

the median parasitemia curves for the two primary attack. James et al (1949) reported that,
groups. It can be seen that peak parasitemia was in their experience with 36 patients infected by
reached in the Caucasian and Negro patients on the bites of mosquitoes, no relapses were
the 8th and 10th days of patent parasitemia with observed. It is not unlikely that asymptomatic
maximum median counts of 4,576 and 3,645 relapses were missed if patients were followed
parasites per mm3, respectively, followed by a up on a strict clinical basis. Sinton (1939)
steady and slow decline to rather low level encountered one relapse in a series of sporozoite
parasitemia by day 40. There is no apparent induced cases of ovale malaria. Jeffery et al
difference between the median parasitemias for (1954) found that relapses were observed
the two groups of patients. This is in great frequently in 38 mosquito induced infections of
contrast with what is generally observed with Donaldson strain ovale malaria. Most of them
vivax malaria infections in Negro and Caucasian were asymptomatic relapses. Only 2 of the 38
patients. The differences between the minimum patients showed symptomatic relapse at 148 and
and maximum parasitemia curves on a day to 235 days after termination of the primary attacks
day basis point out the great variability one can with chloroquine. One of these patients
observe in infections with most human malarias. subsequently experienced an asymptomatic
Duration of parasitemia in sporozoite relapse with low grade parasitemia 152 days
induced infections with the Donaldson strain later.
ranged from 29 to 91 days with a mean of 53.2 Garnham et al (1955) observed relapses in
days or a median of 51 days. During this period, two cases; one relapsed 103 days after
clinical manifestations lasted for 8 to 26 days chloroquine and again 68 days later and the
with an average of 16.9 days. Primary attacks of other relapsed after 98 days, and, again, 101
ovale malaria characteristically terminate days later. Chin and Coatney (1971) found that
spontaneously without the use of antimalarial infections with a West African strain of ovale
drugs even though low level asymptomatic malaria relapsed from 1 to 3 times during a one
parasitemia may continue intermittently for a year period. Relapses occurred as early as 17
time. After cessation of symptoms, in cases of days after treatment of the primary attack in one
Donaldson strain ovale malaria, parasites case and as late as 255 days in another case. All
continued to be present in the peripheral blood of the relapses were symptomatic, except one.
for 11 to 89 days with an average duration of Earlier authors spoke of latent infections
37.5 days. No more than 2 percent required (which we recognize as delayed primary at-
partial suppression or early termination of the tacks), a phenomenon not too uncommon with
infection as compared to 61 to 87 percent for vivax malaria. As mentioned earlier, James et al
some strains of vivax malaria. (1949) reported such a period of 85 days in one
Ovale malaria, in contrast to most strains of of their mosquito induced infections. Jeffery et
vivax malaria, is equally infective to Negroes al (1954), Trager and Most (1963), and Chin and
and Caucasians. Sinton et al (1939) noted the Contacos (1966) reported delayed primary
susceptibility of a Negro patient who had been attacks of 4, 1.8 and 3.5, and 1.3 years,
previously resistant to other human malarias and respectively. These delayed primary infections
suggested that the Negro may show lower racial were probably not true latent infections in the
immunity to ovale malaria than to other classical sense. Rather, they represent late tissue
malarias. Jeffery et al (1954, 1955) found that parasite (relapse) activity.
Negro and Caucasian patients inoculated either Bray (1957) reported that infection of
with Donaldson strain or the Liberian strain of chimpanzees with ovale malaria, can be obtained
ovale malaria were equally susceptible to either by the inoculation of sporozoites or
infection. In fact, they observed no obvious parasitized blood. In the intact animal, there was
differences in prepatent period, incubation evidence of some natural resistance with
period, or clinical activity. spontaneous termination of patent parasitemia
There can be no doubt that ovale malaria is after 8 days. On the other hand, splenectomized
a true relapsing infection; i.e., sporulation from chimpanzees allowed parasitemia to increase for
tissue stages in the liver subsequent to the 10 days with a total duration of patent
182 PRIMATE MALARIAS

parasitemia of 21 days. In addition, the infection SPECIES REFERENCES

in the chimpanzee differs from that in man by its Anopheles albimanus Jeffery, 1954; Jeffery et al,
1954,1955
tendency to produce blood schizonts with more A. atroparvus James et al, 1932, 1933;
than the usual number of merozoites. Sinton et al, 1939; Shute and
Maryon, 1952; Garnham et
al, 1955
Host Specificity A. freeborni Chin et al, 1966
A. maculatus Chin et al, 1966
A. quadrimaculatus Jeffery, 1954; Jeffery et al,
Man is the natural host of this parasite. 1954
Infections have been obtained in intact and A. superpictus Garnham, 1966
splenectomized chimpanzees (Pan troglodytes In addition, we have infected A. stephensi and A.
versus) by the inoculation of sporozoites from A. b. balabacensis mosquitoes with a West African
gambiae mosquitoes (Bray, 1957). Attempts to strain of P. ovale. In a comparative study,
infect rhesus monkeys (M. mulatta) by the Jeffery (1954) showed that A. quadrimaculatus
inoculation of parasitized blood have been mosquitoes were more susceptible than strains
unsuccessful (Christophers, 1934; Jeffery, of A. albimanus from Panama and the Florida
1961). We have made 6 unsuccessful attempts to Keys, to the Donaldson strain of P. ovale. The
infect A. trivirgatus monkeys with P. ovale. relative susceptibility was A. quadrimaculatus,
However, in view of the ease with which 100; A. albimanus (Florida Keys), 77; A.
infections with the other human malarias have albimanus (Panama), 46. In a comparative study
been adapted to this host, its eventual adaptation with the West African strain, (Table 22), we
is thought likely. found that A. stephensi mosquitoes were the
The natural vector of P. ovale is unknown. most susceptible, followed by A. freeborni, A. b.
However, since Bray (loc. cit.) was able to balabacensis, A. quadrimaculatus, A. maculatus,
obtain infections in A. gambiae mosquitoes after and finally, A. albimanus. There was a lesser
they fed on man and on the chimpanzee, this is variation in susceptibility to infection than has
strongly indicative that it is a vector in Africa. been found in studies with the other primate
Vectors of this parasite found in the west Pacific malarias.
have not been determined. Other mosquitoes
which have been experimentally infected are Immunity and Antigenic
shown in the table below:
Relationships
Sinton et al (1939a) showed that a high and
almost equal degree of resistance to reinfection

TABLE 22.—Comparative infectivity of Plasmodium ovale to Anopheles freeborni, A. stephensi, A. b. balabacensis,

A. quadrimaculatus, A. maculatus, and A. albimanus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

F-1 100
6 169 153 59.8 58.2
F-1 : St-1 121.3
8 206 96 73.8 78.1
F-1 : Bal 83.4
14 253 452 69.6 41.2
F-1 : Q-1 43.7
16 238 302 76.9 61.6
F-1 : Mac 43.4
5 23 23 78.3 30.4
F-1 : Alb 30.7

* F-1 = Anopheles freeborni; St-1 = A. stephensi; Hal = A. b. balabacensis; Q-1 = A. quadrimaculatus; Mac = A. maculatus; Alb = A. albimanus.
**GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A.freeborni to another species where the GII
of A. freeborni = 100.
 PLASMODIUM OVALE 183

developed to homologous and to heterologous did not seem to confer appreciable immunity
strains following a primary infection with P. against a subsequent Donaldson strain infection.
ovale. On the other hand, these investigators Sinton (1940), on the basis of his studies on
found that ovale malaria could be successfully the immunity acquired as a result of sporozoite
established in persons who were apparently induced infections as compared to the immunity
immune to the other species of human malaria. acquired by those infections induced by
Jeffery et al (1954, 1955) studied the erythrocytic asexual stages, suggested that
immunologic relationships of several strains of immunity in malaria is more complete if the
ovale malaria. They observed that one infection initial infection is induced by sporozoites.
with the Donaldson strain of ovale malaria Garnham (1966) stated that the antigenic
conferred immunity to reinfection with the same structure of different strains of P. ovale seems to
strain. Moreover, in comparing a Pacific strain be remarkably homogenous. Sinton (1940), in
(Donaldson) and a West African strain studying heterologous strain immunity of ovale
(Liberian) of P. ovale, they found considerable malaria concluded that the heterologous
cross-immunity between them, but in no way antigenic element seemed to be so small in the
was the immunity complete. However, the strains studied by him ''as to make them almost
degree of cross-immunity was greater than identical immunologically."
expected; not only in view of the wide It seems safe to say, that at this writing, no
geographic separation in the origin of the two clear-cut statement can be put forth regarding
isolates, but also on the basis of their experience immunity involving P. ovale.
with various strains of vivax and falciparum Meuwissen (1966) found a high degree of
malaria. cross-reactivity with P. fieldi antigen in sera of
These investigators further reported that patients with P. ovale infections using the
ovale malaria (Donaldson strain) conferred no indirect fluorescent antibody test. In a later study
effective immunity against the other 3 species of (1968), he found that P. ovale antisera would
human malaria. The converse was also ob- cross-react to the P. cynomolgi bastianellii
served; namely, that previous experience with antigen but at a lower level than to the
other species of human malaria did not homologous antigen or to the P. fieldi antigen.
appreciably effect subsequent susceptibility to
infection with the Donaldson strain of P. ovale.
For example, a previous Chesson vivax infection

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Am. J. Trop. Med. & Hyg. 12 : 1-12. Hyg. 17 : 147-155.
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Plasmodium. In Malariology, Vol. I by Boyd. W. B. the human malaria parasites with notes on the principal
Saunders, Philadelphia, Pa. pp. 787. American strains. Human malaria. Am. Assoc.
CHIN, W., CONTACOS, P. G., and BUXBAUM, J. N., 1966. The Advancement of Science. No.15.
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ovale by Anopheles freeborni and Anopheles M. H., and GELL, L. S., 1969. Some observations on
maculatus. Am. J . Trop. Med. & Hyg. 15 : 690-693. the sporogonic cycle of Plasmodium schwetzi, P. vivax
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CHIN, W. and COATNEY, G. R., 1971. Observations on relapse COLLINS, W. E., CHIN, W., and JETER, M. H., 1970.
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 16
Plasmodium fieldi Eyles, Laing, and Fong, 1962

parasitemia was too low to allow for species

ONLY three species of identification. The animal was splenectomized
malaria were known from macaques when Dr. in January, 1961, after which the parasitemia
Eyles embarked on his extraordinary series of increased permitting more careful study of the
studies on simian malaria in peninsular Malaysia parasite which confirmed an earlier assumption
in mid-August of 1960. In September of that that it was a new species. The Eyles group gave
year, he purchased a young pig-tailed macaque it the name Plasmodium fieldi in honor of Dr.
(Macaca nemestrina) from a trapper who said it John W. Field, who has made outstanding
had been taken in the district of Kuala Selangor contributions to our knowledge of malaria in
in the state of Selangor, Malayasia. The monkey general and especially in Malayasia.
was carrying a malaria parasite but the

185
 PLASMODIUM FIELDI 187

Cycle in the Blood enlarged, encloses a red ring of coalesced

eosinophilic stippling (Fig. 23).
PLATE XXVII The mature microgametocytes fill the host
The youngest parasites are ring-shaped and cell and exhibit a dark pink cytoplasm. The
about 3 µ in diameter. Some have double reddish stained nucleus, with a deep red bar-like
chromatin bodies. Multiple infections of the mass, is located eccentrically (Fig. 24). The
erythrocyte are not common; stippling appears pigment granules are heavy and fairly evenly
when the trophozoite is about half-grown (Fig. distributed in the cytoplasm. The host cells show
5). pronounced stippling and some exhibit
The older trophozoites (Figs. 11-13) are fimbriated edges.
compact, rounded or oval, and display very little The asexual cycle is 48 hours.
amoeboidity. The cytoplasm is compact, stains a
deep blue, and the nucleus a deep red; pigment
is dark and made up of fine grains; Schüffner- Sporogonic Cycle
type stippling takes a deep red stain. Some host PLATE XXVIII
cells are oval-shaped (Figs. 7-9); older parasites,
with the vacuole diminished or lost, occur in The sporogonic development of P. fieldi
cells with aggregates of dark eosinophilic has been examined in A. b. balabacensis, A.
masses sometimes larger than their nuclei (Figs. maculatus, and A. freeborni mosquitoes (Table
11-13). The host cell is slightly enlarged. 23). In A. b. balabacensis, at day 5, the mean
Immature schizonts (Figs. 14-20) exhibit diameter was 13 µ, with a range of 8 to 14 µ.
dense blue-staining cytoplasm and relatively The oocysts continued to grow so that by day
large deep red nuclei; pigment is granular, well 13, the mean size was 68 µ, with a range of 32 to
distributed, generally black; stippling is heavy, 96 µ. Sporozoites were present in the salivary
and, as schizogony proceeds, the eosinophilic glands by day 14.
masses come together to form a deep red border Although the oocyst measurements in the
around the developing schizont (Fig. 18). The A. maculatus mosquitoes were limited in
host cell may be appreciably enlarged-- number, it appeared that the mean diameters
ballooned-out--, some of them assume an oval were smaller than in the A. b. balabacensis
shape rather than circular (Figs. 15, 17). The during the period of oocyst differentiation.
mature schizonts (Figs. 20, 21) produce 4 to 16 Sporozoites were present in the salivary glands
large merozoites with a mean number of 12. The of these mosquitoes by day 14. The oocyst
golden brown pigment forms a large mass measurements in the A. freeborni were within
ofttimes in the center of the schizont. The host the ranges of those seen in the other two.
cell may become greatly distorted (Fig. 21 and Although oocyst differentiation appeared to be
earlier); the explanation for this is not known but normal, sporozoites were found only near the
it appears to be distinctive for this parasite. dissected guts of the mosquitoes and there was
The adult macrogametocytes have the no evidence that they had invaded the salivary
nucleus placed off-center; it stains dark red. The glands.
cytoplasm stains a deep blue, and supports A comparison of the P. fieldi oocyst growth
delicate, dark pigment granules scattered in the
cytoplasm. The host cell, which may be slightly

PLATE XXVII.—Plasmodium fieldi.

Fig. 1. Normal red cell. Figs. 14-19. Developing schizonts showing typical host
Figs. 2-4. Young trophozoites. cell distortion.
Figs. 5-10. Growing trophozoites. Figs. 20, 21. Mature schizonts showing ‘ballooned-out’
Figs. 11-13. Nearly mature and mature trophozoites host cell distortion.
with pronounced eosinophilic stippling. Figs. 22, 23. Developing and mature macrogametocytes.
Fig. 24. Mature microgametocyte.
188 PRIMATE MALARIAS
 PLASMODIUM FIELDI 189

curves with P. cynomolgi in A. b. balabacensis fieldi and P. simiovale indicates a close

mosquitoes (Fig. 39), shows that P. fieldi relationship between these two species, almost
requires more time to complete its development, on the same level as those found by Bennett et
than does P. cynomolgi. The oocyst diameters of al, between isolates of P. cynomolgi.
the P. cynomolgi at day 10 were approximately We obtained transmission of P. fieldi to the
equal to those of P. fieldi on day 13. In addition, rhesus monkey via the bites of A. b.
the appearance of P. fieldi sporozoites in the balabacensis (10 times), by A. maculatus (once),
salivary glands required 4 days longer than the and by A. stephensi (once) (see Collins, et al
P. cynomolgi parasite. 1968). In addition, infections have been obtained
In some ways, P. fieldi is similar to P. in our laboratory by the intravenous and/or intra-
simiovale in its sporogonic development. hepatic inoculation of sporozoites from A. b.
However, during the extrinsic incubation period balabacensis (7 times), A. freeborni (twice), and
of 8 to 13 days, oocysts of P. fieldi were slightly A. stephensi (twice). The prepatent periods for
smaller than those of P. simiovale. Also, the the 23 infections ranged from 9 to 18 days with
latter parasite completed its cycle 1 day sooner. a mean of 12.4 days. Coombs et al (1968) also
It was shown by Bennett et al (1966) that there obtained transmission of this parasite to the
are differences in the growth rate and time of the rhesus monkey by the intravenous inoculation of
appearance of sporozoites in the salivary glands sporozoite from A. b. balabacensis mosquitoes.
between different sub-species and isolates of P. The prepatent period was 13 days.
cynomolgi. It is possible that this minor Our attempts to transmit the infection to
difference between the sporogonic cycles of P. man (10 volunteers) via the bites of infected

TABLE 23.—Oocyst diameters of Plasmodium fieldi in Anopheles b. balabacensis, A. maculatus, and

A. freeborni mosquitoes.

Days after A. b. balabacensis A. maculatus A. freeborni

Infection No. Range Mean* No. Range Mean No. Range Mean
4 39 8-14 11
5 59 8-14 13 84 9-18 13
6 62 11-22 16 112 9-19 15
7 115 12-27 19 4 18-30 23 118 11-37 21
8 136 12-35 25 16 15-31 23 126 13-34 24
9 132 17-45 32 97 17-44 30
10 51 18-65 40 55 24-59 44 119 15-59 34
11 59 31-64 52† 31 31-74 49 95 24-72 47
12 160 17-92 59† 144 23-83 50† 15 26-71 53†
13 187 32-96 68† 101 18-79 50† 67 34-83 59†
14 135 25-104 64†** 109 30-85 57†** 23 30-68 50†
Totals 1096 8-104 460 15-85 895 8-83

* Measurements expressed in microns.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.

PLATE XXVIII.—Developing oocysts of Plasmodium fieldi in Anopheles b. balabacensis mosquitoes. X 580 (Except Figs. 1 & 2).
Fig. 1. 5-day oocysts. X 740. Fig. 7. 11-day oocyst showing some small vacuoles.
Fig. 2. 6-day oocyst showing clumped pigment. X 740. Fig. 8. 12-day oocyst.
Fig. 3. 7-day oocyst. Fig. 9. 13-day oocyst showing numerous small vacuoles.
Fig. 4. 8-day oocyst. Fig. 10. 13-day differentiating oocyst.
Fig. 5. 9-day oocyst. Fig. 11. 13-day fully differentiated oocyst.
Fig. 6. 10-day oocyst.
190 PRIMATE MALARIAS

FIGURE 39.—Range in oocyst diameters and mean oocyst diameter curve of Plasmodium fieldi and P. cynomolgi in Anopheles b.
balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

mosquitoes were unsuccessful. The sporozoites stain and, in some sections, showed dark blue
were viable because control rhesus monkeys cytoplasmic aggregates up to 3 µ in diameter.
became infected. Certain of the exoerythrocytic bodies stained a
deeper blue than others which suggested that
Cycle in the Tissue they were more compact. Some sections
exhibited pale pink staining vacuoles up to 3 µ
PLATE XXIX in diameter. In many preparations, there was a
With the exception of P. cynomolgi, it is
partial separation, measuring 1 to 4 µ, between
doubtful if the tissue phase of any simian
the parasite body itself and the surrounding host
malaria parasite has been studied more than has
tissue which was interpreted as shrinkage
P. fieldi in the rhesus monkey (Held et al, 1967;
following fixation.
Coombs et al, 1968; and Sodeman et al, 1970).
Coombs et al (1968) supplied data on the 6-
Held and his co-workers described the 8-,
and 12-dayforms of the parasite. Two 6-day old
10-, and 12-day forms and their illustrations are
forms were spheroid in shape and measured 18
models for future investigators to emulate.
and 20 µ in maximum length. One complete 12-
Generally, the exoerythrocytic bodies were
day form was ovoid in shape and measured 40 to
circular to elliptical in section, the body edges
60 µ which agrees with the 12-day form
were smooth, although some exhibited slight
described by Held et al.
indentations giving the impression of scalloping.
Sodeman et al (1970) studied the 5, 6, 7,
The nuclei, 0.5 to 1.0 µ in diameter, stained
and 9-day forms and they agree with the opinion
magenta and were generally circular; some
of Held et al to the effect that the tissue stages of
forms suggested a diploid or tetrad
configuration. The cytoplasm took a pale blue
 PLASMODIUM FIELDI 191

PLATE XXIX.—Exoerythrocytic bodies of Plasmodium fieldi in liver tissue of Macaca mulatta monkeys. X 580.
Fig.1. 8-day body showing small flocculi. Fig. 4. 10-day body.
Fig. 2. 8-day body shwing vacuoles. Fig. 5. 12-day body.
Fig. 3. 10-day body. Fig. 6. 12-day body.

P. fieldi exhibit no morphological characteristics period of one month. After the animal was
which would separate them from the other splenectomized, the parasite count reached a
species of Plasmodium. peak of 196,000 per mm3 21 days later, after
which it declined. Two pig-tailed macaques,
Course of Infection splenectomized prior to receiving the infection
via inoculation of parasitized blood, developed
counts of 51,000 and 127,000 per mm3 after
The course of the natural infection in the
which their parasitemias declined. Each of the
original pig-tailed macaque was followed by
animals continued to show a patent infection,
Eyles et al (loc. cit.) for some 3 months, prior to
with low to moderate counts, for months.
splenectomy, during which it exhibited a low
The same group of workers induced
parasitemia ranging from zero to 33 parasites per
infections with P. fieldi in two intact and two
mm3 of blood. After splenectomy, the parasite
splenectomized rhesus (M. mulatta) monkeys by
count increased rapidly to some 9,000 parasites
the inoculation of parasitized blood. The intact
per mm3 of blood and then declined, to persist at
animals displayed parasitemias of 11,000 to
a low level for several weeks. When the parasite
20,000 per mm3 of blood while, in the
was introduced into the intact natural host (M.
splenectomized animals the peak counts ranged
nemestrina), the parasite count did not exceed
from 50,000 to 100,000 per mm3 of blood.
737 per mm3 of blood during an observation
192 PRIMATE MALARIAS

A summary of our studies with P. fieldi The phenomenon of relapse had intrigued
(Fig. 40) shows that the parasitemias in blood- malariologists even before Thayer (1899)
induced infections, in intact M. mulatta published his series of lectures with illuminating
monkeys, reached a peak of approximately references to relapse in vivax malaria. Since
9,000 per mm3 by day 7 and declined rapidly to then, a prodigious literature has accumulated
a level of approximately 500 per mm3 by day 15. which can not be gone into here except to point
This level was maintained for the next 30 days, out that the phenomenon has received only
after which, the parasitemia receded slowly to cursory examination among the simian malarias.
minimal levels. In the splenectomized M. True relapses, in contrast to recrudescences, do
mulatta monkeys, the median peak parasitemia occur in sporozoite-induced P. cynomolgi
was almost 73,000 per mm3. There was infections. Our studies with P. fieldi lead us to
considerable variation in the parasitemia curve consider it related to P. ovale, a 'relapser' in
in these animals with four separate peaks of man, and a life-pattern study was set up to test
parasitemia during the 60-day observation its relapse potential. Each of seven rhesus
period. At 60 days, the median parasite count monkeys with sporozoite-induced infections was
was less than 500 per mm3. In the monkeys allowed to experience an initial parasitemia,
infected by sporozoites, the peak parasitemia which was treated early, as was each succeeding
was the same as in the blood-induced series, but attack, with either quinine sulfate, at a dosage of
was delayed by about 3 days. The parasitemia 300 mg daily for 5 or 7 days, or chloroquine
then rapidly declined to a minimal level by day phosphate 150 mg (base) daily for 2 days or 50
30. The secondary rise to a peak of mg daily for 3 days (see Fig. 41). These dosages
approximately 100 per mm3 by day 42, possibly in our hands were known to be curative of
represents relapse activity or the appearance of a blood-induced infections, and would, on that
new antigenic variant. In the 6 M. nemestrina basis, eradicate the blood forms in the
monkeys infected by inoculation of parasitized sporozoite-induced infections under study. As
blood, the peak of the median curve was 475 per may be seen by perusal of Figure 41, each
mm3 which obtained on day 9. Although there infection exhibited two or more relapses at
was a secondary rise in the parasitemia at varying intervals. The infection in one animal (T
approximately day 25, the levels were generally 688) exhibited 14 relapses during a period of 12
minimal. months. The relapses did not fall into a distinct
pattern, which was not unexpected, but one may

FIGURE 40.—Median parasitemia curves of Plasmodium fieldi as seen in 76 Macaca mulatta and in 6 M. nemestrina monkeys.
 PLASMODIUM FIELDI 193

FIGURE 41.—Relapse pattern of Plasmodium fieldi as seen in seven Macaca mulatta monkeys.

note, that as the time from initial infection mosquitoes, Anopheles hackeri and A.
increased the tendency for longer intervals balabacensis introlatus (Warren and Wharton.
between relapses increased also, which was 1963). Warren and Wharton (1963) reported that
expected. The main point was answered, A. donaldi, A. freeborni, A. hackeri, A. letifer, A.
namely, that P. fieldi does relapse and that maculatus, and A. philippinensis were
relapse producing infections may last for at least susceptible to infection, all at a low level.
one year. Additionally, A. b. balabacensis, A. kochi, A.
vagus, A. sinensis, A. albimanus, A. argyropus,
Host Specificity A. peditaeniatus, A. atroparvus, and A.
quadrimaculatus were shown to be susceptible,
The type host of P. fieldi is M. nemestrina,
at least to the production of oocysts. Anopheles
from which a single isolation was made by Eyles
freeborni was the most susceptible (Table 24)
et al (1962a). If the parasite was looked for more
followed by A. b. balabacensis, A. kochi, and A.
carefully in this host, it probably would be
vagus. However, A. freeborni did not readily
found, as it was in the kra monkey (M. irus (=
support P. fieldi infections to completion and
fascicularis) ) by Warren and Wharton (1963) in
therefore A. b. balabacensis is considered the
one of twenty of these animals taken in the
best experimental vector.
Kuang forest, north of Kuala Lumpur, Malaysia.
The parasite will also grow in rhesus
monkeys (M. mulatta) but as Warren et al Immunity and Antigenic
(1964) pointed out the parasite's unique staining Relationships
characteristics, i.e., large eosinophilic masses
Two M. mulatta monkeys infected with P.
and an intense red ring around the parasite, are
fieldi were allowed to have patent parasitemia
modified in M. fascicularis and in M. mulatta.
for 32 and 33 days, respectively, with peak
However, these hosts do display the enlarged
parasitemias of 15,100 and 21,300 per mm3.
parasitized host cell. Low level infections have
They were then given curative treatment with
been obtained by us in the baboon (Papio
chloroquine. When their blood was parasite-
doguera) and in M. radiata.
free, one animal was blood-inoculated with P.
Plasmodium fieldi has been isolated from
fragile and the other with P. cynomolgi.
two members of the Leucosphyrus group of
194 PRIMATE MALARIAS

Neither animal displayed any evidence of reciprocal titer ratios against the different
immunity since their peak counts reached 5/100 antisera were as follows: P. inui, 100:107; P.
and 11/100 RBC, respectively. In a reverse shortti (= OS strain P. inui), 100:47; P.
study, a rhesus monkey was allowed to brasilianum, 100:24; P. cynomolgi, 100:76; P.
experience a patent infection with P. cynomolgi coatneyi, 100:107; P. gonderi, 100:41; P.
for 62 days. It was then treated, as above, and fragile, 100:93; and P. knowlesi, 100:87.
later challenged with P. fieldi. The fieldi Plasmodium fieldi antigen has also been
infection was higher than normally expected, a shown to react at a high level to P. malariae, P.
peak count of 89,000 per mm3. On the basis of falciparum, and P. ovale antisera (Collins et al,
these limited data, it appears that there is no 1966a; Meuwissen, 1966, 1968). It appears,
cross-immunity among P. cynomolgi, P. fragile, therefore, that the P. fieldi antigen contains a
and P. fieldi. fraction which is common to most, if not all, of
Antisera to P. fieldi gave a fluorescent the primate malarias. Because of this
antibody cross-reaction at only a low level to P. phenomenon, the P. fieldi antigen has been used
cynomolgi (mean reciprocal titer ratio of 100:31) with human antigens successfully in several
and at much lower levels to other primate serologic surveys to determine the presence of
malaria antigens (Collins et al, 1966). In the antibodies to malaria in endemic populations
reverse procedure, however, P. fieldi antigen (Collins et al, 1967, 1968a).
cross-reacted at a consistently high level. Mean
TABLE 24.—Comparative infectivity of Plasmodium fieldi to Anopheles b. balabacensis, A. freeborni, A. kochi, A. vagus, A. maculatus, A.
sinensis, A. albimanus, A. argyropus, A. atroparvus, A. peditaeniatus, A. stephensi, and
A. quadrimaculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

Bal 100
Bal : F-1 71 1495 1498 40.5 50.7 129.9
Bal : Kochi 2 42 11 38.1 27.3 55.4
Bal : Vagus 2 28 4 92.9 75.0 39.8
Bal : Mac 16 273 247 49.8 15.2 16.5
Bal : Sin 2 26 13 92.3 53.8 6.3
Bal : Alb 4 113 89 54.0 4.5 4.8
Bal : Arg 1 17 13 88.2 100.0 4.1
Bal : Ped 3 30 20 90.0 25.0 3.6
Bal : Atro 15 337 347 54.6 8.4 3.1
Bal : St-1 10 234 199 53.4 7.6 1.8
Bal : Q-1 17 375 406 52.3 3.9 1.4

* Bal = Anopheles b. balabacensis, F-1 = A. freeborni, Kochi = A. kochi, Vagus = A. vagus, Mac = A. maculatus, Sin = A. sinensis, Alb = A.
albimanus, Arg = A. argyropus, Atro = A. atroparvus, Ped = A. peditaeniatus, St-1 = A. stephensi, and Q-1 = A. quadrimaculatus.
**GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. b. balabacensis to
another species where the GII of A. b. balabacensis = 100.

REFERENCES
BENNETT, G. F., WARREN, McW., and CHEONG, W. H., 1966. Response of sera from Nigerians to five Plasmodium
Biology of simian malarias of Southeast Asia. IV. antigens. Am. J. Trop. Med. & Hyg. 16 : 568-571.
Sporogony of four strains of Plasmodium cynomolgi. J. COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD,
Parasit. 52 : 639-646. J. R., 1968. Transmission of Plasmodium fieldi by
COLLINS, W. E., SKINNER, J. C., and GUINN, E. G., 1966. Anopheles maculatus, A. stephensi, and A. balabacensis
Antigenic variations in the plasmodia of certain balabacensis. J. Parasit. 54 : 376.
primates as detected by immuno-fluorescence. Am. J. COLLINS, W. E., WARREN, McW., SKINNER, J. C., and
Trop. Med. & Hyg. 15 : 483-485. FREDERICKS, H. J ., 1968a. Studies on the
COLLINS, W. E., JEFFERY, G. M., GUINN, E., and SKINNER, relationship between fluorescent antibody response and
J. C., 1966a. Fluorescent antibody studies in human ecology of malaria in Malaysia. Bull. Wid. Hlth. Org.
malaria. IV. Cross-reactions between human and simian 39 : 451-463.
malaria. Am. J. Trop. Med. & Hyg. 15 : 11-15. COOMBS, G. L., FREDERICKS, H. J., CHEONG, W. H.,
COLLINS, W. E., SKINNER, J. C., and COIFMAN, R. F., 1967. SANDOSHAM, A. A., and STAMARIA, F. L., 1968.
Fluorescent antibody studies in human malaria. V.
 PLASMODIUM FIELDI 195

REFERENCES—Continued
The exoerythrocytic schizonts of Plasmodium fieldi. MEUWlSSEN, J. H. E. TH., 1968. Antibody responses of patients
Med. J. Malaya 22 : 225-227. with natural malaria to human and simian Plasmodium
EYLES, D. E., LAING, A. B. G., and FONG, Y. L., 1962. antigens measured by the fluorescent antibody test.
Plasmodium fieldi sp. nov., a new species of malaria Trop. geogr. Med. 20 : 137-140.
parasite from the pig-tailed macaque in Malaya. Ann. SODEMAN, T. et al, 1970. (In manuscript).
Trop. Med. Parasit. 56 : 242-247. THAYER, W. S., 1899. Lectures on the malarial fevers. D.
EYLES, D. E., LAING, A. B. G., and DOBROVOLNY, C. G., Appleton & Co., New York. pp. 326.
1962a. The malaria parasites of the pig-tailed macaque, WARREN, McW. and WHARTON, R. H., 1963. The vectors of
Macaca nemestrina (Linnaeus), in Malaya. Ind.J. simian malaria: Identity, biology, and geographical
Malariol. 16 : 285-298. distribution. J. Parasit. 49 : 892-904.
HELD, J. R., CONTACOS, P. G., and COATNEY, G. R., 1967. WARREN, McW., ALl, K., BENNETT, G. F., and
Studies on the exoerythrocytic stages of simian malaria. SANDOSHAM, A. A., 1964. Morphology of
I. Plasmodium fieldi. J. Parasit. 53 : 225-232. Plasmodium fieldi in different species of the Macaca.
MEUWlSSEN, J. H. E. TH., 1966. Fluorescent antibodies in Med. J. Malaya 19 : 31.
human malaria, especially in Plasmodium ovale. Trop.
geogr. Med. 18 : 250-259.
This page intentionally left blank.
 17
Plasmodium simiovale Dissanaike, Nelson, and
Garnham, 1965

and a "new" parasite. When blood from the dual-

DURING the course of infected monkey was inoculated into a rhesus
an extensive survey of simian malaria in Ceylon, monkey, both parasites appeared. The "new"
Dissanaike obtained blood from two toque parasite was obtained as a pure line in rhesus
monkeys, Macaca sinica, shot near the Mi-Oya monkeys by passage through mosquitoes,
river, near the 7-mile post on the Puttalam- Anopheles atroparvus, at the London School of
Anuradhapura road in N. W. Ceylon. Hygiene and Tropical Medicine. Dissanaike,
Examination of blood films did not disclose Nelson and Garnham (1965) described it as a
parasites of malaria, but when heart blood from new species under the name Plasmodium
the animals was inoculated into two parasite-free simiovale to call attention to the parasite as "a
M. sinica, each became infected. One of them simian counterpart of the human P. ovale."
exhibited a double infection: Plasmodium shortti

197
 PLASMODIUM SIMIOVALE 199

Cycle in the Blood Spherical gametocytes appear early during

the initial parasitemia. The macrogametocytes
PLATE XXX far outnumber microgametocytes. The growing
female parasites are difficult to distinguish from
The first parasites to appear in the
the mature trophozoites because the latter often
peripheral blood are delicate ring forms with
fill the corpuscle and their pigment too is
prominent nuclei. As the parasite grows, the ring
scattered in the cytoplasm. The young adult
form may display an accessory chromatin dot,
distaff parasites are circular and occupy a fair
ofttimes within the vacuole, and occupies a fair
portion of the enlarged host cell which shows
portion of the host cell which has increased in
delicate diffuse stippling (Fig. 22). The fully
size (Fig. 3). With further growth, the
mature macrogametocyte is compact, stains a
erythrocyte may become distorted. The
deep blue and has scattered granular dark
stippling, at first small and delicate, becomes
pigment. The nucleus is marginal, elongated,
granular and prominent. In heavy infections,
and stains a deep red. The parasite may not fill
especially, multiple invasion of the host cell is
the depleted host cell, which is distorted;
common (Figs. 5, 12). The vacuole of the early
stippling is prominent and located near the
stages becomes much larger and may occupy a
periphery (Fig. 23). The microgametocyte stains
third or more of the parasite. The cytoplasm of
red to pink. In the oldest forms, the pigment is
the young non-amoeboid parasite becomes more
granular, and scattered in the cytoplasm. The
dense, sometimes enclosing small vacuoles.
nucleus which occupies about a third of the
Dark granular pigment is scattered in the
parasite is without pigment and embraces a
cytoplasm. The nucleus is large and stains a dark
darker staining bar- or skein-like area. The adult
red. By the end of the trophozoite stage, the
parasite fills the host cell (Figs. 24, 25).
large vacuole has disappeared and the parasites
The asexual cycle occupies 48 hours.
begin nuclear division. As schizogony proceeds,
small cleft-like or circular vacuoles appear in the
cytoplasm (Figs. 13, 14); the pigment remains Sporogonic Cycle
granular but definite. Some of the host cells are PLATE XXXI
greatly enlarged and display an eosinophilic
ring. The cytoplasm is discolored but the Mosquito infections were reported by
enclosed parasite appears normal (Fig. 16). Dissanaike et al (1965) in Anopheles atroparvus
Schizogony continues, finally producing 12 to and A. stephensi. In the former, oocysts
16 merozoites. The enlarged host cell is containing dark discrete grains of pigment,
generally distorted and the parasite tends to be arranged in irregular lines, measured 23 to 35 µ
oval or roughly circular. Vacuoles are in after 7 days of extrinsic incubation at 26° C. On
evidence; the pigment granules come together in the 9th day, oocysts measured 35 to 45 µ. At this
loose aggregates and finally form a solid time, the pigment grains were concentrated over
greenish-yellow mass. During early schizogony, a small area or were obscure. By the 11th day,
the stippling in the host cell remains prominent, oocysts measured 40 to 50 µ. Sporozoites
but toward the end, it collects toward the appeared in the salivary glands 13 days after
periphery; the cell is discolored and generally feeding. In A. stephensi, one oocyst was seen 5
distorted (Figs. 13-21). days after feeding. This oocyst measured 15 µ in

PLATE XXX.—Plasmodium simiovale.

Fig. 1. Normal red cell. Figs. 11, 13-18. Developing schizonts.
Figs. 2-4. Young trophozoites. Figs. 19-21. Mature schizonts.
Figs. 5-9. Growing trophozoites. Figs. 22-23. Young adult and mature macrogametocytes.
Figs. 10, 12. Nearly mature and mature trophozoites. Figs. 24-25. Mature microgametocytes.
200 PRIMATE MALARIAS

diameter and the pigment was in lines on the by day 12, their size ranged from 26 to 83 µ,
periphery. with a mean of 67 µ. Sporozoites were present in
In our studies, oocyst development was the salivary glands on day 13. In the A.
studied in A. b. balabacensis, A. maculatus, A. maculatus, although the oocyst diameters were
freeborni, and A. stephensi mosquitoes within the range of those seen in the A. b.
incubated at 25° C (Table 25). balabacensis, the mean diameters, after 10 or
In A. b. balabacensis, at 4 days, the oocysts more days of extrinsic incubation, were smaller.
ranged in diameter from 9 to 12 µ with a mean Sporozoites did not appear in the salivary glands
of 10 µ. The oocysts continued to grow so that until day 14. Although oocysts developed and

PLATE XXXI.—Developing oocysts of Plasmodium simiovale in Anopheles b. balabacensis mosquitoes. X 580 (Except Figs. 1 & 2).
Fig. 1. 4-day oocyst showing abundant scattered pigment. Fig. 6. 9-day oocyst.
X 740. Fig. 7. 10-day oocyst.
Fig. 2. 5-day oocyst showing scattered pigment. X 740. Fig. 8. 11-day oocyst.
Fig. 3. 6-day oocyst. Fig. 9. 12-day differentiating oocyst.
Fig. 4. 7-day oocyst. Fig. 10. Rupturing 12-day oocyst showing release of
Fig. 5. 8-day oocyst still showing pigment. sporozoites.
PLASMODIUM SIMIOVALE 201
202 PRIMATE MALARIAS

showed differentiation in the A. freeborni relationship between them. The P. fieldi parasite
mosquitoes, infection of the salivary glands was requires one day longer to complete its
much later and then only at a very low level. sporogonic cycle in A. b. balabacensis
Measurement of oocysts in the A. stephensi mosquitoes than does P. simiovale.
mosquitoes gave diameters in the range of those Garnham (1966) reported that sporozoites
seen in the A. b. balabacensis. An insufficient of P. simiovale in dried preparations are 12 to 14
number of the A. stephensi mosquitoes survived µ in length. Although few sporozoites survived
to determine if sporozoites would or would not in A. atroparvus, transmission was obtained via
invade the salivary glands. the bites of infected mosquitoes of this species.
A comparison of the oocyst growth curves The prepatent period in the recipient M. mulatta
of P. simiovale and P. cynomolgi in A. b. monkey was 24 days, which agrees with
balabacensis mosquitoes (Fig. 42) discloses that Dissanaike et al (1965).
P. simiovale takes 3 days longer to complete its In our studies, transmission was obtained
development than does P. cynomolgi. The mean on two occasions. Dissected salivary glands
oocyst diameter of P. simiovale on day 11 was from A. b. balabacensis were inoculated intra-
approximately the same as that of P. cynomolgi hepatically into an intact M. mulatta monkey.
on day 9. However, the former parasite develops The animal developed an infection with P.
to a maximum size slightly larger than does the simiovale with a prepatent period of 11 days.
P. cynomolgi parasite. A comparison of the Another monkey was bitten by infected A.
oocyst growth curves between P. simiovale and maculatus mosquitoes. The prepatent period in
P. fieldi (Fig. 43) indicates a very close this animal was 17 days. Attempts to transmit

FIGURE 42.—Range in oocyst diameters and the mean oocyst diameter curves of Plasmodium simiovale and P. cynomolgi in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
 PLASMODIUM SIMIOVALE 203

FIGURE 43.—Range in oocyst diameters and the mean oocyst diameter curves of Plasmodium simiovale and P. fieldi in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

the infection by the bites of infected A. b. conclusions can be made concerning differences,
balabacensis mosquitoes to two volunteers were if any, which may exist between the EE stages
unsuccessful. of this parasite, and the other primate malarias.

Cycle in the Tissue Course of Infection

PLATE XXXII
In the normal host, M. sinica, the infection
The exoerythrocytic stages of Plasmodium is mild (Dissanaike et al, 1965). In the M.
simiovale have been studied only recently. Thus mulatta monkey, the parasitemia never reaches a
far, we have seen only the 7-day forms (Figs. 1- high level, even after splenectomy (Garnham,
3). Although smaller, these parasites are similar 1966). An examination of the parasitemia curves
to the 8-day forms of P. fieldi described by Held in the two monkeys infected in our laboratory by
et al (1967). Like the P. fieldi, the P. simiovale sporozoite inoculation confirms this observation
parasites contained numerous small flocculi and (Fig. 44). The maximum parasite counts during
peripheral indentations (Fig. 2) described by the 60-day observation period were 21,300 and
Held et al (loc. cit.) as scalloping. The space 31,740 per mm3. After the initial peak, the
occupied by 25 P. simiovale parasites ranged parasitemias quickly dropped to very low levels
from 14 to 25 µ in width and from 16.5 to 29.5 µ which were maintained for the remainder of the
in length. The average dimensions were 18.9 by 60-day period of observation.
23.8 µ. Further studies will be needed before any
204 PRIMATE MALARIAS

PLATE XXXII.—Exoerythrocytic bodies of Plasmodium simiovale in liver tissue of Macaca mulatta monkeys. X 580.
Fig. 1. 7-day body showing very small flocculi. Fig. 3. 7-day body showing host cell nucleus.
Fig. 2. 7-day body.

FIGURE 44.—Parasitemia curves of Plasmodium simiovale in two Macaca mulatta monkeys infected via the bites of infected
mosquitoes.

Host Specificity quadrimaculatus mosquitoes (Table 26). Only

A. b. balabacensis and A. maculatus readily
support development to the presence of
The natural host of Plasmodium simiovale
sporozoites in the salivary glands. Because the
is M. sinica (Dissanaike et al, 1965).
morphology of the blood forms of P. fieldi and
Experimentally, M. mulatta is also susceptible to
P. simiovale are similar, it was expected that
infection either by blood- or sporozoite-
these species would also show a similarity in
inoculation.
their infectivity to different species of
The natural vector of this parasite is
mosquitoes. When such a comparison was made,
unknown. Dissanaike et al (1965) reported the
however, it was found that the average number
experimental infection of A. stephensi and A.
of oocysts per gut in A. b. balabacensis was
atroparvus); the latter species being apparently
higher than in A. freeborni infected with P.
more susceptible to the infection. We have
simiovale. The reverse was true with P. fieldi
infected A. b. balabacensis, A. freeborni, A.
(see Chapter 16). Because the A. freeborni
maculatus, A. stephensi, A. atroparvus, and A.
 PLASMODIUM SIMIOVALE 205

mosquitoes are not co-indigenous with either of Immunity and Antigenic

these malarias, it might serve as the standard for
comparison. The A. b. balabacensis are co- Relationships
indigenous with P. fieldi and would be expected
to demonstrate a high level of susceptibility to it. Apparently, a prior infection with P. fieldi
However, the higher ratio of susceptibility was in a rhesus monkey confers no immunity against
to the simiovale parasite indicating a true an infection with P. simiovale. In our hands, an
difference between these 2 parasites. If A. b. animal was infected with P. fieldi and allowed to
balabacensis is used as the standard, as in Table run a course of patent parasitemia for 61 days.
26, there is little difference between the relative The maximum parasite count was 18,300 per
susceptibility of these parasites to A. maculatus mm3. The infection was then eliminated by
(100:11.3) for P. simiovale and (100:16.5) for P. treatment with chloroquine. Seventy-eight days
fieldi. However, if the non-co-indigenous later, the monkey was inoculated with blood
mosquito, A. freeborni, is used as the standard, containing parasites of P. simiovale. The
then the difference in mosquito susceptibility is infection became patent and followed a course
apparent. Thus it can be said that these parasites normal for a blood-induced infection with a
do exhibit differences in their infectivity to maximum parasitemia of 12,000 per mm3 11
mosquitoes which emphasizes their specificity. days after inoculation.

TABLE 26.—Comparative infectivity of Plasmodium simiovale to Anopheles b. balabacensis, A. freeborni, A. maculatus,

A. stephensi, A. atroparvus, and A. quadrimaculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

Bal 100
Bal : F-1 12 127 377 67.7 48.8 55.4
Bal : Mac 14 193 465 54.9 34.8 11.3
Bal : St-1 8 168 161 44.6 21.7 10.9
Bal : Atro 8 50 236 76.0 16.5 4.1
Bal : Q-1 15 235 417 50.2 6.2 1.4

* Bal = Anopheles b. balabacensis, F-1 = A. freeborni, Mac = A. maculatus, St-1 = A. stephensi, Atro = A. atroparvus, Q-1 = A.
quadrimaculatus.
**GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. b. balabacensis to
another species where the GII of A. b. balabacensis = 100.
REFERENCES
DISSANAIKE, A. S., NELSON, P., and GARNHAM, P. C. C., HELD, J. R., CONTACOS, P. G., and COATNEY, G. R., 1967.
1965. Plasmodium simiovale sp. nov., a new simian Studies of the exoerythrocytic stages of simian malaria.
malaria parasite from Ceylon. Ceylon J. Med. Sci. 14 : I. Plasmodium fieldi. J. Parasit. 53 : 225-232.
27-32.
GARNHAM, P. C. C., 1966. Malaria parasites and other
haemosporidia. Blackwell Scientific Publications,
Oxford.
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 SECTION 4
Malariae-Type Parasites
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 18
Plasmodium malariae (Grassi and Feletti, 1890)
malariae should be declared valid as the de facto
SYNONYMS: name and credited to Grassi and Feletti, 1890.
Haemamoeba malariae Feletti and Grassi, 1889; Part of this was accomplished, Opinion 283,
Plasmodium malariae M. and C. var. quartanae under the Plenary Powers of the Commission on
Celli and Sanfelice, 1891; Plasmodium malariae Zoological Nomenclature (Hemming, 1954), but
quartanae Kruse, 1892; Haemamoeba laverani credit for the name was given to Grassi and
var. quartanae Labbe, 1894; Haemosporidium Feletti, 1889. It now appears that the 1889
tertianae Lewkowicz, 1897; ? Plasmodium pamphlet, which is said to have given the
rodhaini Brumpt, 1939. authorship in reverse, is no longer extant and
There is little doubt that Laveran saw this therefore the order of authorship cannot be
parasite in fresh blood of patients in Algeria in verified. In 1890, Grassi and Feletti gave
1880 because his illustrations show schizonts malariae as the specific name for the quartan
with 8 merozoites arranged in the typical rosette parasite in the genus Haemamoeba, and on the
and a central body of pigment. In his opinion, basis of priority, that date is valid. Following
the human malaria parasites belonged to one this, Grassi and Feletti (1892) described and
species and, possibly for that reason, he did not illustrated the quartan parasite, as seen in the
propose a name. He recognized the phenomenon peripheral blood, and separated it from the other
of periodicity but rejected the idea that it might blood-inhabiting forms in man and birds under
be a clue toward the taxonomy of the malarias. the name H. malariae Grassi and Feletti.
Golgi in a short note to the Royal Academy Garnham (1966) holds that "in view of the
of Medicine in Turin (1885) first recognized the authority of this paper, and the disappearance of
periodic succession of fever attacks and in 1889 all traces of the original 1889 pamphlet", the
he clearly showed how to separate tertian and name should be credited to Grassi and Feletti
quartan fevers by linking the development of a 1892. The tenet that authority is a valid basis for
particular brood of parasites to the fever episode. assigning credit is unacceptable, and in view of
He was definite in pointing out that if only the loss of the 1889 paper and the fact that the
young forms are present in the circulating blood Regles had to be suspended in order to validate
there will be 1 or 2 days, depending on the the de facto name, it appears to us, therefore,
species, free of fever. Golgi was also able to that the correct name for the human quartan
demonstrate quotidian fevers in both tertian and parasite is Plasmodium malariae (Grassi and
quartan infections and postulated, correctly, that Feletti, 1890).
the phenomenon was due to double and triple Plasmodium malariae is a cosmopolitan
broods of parasites. This important contribution parasite which develops where the summer
to the biology of these parasites was more or less isotherm does not fall below 15° C (59° F). Its
ignored at the time and then virtually forgotten distribution is variable and spotty. Why this is
until well after the turn of the century. true no one has been able to explain although
In attempting to arrive at the correct name numerous theories have been advanced; it is
and credit for the quartan parasite of man, one still, one of the unsolved problems in the
runs into considerable difficulties. Coatney and biology of human quartan malaria.
Young (1941) in reviewing the problem arrived One theory to account for its unique
at the conclusion that the name Plasmodium distribution was that because the parasite

209
210 PRIMATE MALARIAS

requires an extended sporogonic cycle, its It may well be that one or more of these
greatest prevalence would be in areas where the factors, along with changes in the environment
vector was able to survive for the longest time. which might facilitate transmission, play a part
This might obtain in some areas, but it in the unique distribution of quartan malaria
sometimes reaches its highest incidence in parts throughout the world. But whatever the
of the tropics where the vector has only a short explanation may be, it appears that P. malariae
life. is probably the oldest parasite in terms of time,
Another theory was that quartan malaria and although Knowles et al (1930) considered it
demands a special vector. It is true, that a disappearing parasite, it might well be that
experimental infections in mosquitoes are instead of dying out, it has 'learned' during its
difficult to produce and when obtained, the long association with man how to cope with
oocysts fail to develop at a uniform rate which adversity and, when conditions permit, to enjoy
probably accounts for the varied results obtained prosperity.
by such workers as Mayne (1932), Mer (1933), This is illustrated in the work of Field and
and others. However, the special vector theory Shute (1956) where they site that in West
hardly seems to cover the problem either Malaysia "quartan infections are not commonly
because many species transmit the parasite: A. found in hospital patients--less than five per cent
atroparvus, A. sacharovi, A. stephensi et al. of the admissions . . .--but there are localized
The third theory, rested on the presence of areas where P. malariae is the dominant
an animal reservoir in certain tropical regions. species." In areas of high transmission of all
This might be applicable if one is concerned species, P. falciparum is dominant for a few
with certain areas in tropical Africa where months, whereupon, it is succeeded by P. vivax,
chimpanzees harbor P. rodhaini. In man, that which holds center stage for a time only to be
parasite takes on the attributes of P. malariae replaced by P. malariae which has 'learned' to
and, in some quarters, is considered to be P. wait in the wings. Field and Shute call it a
malariae. The inui parasites of India and the "residual infestation" which pretty well sums up
Malaysian area are morphologically distinct the life-cycle of P. malariae. It is a highly
from human quartan and so far only one strain successful parasite in that it can live in a host
has been established in man. The rest of the Old longer than any other malaria and without
World is without any zoonotic connection to renewed activity from fixed tissue parasites, it
explain the persistence of quartan malaria. In the causes the host relatively little inconvenience
New World, the situation is altogether different because of its symbiotic nature and, it is always
than it is in Africa or the Far East, because it ready, during periods of recrudescence, to gain
appears relatively clear that the quartan malaria access to a cooperative vector and, hence,
of monkeys (P. brasilianum) came from man, perpetuate the species.
hence an anthroponosis, rather than the other
way around.
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212 PRIMATE MALARIAS
 PLASMODIUM MALARIAE 213

Cycle in the Blood clumped in a loose mass in the center of the cell,
surrounded by more or less symmetrically
PLATE XXXIII arranged merozoites, to give the "rosette" effect
The first stage to appear in the peripheral
so characteristic of the species. The number of
blood is the familiar ring-stage, not shown as a
merozoites may be from 6 to 12, sometimes 14;
true ring on the plate, a circle of cytoplasm, and
the average is 8 (Figs. 17-22).
a spherical bit of chromatin enclosing a vacuole.
The young gametocytes are very similar to
It very shortly occupies one-fourth to one-third
the asexual forms which makes it virtually
of the parasitized cell and sometimes exhibits an
impossible to distinguish them with certainty
accessory chromatin dot (Figs. 4, 5). Although
until about the 54th hour when the asexual forms
this parasite generally displays denser cytoplasm
begin segmentation (Fig. 23). The mature
and chromatin than P. vivax, it is difficult to
macrogametocyte exhibits a heavy deeply-
separate them at this stage because they are
staining blue cytoplasm with a small, eccentric,
about the same size. The parasite grows more
well-defined deep red-staining nucleus. The
slowly than any of the other human malarias.
pigment is scattered. The parasite completely
The vacuole disappears after a few hours. In the
fills the host cell (Fig. 24). The cytoplasm of the
living condition, the movement is sluggish.
adult microgametocyte takes a light bluish-pink
Pigment granules appear early in its growth and
stain. The pigment is limited to this area of the
sometimes a granule may appear in the late ring-
parasite. The nucleus is diffuse, takes a pinkish-
stage. The pigment increases rapidly; half grown
blue stain, and may occupy about half the cell.
parasites may exhibit 30 to 50 jet-black granules
The parasite fills the entire host cell (Fig. 25).
(Figs. 12, 13) in contrast to the rod-like pigment
Ordinarily, microgametocytes outnumber the
found in P. vivax.
macrogametocytes but this may vary with
As the parasite grows, it assumes various
different strains.
shapes. Some appear stretched out like a ribbon
The asexual cycle requires 72 hours.
across the host cell and are known as band forms
(Figs. 6, 10, 11). Band forms are found in other
species, but are more frequent in P. malariae Sporogonic Cycle
and hence are considered diagnostic. These PLATE XXXIV
forms may be seen at any time until the parasite Observations have been made by a number
virtually fills the host cell which is not enlarged of workers concerning the sporogonic cycle of
or blanched. At about the 54th hour, Plasmodium malariae, but Shute and Maryon
segmentation begins, and by the 65th hour, the (1952) carried out the first definitive studies.
host cell is completely filled, or nearly so, and These investigators observed its development in
the parasite contains 5 to 6 chromatin masses; Anopheles atroparvus mosquitoes incubated at a
the pigment is scattered (Figs. 14-16). During temperature of 25°C. They found that the
further growth, the definitive number of nuclei pigment, which seldom consisted of more than
are formed and with the final number, the 30 granules, was very dark brown and variable
cytoplasm divides to give each nucleus a small in size. During the first 7 to 8 days, the granules
amount of cytoplasm. During this stage, the were distributed over the oocyst, but, from the
pigment may appear segregated and then

PLATE XXXIII.—Plasmodium malariae.

Fig. 1. Normal red cell. Figs. 21, 22. Mature schizonts.
Figs. 2-5. Young trophozoites. Fig. 23. Developing gametocyte.
Figs. 6-11. Growing trophozoites. Fig. 24. Mature macrogametocyte.
Figs. 12, 13. Nearly mature and mature trophozoites. Fig. 25. Mature microgametocyte.
Figs. 14-20. Developing schizonts.

213
214 PRIMATE MALARIAS

Plate XXXIV.—Developing oocysts and sporozoites of Plasmodium malariae in Anopheles freeborni mosquitoes. X 580.
Fig. 1. 10-day oocyst showing small clump of pigment. Fig. 7. 14-day oocyst showing differentiation.
Fig. 2. 11-day oocyst showing pigment clumped within Fig. 8. 15-day oocyst.
small vacuole. Fig. 9. 17-day fully differentiated oocyst.
Fig. 3. 12-day oocyst. Fig. 10. Sporozoites present near salivary gland tissue
Figs. 4, 5. 13-day oocysts. 19 days after feeding.
Fig. 6. 14-day oocyst showing early differentiation.

9th day onwards, the pigment was clumped in a for completion of the cycle was 15 days, the
mass at the periphery. From the 3rd to the 9th maximum, 21 days.
day, daily increase in size of the oocyst was less In our studies (Collins and Contacos, 1969),
than 2 µ; from the 9th day onward, the daily A. freeborni was the most suitable mosquito for
increase varied from 5 to 8 µ. Oocysts ranged in studying the sporogonic cycle (Table 27). On
size from 5 to 44 µ. The minimum time required day 6, at an incubation temperature of 25° C, the
 PLASMODIUM MALARIAE 215

mean oocyst diameter was 12 µ with a range of TABLE 27.—Oocyst diameters of Plasmodium malariae in
Anopheles freeborni mosquitoes.
9 to 14 µ. The oocysts continued to grow so that
by day 14, the mean size was 38 µ with a range Days post A. freeborni
infection
of 20 to 65 µ. The first signs of oocyst No. Range Mean*
differentiation were apparent by day 14;
sporozoites were present in the salivary glands 6 104 9-14 12
7 105 11-19 14
on day 17. 8 185 12-21 17
A comparison of the oocyst growth rate of 9 169 14-27 20
10 146 13-32 21
P. malariae with that of P. cynomolgi (Fig. 45) 11 268 13-45 27
points-up the marked difference between the 2 12 218 14-51 29
parasites. The mean oocyst diameter of P. 13 195 19-59 37
14 260 20-65 38†
cynomolgi on day 8 is about the same as that of 15 260 17-77 50†
P. malariae on day 15. Sporozoites appear in the 16 348 19-88 48†
salivary glands 6 days sooner with P. cynomolgi 17 168 15-86 53†**

than with P. malariae. The comparison of the Totals 2426 9-88

sporogonic cycle of this parasite with P.
brasilianum is presented elsewhere (Chapter * Measurements expressed in microns.
† Oocyst differentiation.
19). ** Sporozoites present in the salivary glands.

Cycle in the Tissue The 8-day tissue schizont was described as

lying in the liver parenchyma cell usually, but
The first tissue stages of Plasmodium not always, in a vacuole. The host cell was
malariae were seen and described by Bray enlarged and the cytoplasm pushed aside in a
(1959, 1960). Liver biopsy specimens (8, 9, 10, crescent-shape around the parasite. The host cell
11, 12, and 12.5 days) were taken from 3 nucleus was enlarged and pushed to one side. In
different chimpanzees which had received 72 to over 50 percent of the parasitized parenchymal
110 salivary glands of Anopheles Gambia; 33 to cells, 2 or more enlarged nuclei were present.
50 percent of them were infected with The tissue schizont usually had a distinct
sporozoites of a Liberian strain of P. malariae.

FIGURE 45.—Range in oocyst diameters and the mean oocyst diameter curves of Plasmodium cynomolgi and P. malariae in
Anopheles freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

215
216 PRIMATE MALARIAS

envelope with no clefts. Its cytoplasm was all ages; namely, the nuclei were always
generally homogeneous but was sometimes randomly distributed, there were no
tenuous or in strands. Occasionally, the pseudocytomeres, no evidence of septal
periphery showed tiny vacuoles. The nuclei were formation, or plasmotomy. He saw no mature
usually small dots measuring about 0.5 µ or schizonts or any evidence of sporulation. Bray's
round compact masses of chromatin measuring studies showed quite distinctly that the tissue
about 1 µ in diameter. The average size of the 8- stages of P. malariae in the chimpanzee differ
day tissue schizont was 12.5 µ (range of 9.5 to markedly from the other species of human
17 µ). The average number of nuclei was 25 malaria. It differs from vivax and falciparum
(range of 7 to 49). malaria in that it causes enlargement of the host
The 9-day schizonts were generally like the cell nucleus, the older forms show considerable
8-day forms except for the scatter in size, the vacuolation of the peripheral cytoplasm, it
range was 8.5 to 21 µ (average 12.7 µ). The produces fewer merozoites, and the growth rate
average number of nuclei was 27 (range of 10 to is considerably slower. It differs from P. ovale
83). in that the nuclei of malariae tissue stages are
The 10-day schizonts were usually within a smaller, fewer in number, and its growth rate is
vacuole in the parenchyma cell. The parasite had slightly slower. The first evidence of parasitemia
pushed the enlarged nucleus, or nuclei, of the in the chimpanzees was seen 16 days after the
enlarged host cell, to one side. The cytoplasm inoculation of sporozoites.
was homogeneous and reasonably dense. In Lupascu et al (1967) determined the "exact
about half of the parasites, the cytoplasm was duration" of the "prepatent period" of the VS
pulled slightly away from the distinct envelope; (Roumanian) strain of P. malariae by the
in about 40 percent, it had not contracted away subinoculation of blood into new patients. Only
from the envelope; in the remaining 10 percent, the recipients given blood 15 days or more after
the cytoplasm was irregularly vacuolated. Some inoculation of sporozoites became infected. In
showed, what was described as, 3- or 4-point other words, with this strain of P. malariae, the
type nuclei. The size of the 10-day stage exoerythrocytic cycle requires exactly 15 days
averaged 22.1 µ (range of 15 to 29 µ); the and, therefore, the prepatent period could be as
average number of nuclei was 100 (range of 33 early as day 15.
to 201). Lupascu et al also described the later tissue
The 11-day schizonts were smaller than the stages of P. malariae. They inoculated a 3 year-
8- or 9-day stages, the average being 11.7 µ old chimpanzee with a total of 110 salivary
(range of 9.5 to 14 µ); the average number of glands from Anopheles labranchiae atroparvus
nuclei was approximately 15 (range of 6 to 30). mosquitoes, 86 to 100 percent infected with the
The 12-day tissue parasite had a distinct VS strain. Liver biopsy material was obtained at
envelope with dense cytoplasm in the central 12, 13, 14, and 15 days. All 4 biopsy specimens
portions of the parasite. The periphery consisted contained tissue schizonts. The 12-day stages
of vacuoles measuring 3 to 6 µ in diameter. The were usually oval, and, less often, spherical in
cytoplasm contained a number of small shape. The contour was usually smooth but
basophilic aggregates. The schizont was occasionally disturbed by the presence of
sometimes slightly lobulated. The average size peripheral vacuoles. The parasitized
was 41 µ (range of 34 to 47 µ). The average perenchymal cells were greatly enlarged. The
number of nuclei was 712 (range of 435 to host cell nucleus was pushed to one side and
1151). The 12.5-day schizonts resembled the 12- much enlarged, sometimes twice its normal size,
day forms except for 2 characteristics; namely, and frequently pyknotic. The mean diameter of
the parasite was markedly lobulated and the the 12-day schizont was 30 µ. The nuclei were
peripheral vacuolations were more marked-- round and interspersed throughout the
large peripheral vacuoles measuring 5 to 8 µ cytoplasm, being somewhat scantier towards the
were commonly observed. margin of the parasite. The diameter of the
Bray mentioned several general nuclei was approximately 1.5 µ. The cytoplasm
observations concerning the tissue schizonts at tended to accumulate either as individual
 PLASMODIUM MALARIAE 217

flocculi or as larger masses. Numerous vacuoles number of merozoites was estimated to be

of all sizes were present. The largest vacuoles 18,650 and the smallest 7,500. The merozoite
were 10 µ. The parasite had a definite was a sphere measuring 2 µ in diameter,
membrane. consisting of cytoplasm which indents the
The 13-day forms had an average size of 44 nucleus to render it characteristically crescentic
µ, indicating considerable growth in 24 hours. in shape.
This rapid growth probably accounted for the Lupascu et al (1967) in their summary gave
pronounced lobulation. The internal structure of the main characteristics of the tissue schizonts of
the parasite was much the same as that of the 12- the VS strain of P. malariae as: enlargement of
day schizont except that the nuclei were "less the host cell nucleus, many peripheral and
loosely packed." Cytoplasmic clumps and internal vacuoles, no cytomeres, large clefts,
vacuoles persisted and clefts were usually seen. red-staining strands, and plaques in the mature
The 14-day schizonts reached a maximum schizonts.
mean diameter of 47 µ. They were more difficult
to measure because of the lobulated surface. The Course of Infection
most striking feature was the extensive cleavage
of the cytoplasm in some of the schizonts. The
As mentioned earlier, the shortest prepatent
cytoplasm was condensed into long strands,
period for P. malariae could be as early as 15
studded with nuclei, and sometimes smaller
days, on the basis of the findings of Lupascu's
clefts were present throughout the parasite. The
subinoculation experiments. However, in
nuclei were "tightly packed" and measured
actuality, the earliest reported prepatent period
about 2 µ in diameter; vacuoles were obvious.
has been 16 days in a West African strain (Shute
The limiting membrane of the parasite was
and Maryon, 1951).
distinct and occasionally thrown into
Generally, the prepatent periods, with this
pronounced folds. Only the 14-day schizonts
species, have been longer. Boyd and Stratman-
exhibited merozoites.
Thomas (1933) transmitted 2 strains in which
The 15-day schizonts were considered
the prepatent periods were 27, 32, and 37 days.
mature and remnants of ruptured schizonts were
Mer (1933) transmitted a Palestinian strain to 3
readily observed. The mature forms had a mean
patients; prepatent periods were 26, 28, and 31
diameter of about 51 µ. Their shape was mostly
days. Prepatent periods of 23 to 26, days were
oval, although irregular processes (lobulations)
reported by de Buck (1935) for 4 patients
were observed. The nuclei, of the near-mature
infected with a Vienna strain. Boyd and
schizonts, lost their spherical shape just prior to
Stratman-Thomas (1936) reported a prepatent
the final division, became triangular, and
period of 37 days (thin smear examination); the
measured as much as 3.3 µ in their greatest
incubation period was 40 days. This patient had
dimension. The cytoplasm formed patterns
only 5 paroxysms with 4 chills. The paroxysms
although the nuclei did not enter this material
subsided spontaneously although parasites were
and no cytomeres or any form of aposchizogony
found as late as 173 days after exposure to
was observed. Large vacuoles were present but
infection. They also reported a prepatent period
were less common than in the nearly-mature
of 28 days and an incubation period of 30 days
stage. The authors described curious reddish
in another patient. Marotta and Sandicchi (1939)
strands in the cytoplasm of some of the
reported incubation periods of 23 and 29 days in
schizonts and considered this feature unique
2 patients.
among tissue forms of malaria parasites. The
Boyd (1940) reported on 3 different strains:
mature schizont, except for the strands and a few
USPHS, Jones, and Weaver. The prepatent
vacuoles, consisted, almost entirely, of
periods ranged from 28 to 37 days (median of 34
merozoites within the outer limiting membrane
days) and the incubation periods from 30 to 49
of the parasite. The largest mature schizont
days (median of 36 days). Siddons (1944)
measured 41 by 85 µ, the average was 56 µ. The
transmitted an Indian strain of P. malariae to a
number of merozoites was directly dependent
upon the size of the mature schizont. The largest

217
218 PRIMATE MALARIAS

patient who exhibited a prepatent period of 30 pattern (Kitchen, 1949) because the sporulation
days and an incubation period of 36 days. Young of this species is highly synchronous.
and Burgess (1947) observed prepatent periods Young et al (1940) studied the periodic
of 29 and 59 days in 2 patients whose incubation phenomenon of the asexual cycle of quartan
periods were 28 and 69 days. These authors malaria in Negro paretics. They found that their
considered the prepatent period of 59 days to be strain of malariae malaria (USPHS) exhibited a
due, possibly, to some immunity on the part of high degree of synchronicity, the asexual
the patient which was substantiated by a short erythrocytic cycle repeating itself every 72
duration of parasitemia and extremely low hours, but being more exact in some patients
parasite counts. Mackerras and Ercole (1948) than in others. The length of time consumed by
reported a prepatent period of 24 days for a the different growth stages was: 54.2 hours for
Melanesian strain. Kitchen (1949) reported the trophozoites, 10.4 hours for the young
mean prepatent periods of 32.2 days (range 27 to schizonts, and 7.4 hours for the segmenters or
37 days) and mean incubation periods of 34.8 late schizonts. The process of segmentation
days (range 29 to 40 days) for naturally induced required roughly 6 hours. The rise in
infections of American strains of P. malariae. temperature closely followed the progress of
Young and Burgess (1961) transmitted the segmentation and reached its height
USPHS strain of P. malariae to 2 patients and approximately at the end of the process.
observed prepatent periods of 33 and 36 days Young et al (1940a) showed that modifying
and incubation periods of 28 and 43 days. Ciuca the external conditions of the host affected the
et al (1964) reported prepatent periods ranging time of sporulation of P. malariae in man when
from 18 to 25 days for a local Roumanian strain, patients were placed under reversed conditions
now known as the VS strain. Lupascu et al of activity; the segmenter-number peaks in one
(1968) reported incubation periods of 18 to 19 changed from the normal 9:00 a.m. hour to 9:00
days with the VS strain of P. malariae. As these p.m. In the other, the cycle was shortened until
data show, there is a wide range in the length of the segmenters peaked about 22 hours before
the prepatent periods in naturally transmitted P. normally expected. Two other patients were
malariae (18 to 59 days) and an equally wide placed under reversed conditions, except lighted
range in the incubation periods (28 to 69 days). continuously. In these, the segmenter-peak time
In our transmission studies with a Nigerian changed from 9:00 a.m. to 9 :00 p.m. When one
strain of P. malariae, involving 4 volunteers, we of them was returned to a normal schedule, the
have observed prepatent periods ranging from segmenter-number peaks returned to the normal
24 to 33 days (Contacos and Collins, 1969); time, 9:00 a.m.
with the VS strain, in 4 patients, the pre-patent Young et al (1941) presented data on 420
periods ranged from 21 to 30 days. paroxysms occurring in 15 patients infected with
Malariae malaria infections are considered the USPHS strain of P. malariae. Chills were
to be relatively benign when compared to observed 102 times (24 percent). Some patients
falciparum malaria but more severe than vivax experienced chills more often than others; one
infections. Probably its most peculiar had 9 chills in 14 paroxysms (64 percent), while
characteristic is its pronounced chronicity, another had only 1 chill in 45 paroxysms (2
especially the unusually long period of time percent). Temperatures at the beginning of chills
during which parasites can remain 'dormant' were found to range from 97.6° F to 106.0° F
within the host. A less constant characteristic is rectally, with an average of 101.8° F.
the tendency to cause renal damage which was Temperatures at the end of the chills ranged
dealt with in some detail by Giglioli (1930). from 97.8° F to 106.0° F, with an average of
The onset of P. malariae attacks is 103.5° F. The greatest increase in temperature
generally more gradual than observed with many during a chill was observed in a patient whose
vivax and falciparum infections. The initial temperature rose from 98.0° F to 103.0° F
remittent fever pattern characteristic of vivax during a chill lasting 45 minutes. Two patients
infections is seen less frequently in malariae experienced the opposite; in other words, a drop
infections. Rather, they exhibit an intermittent in temperature during a chill. One had a
 PLASMODIUM MALARIAE 219

temperature of 106.0° F at the beginning and malariae paroxysms are longer in duration than
105.0° F at the end of the chill which lasted 50 those of P. vivax.
minutes. Another patient dropped from 104.2° F In the Young et al series, the average fever
at the beginning of the chill to 103.2° F at the peak for the 420 paroxysms was 104.1°F
end of the chill which lasted 15 minutes. It is rectally, with the highest temperature recorded,
interesting that in both these cases, very high 106.4° F. The duration of the fevers ranged from
temperatures had already been reached by the 5 to 32 hours with an average of 10 hours, 58
time the chill began. The durations of all chills minutes. The average time for the interval from
ranged from 13 to 195 minutes with an average the beginning of fever to the fever peak was 4
of 53 minutes (Fig. 46). hours, 45 minutes and from the fever peak to the
According to Kitchen (1949), the malariae end of the fever was 6 hours, 13 minutes. These
paroxysm is less often introduced by chills than authors also observed that some paroxysms were
are vivax paroxysms. Boyd (1940) found that introduced by a chill, while others were devoid
the initiation of the malariae paroxysm by chills of chills. When a chill accompanied the
was extremely variable. Indeed, one of 5 paroxysm, the fever was significantly higher
naturally induced cases studied by him had no (104.6° F as compared to 104.0° F without a
chills at all. In Kitchen's observations, on chill) and the duration of the fever was 1 hour,
patients infected with the Trinidad strain, the 16 minutes shorter (10 hours duration for the
temperature at the onset of the chill varied paroxysms with chills as compared to 11 hours,
between 97.4°F and 104.4°F (mean of 100.3° F). 16 minutes for the paroxysms without chills). In
The mean duration of the chills was 55.97 addition, it was obvious that this shortening of
minutes. The mean temperature peak was the fever period occurred almost entirely in the
104.98° F reached, on an average, 2 hours, 54 period between the onset of the paroxysm and
minutes after the onset. The interval from peak the peak of the fever. They believed that the
temperature until the temperature returned to chill exerted a definite influence on the character
normal was 10 hours, 23 minutes. of the paroxysm. When a chill occurred, the
The entire paroxysm, therefore, occupied a duration of the fever was shorter (1 hour, 16
period of 13 hours and 17 minutes. Plasmodium minutes) which was due, apparently, to the

FIGURE 46.—The temperature curve in relation to the chill in 102 paroxysms in Plasmodium malariae infections (after Young,
Coatney, and McLendon, 1941).

219
220 PRIMATE MALARIAS

shortening of the period when the temperature asymptomatic cases. Ciuca et al (1964) reported
was rising, and, that the maximal temperature maximum parasitemias of 6,700 and 8,852 per
was significantly higher than in paroxysms mm3 for sporozoite-and parasitized blood-
without chills. The greatest proportionate induced infections, respectively. In our studies
increase in temperature occurred during the with a Nigerian strain of P. malariae, a
chilling period (Fig. 46). maximum parasite count of 11,200 per mm3 has
Generally, the degree of parasitemia is been observed in a blood-induced infection
lower for P. malariae than for either vivax or (Collins and Contacos, 1969).
falciparum malaria. Boyd (1940) reported mean Figure 47 shows the median parasitemia
parasite densities ranging up to 12,500 per mm3. curves for Caucasian and Negro patients
However, there was one patient in his series, infected with the USPHS strain of Plasmodium
whose infection resulted in a fatal outcome, in malariae, as well as curves for the minimum and
whom the count exceeded 100,000 parasites per maximum parasitemia. It can be readily seen
mm3. Young and Burgess (1947) observed that the median parasitemia curves for the
parasites at least through 58 days in a patient Caucasian and Negro patients show only minor
having a maximum parasite count of 3,280 per differences. Median peak parasitemia occurs
mm3 of blood. Mackerras and Ercole (1948) did some time between day 15 and day 20. The
not observe parasitemias which exceeded 2,300 parasitemia subsequently persists at an almost
per mm3. constant level, tapering off very gradually during
Kitchen (1949) stated that the parasitemias the next 40 days, in the Negro patients and only
observed in his P. malariae infections were a very little, if at all, in the Caucasians. The
comparatively low. Counts above 20,000 per median maximum parasitemias were
mm3 were unusual and counts above 50,000 per 3
approximately 5,000 per mm at day 25 and
mm3 were rare. He indicated that most patients 6,000 at day 28 for the Negro and Caucasian
do not attain counts above 10,000 per mm3 patients, respectively. The maximum
during their entire attack. After the first few parasitemia attained by a Caucasian patient was
weeks, the counts tend to drop and remain below 22,000 parasites per mm3 of blood at day 17,
5,000 per mm3. Young and Burgess (1961) and, for Negro patients, 25,000 per mm3 at day
reported parasitemias up to 17,580 parasites per 18, and 26,500 per mm3 at day 40. There were
mm3, with an average of 3,799 per mm3 in slight differences between the parasitemias in
symptomatic cases and up to 4,920 per mm3, the Caucasian and Negro patients from day 30
with an average of 999 per mm3 in

FIGURE 47.—Minimum and maximum parasitemia curves in 54 human volunteers infected with Plasmodium malariae and
median parasitemia curves for 29 Caucasian and 25 Negro volunteers, all infected by the inoculation of parasitized blood.
 PLASMODIUM MALARIAE 221

on. By day 30, the median parasite count for the Finally, a comment about true relapse
Caucasian patients was approximately 5,000 per activity in P. malariae. Ciuca et al (1964) did
mms whereas for the Negro patients, it was not observe relapse activity in any of their
approximately 3,000 per mm3. Between day 30 patients whose infections were induced by bites
and 50, the median parasite counts for the of infected mosquitoes. They concluded,
Caucasian patients ranged between 3,000 and therefore, that there was no secondary
6,000 per mm3 whereas for the Negro patients, it exoerythrocytic cycle. In this connection, we
was between 2,000 and 3,500 per mm3. After also have observed no relapse activity after
day 50, the median parasite counts for the adequate blood schizonticidal therapy in our
Caucasian patients ranged between 3,000 and volunteers exposed to infection with a Nigerian
4,000 per mm3 whereas in the Negro patients, strain of P. malariae. It seems safe to say that, in
the counts ranged between 1,000 and 3,000 per this respect, P. malariae resembles P.
mm3. In other words, there appeared to be a falciparum more than it does the human tertian
slightly earlier trend toward chronicity in the malarias which do relapse.
Negro patients than in the Caucasian patients Plasmodium malariae can infect the
infected with the same strain. chimpanzee (Rodhain, 1948; Garnham et al,
The duration of P. malariae infections can 1956). Rodhain observed maximum parasitemias
be extremely long, especially in the form of of 7,000 per mm3. Garnham et al observed a
subpatent parasitemias. Boyd and Stratman- maximum parasitemia of 160,000 per mm3 in a
Thomas (1936) observed malariae parasites as splenectomized chimpanzee. Bray (1960) ob-
late as 173 days after exposure to infection. served parasitemia of P. malariae in
Boyd (1940) found that in infections induced by splenectomized chimpanzees, between 25,000
mosquito bites, P. malariae attacks, including and 50,000 parasites per mm3 of blood. Some
recurrences, had a mean duration of 170 days in consider P. malariae of man and P. rodhaini in
Caucasians and 76 days in Negroes. The mean the chimpanzee to be one and the same species.
duration of all naturally induced clinical attacks, Maybe they are one and the same but, in our
regardless of race, was 132 days. Boyd (1947) opinion, adequate proof is lacking.
reported that a naturally induced infection with The first adaptation of Plasmodium
P. malariae had persisted in a latent-chronic malariae to Aotus trivirgatus monkeys was
state for a period of 4,305 days (11 years, 9 reported by Geiman and Siddiqui (1969). One
months, 14 days); this was established when the hundred and thirty days after inoculation, the
patient, who had received no specific therapy, parasitemia reached a peak--of 97,920 per mm3
gave 250 ml of blood for a transfusion. Shute of blood. During the succeeding one year of
and Maryon (1955) reported "true relapse" of P. observation, the parasitemia fluctuated between
malariae infection in 2 patients 12 and 32 years 200 and 26,000 per mm3. The infection was
after infection. What they reported, probably, subsequently passaged to splenectomized and to
were recrudescences since it is not clear whether intact A. trivirgatus monkeys; their parasitemias
the therapy given was adequate to totally ranged from 100 to 23,000 per mm3.
eliminate all the blood stages. Ciuca et al (1956) In our own studies (Contacos and Collins,
published on a case in which parasites were 1969; Collins and Contacos, 1969), we have
observed after 18 years. Lentini and Tecce been able to transfer the infection from man to
(1955) observed a patient whose infection splenectomized A. trivirgatus monkeys on 2
relapsed (recrudesced) 45 years after leaving an occasions (Fig. 48). The parasitemia was slow to
endemic area. Guazzi and Grazi (1963) reported rise and reached a mean parasite level of
an infection which relapsed, subsequent to a approximately 1,000 per mm3 on the 25th day of
splenectomy, approximately 53 years after the patent parasitemia. Subsequent passage to 10
primary attack. Diamond (1966) found P. other splenectomized monkeys resulted in a
malariae parasites in a blood film from a patient peak median parasitemia of approximately 2,500
who had immigrated from Antigua to England per mm3 by day 15. Thereafter, the parasitemia
12 years earlier and had no previous history of was maintained at approximately the same level
malaria. as that in the primary passages.
222 PRIMATE MALARIAS

Plasmodium malariae grows well in this malariae has been transmitted to 6 volunteers by
monkey as indicated by the nearly normal the bites of infected A. freeborni mosquitoes
appearance of the parasites in the blood, (Plate and, the Roumanian VS strain, to 4 volunteers
XXXV) the long persistence of the infection, via the bites of A. atroparvus.
and its ready infectivity to mosquitoes (Fig. 49).
In one monkey (AO-74), A freeborni mosquitoes Host Specificity
were allowed to feed beginning day 11 and
through day 108 of parasitemia (no feedings
Plasmodium malariae is found naturally in
were made on days 48 through 56). Infections
man and, possibly, in chimpanzees.
were obtained on 74 of the 89 feeding days.
Experimentally, infections have been obtained in
Although, in general, the percentage of infection
owl monkeys, Aotus trivirgatus, although, to
was low, there were indications, at least, of an
date, only blood induced infections have been
every-third-day peak in the mosquito infection
reported.
rate during certain periods, such as, on days 16,
Mosquitoes which have been reported as
19, 25, 28, 34, 37, 40, 43, and 46. Although not
susceptible to infection with P. malariae are
as pronounced as with some of the tertian
listed in Table 28. In addition, we have obtained
malarias (see Chapters 6 and 23), it suggests that
infections in A. b. balabacensis mosquitoes. A
infectivity may be correlated with the
comparative infectivity study with a strain of
schizogonic cycle in the vertebrate host.
this parasite from Nigeria (Table 29), indicates
Experimental transmission of P. malariae
that A. freeborni was the most susceptible
by the bites of infected mosquitoes has been
followed by A. b. balabacensis, A. stephensi, A.
demonstrated by a number of workers as listed
atroparvus, and finally, A. maculatus.
in Table 28. To that list we can add our
experiences in which a Nigerian strain of P.

FIGURE 48.—Median parasitemia curves of Plasmodium malariae in Aotus trivirgatus monkeys after primary inoculation of
parasitized blood from man and secondary inoculation into additional monkeys.
 PLASMODIUM MALARIAE 223

TABLE 28.—Anopheline mosquitoes tested for susceptibility to infection with Plasmodium malariae and those which effected transmission.

Mosquito species Transmission References Mosquito species Transmission References

Anopheles acontius -- Schuurman and Huinink, 1929 A. melas -- Garnham, 1966.

A. annulipes -- Garnham, 1966* A. messeae + Marotta and Sandicchi, 1939

A. atroparvus -- Jancso, 1921 A. minimus -- Anazawa, 1931

A. atroparvus -- James, 1931
A. atroparvus + de Buck, 1935 A. moucheti -- Garnham, 1966
A. atroparvus -- Cambournac, 1942
A. atroparvus -- Shute,1951 A. plumbeus -- Gendel'man, 1936
A. atroparvus + Shute and Maryon, 1955
A. atroparvus + Ciuca et al, 1964 A. punctulatus + Mackerras and Roberts, 1947
Constantinescu and Negulici, A. punctulatus -- Mackerras and Ercole, 1948
A. atroparvus --
1967
A. punctipennis -- Mayne, 1932
A. aztecus -- Young and Burgess, 1961 A. punctipennis -- Young and Burgess, 1961

A. claviger -- Grassi et al, 1899 Boyd and Stratman-Thomas,

A. quadrimaculatus +
1933
A. culicifacies -- Stephens and Christophers, 1902 Boyd and Stratman-Thomas,
A. quadrimaculatus --
A. culicifacies -- Iyengar, 1933 1934
A. culicifacies -- Siddons, 1944 Boyd and Stratman-Thomas,
A. quadrimaculatus +
1936
A. darlingi -- Garnham, 1966* A. quadrimaculatus + Young and Burgess, 1947
A. quadrimaculatus -- Young et al, 1948
A. fluviatilis -- Garnham, 1966* A. quadrimaculatus + Young and Burgess, 1961

A. freeborni + Young and Burgess, 1947 A. sacharovi -- Khodukin and Lisova, 1930
A. freeborni -- Young and Burgess, 1961 A. sacharovi + Averbouch, 1930
A. freeborni + Contacos and Collins, 1969 A. sacharovi + Mer, 1933

A. fuliginosus -- Stephens and Christophers, 1902 A. splendidus -- Anazawa, 1931

A. fuliginosus -- Anazawa, 1931
A. fuliginosus -- Sur et al, 1932 A. stephensi -- Sur et al, 1932
A. fuliginosus -- Basu,1943 A. stephensi -- Iyengar, 1933
A. stephensi -- Russell and Mohan, 1939
A. funestus -- Garnham, 1966 * A. stephensi -- Knowles and Basu, 1943
Shute, 1951; Shute and Maryon,
A. stephensi +
Gordon and Davey, 1932 1951
A. gambiae --
A. gambiae -- Muirhead-Thompson, 1957
A. sundaicus -- Swellengrebel et al, 1919
A. sundaicus -- Hylkema, 1920
A. hyrcanus sinensis -- Swellengrebel et al, 1919
Anazawa, 1931 A. sundaicus -- Anazawa, 1931
A. hyrcanus sinensis --
Khaw and Kan, 1934 A. sundaicus -- Iyengar, 1933
A. hyrcanus sinensis --
A. sundaicus + Brumpt, 1949
A. jeyporiensis -- Garnham, 1966 *
A. tessellatus -- Anazawa, 1931
A. lindesayi -- Anazawa, 1931
A. varuna -- Sur et al, 1932
Green, 1929 A. varuna -- Iyengar,1933
A. maculatus --
A. maculatus -- Anazawa, 1931
A. maculatus -- Strickland et al, 1940
*Listed by Garnham as experimental hosts; reference not given.

223
 PLASMODIUM MALARIAE 225

FIGURE 49.—Course of parasitemia and infectivity to Anopheles freeborni mosquitoes of an infection of Plasmodium malariae in
an Aotus trivirgatus monkey (AO-74).

Immunity and Antigenic them were not infective). They also reported,
that in patients reinoculated repeatedly with the
Relationships same strain, the percentage of takes decreased.
They showed, also, that homologous immunity
Natural infections of Plasmodium malariae was not only clinical but also parasitologic.
give rise to a strong immunity but apparently not Reinoculations with the same strain of parasite,
as strong as those resulting from repeated blood- after cessation of clinical manifestations,
induced infections. Ciuca et al (1934) showed resulted in varying degrees of immunity to
that many patients convalescing from blood- clinical symptoms and to parasitemia. By the 5th
induced quartan malaria acquire a sterile reinoculation, no patients showed fever and
immunity (i.e., large volumes of blood from

PLATE XXXV.—Plasmodium malariae in the owl monkey, Aotus trivirgatus.

Fig. 1. Normal red cell. Figs. 14-20. Developing schizonts.
Figs. 2-5. Young trophozoites. Fig.21. Mature schizonts.
Figs. 6-10. Growing trophozoites, note double infection Figs. 22, 23. Developing and mature macrogametocytes.
(Fig. 6). Figs. 24, 25. Developing and mature microgametocytes.
Fig. 11-13. Nearly mature and mature trophozoites.

225
226 PRIMATE MALARIAS

TABLE 29.—Comparative infectivity of Plasmodium malariae to Anopheles freeborni, A. b. balabacensis, A. stephensi,

A. atroparvus, and A. maculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

F-1 100
F-1 : Bal 13 223 116 9.4 17.2 80.4
F-1 : St-1 5 134 65 9.7 4.6 20.3
F-1 : Atro 52 996 238 37.1 12.2 14.9
F-1 : Mac 33 554 865 43.7 11.3 3.5

* F-1 = Anopheles freeborni, Bal = A. b. balabacensis, St-1 = A. stephensi, Atro = A. atroparvus, Mac = A. maculatus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. freeborni to another
species where the GII of A. freeborni = 100.

parasites. Twenty-three percent had parasites immunity were considered by Ciuca et al to

without fever and 77 percent had no fever and develop in parallel. The duration of such
no parasites. By the 7th reinoculation, immunities was observed to persist from 5 to 14
homologous immunity was complete, i.e., 100 years.
percent with no fever and no parasites. Antibodies to P. malariae were shown to
In naturally acquired or induced infections, cross-react to Plasmodium fieldi, P. gonderi, P.
parasites rarely disappear entirely and/or inui, P. coatneyi, P. knowlesi, P. cynomolgi (B
permanently; rather, there is a tendency for strain), and P. brasilianum antigens (Collins et
extreme chronicity of infection. Garnham (1966) al 1966). The highest heterologous reactions
stated that the assumption by the Roumanian were to the P. brasilianum antigen and
workers that "this 'resistance' is of the nature of secondarily to P. fieldi. Such cross-reactions
premunition" was probably correct. Ciuca et al were also reported by Meuwissen (1968) using
(1955) reported that the forms of immunity P. falciparum, P. vivax, P. ovale, P. cynomolgi
acquired as a result of P. malariae infection (B strain), and P. fieldi antigens. In a further
present characteristics entirely different from study, Sulzer et al (1969) found that a P.
those for P. vivax and P. falciparum in the sense malariae antiserum gave a high level of reaction
that the proportion of residual immunity to P. brasilianum, followed by lower reactions
(immunity without premunition) is superior to to P. vivax, P. falciparum, and, finally, to P.
that of "concomitante" immunity (immunity fieldi antigens. Dranga et al (1969) in testing the
with premunition), being, respectively, 72.8 and cross-reactions between P. vivax and P.
27.2 percent. malariae antisera showed that a cross-reaction
Plasmodium malariae offers better could always be observed between the 2
antigenic quality than the other human malarias antigens but that the homologous responses were
for stimulating a more durable immunity which higher than the heterologous responses.
maintains itself even after the disappearance of
parasites. Homologous and heterologous
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Formosa 30 : 609-632. (NS). December 99-101.
AVERBOUCH, I., 1930. Experiences sur la transmission a BOYD, M. F. and STRATMAN-THOMAS, W. K., 1933. A note
l'homme de la fievre quarte par les piqûres d' Anopheles on the transmission of quartan malaria by Anopheles
maculipennis var. saccharovi. Rev. Micro. Epid. quadrimaculatus. Am. J. Trop. Med. 13 : 265-271.
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consecutive human-anopheline passages. Am. J. Trop. DE BUCK, A., 1935. Infection experiments with quartan malaria.
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BRAY, R. S., 1959. Pre-erythrocytic stages of human malaria DIAMOND, M. P., 1966. Blood film containing Plasmodium
parasites: Plasmodium malariae. Brit. Med. Jour. 2 : malariae from patient who came to this country 12
679-680. years ago. Trans. Roy. Soc. Trop. Med. & Hyg. 60 :
BRAY, R. S., 1960. Studies on malaria in chimpanzees. VIII. The 425.
experimental transmission and pre-erythrocytic phase DRANGA, A., MARINOV, R., ROMANESCU, C., and
of Plasmodium malariae, with a note on the host-range TUDOSE, M., 1969. L'evolution des anticorps
of the parasite. Am. J. Trop. Med. & Hyg. 9 : 455-465. fluorescents dans le paludisme provogue par
BRUMPT, E., 1939. Les parasites du paludisme des chimpanzes. inoculation palustre primaire. (Personal communication
C. R. Soc. BioI. 130 : 837-840. and WHO /Mal/69.697.)
BRUMPT, E., 1949. The human parasites of the genus FELETTI, R. and GRASSI, B., 1889, 1890. Sui parassiti della
Plasmodium. Malariology, Vol. I, edited by Mark F. malaria. Rif. Med. 6 : 62-64. (The 1889 paper was a
Boyd. W. B. Saunders Co., Phila. preprint of the 1890 paper according to Hemming,
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sezonismo em Portugal. Soc. Ind. de Tipografia, Lda., FIELD, J. W. and SHUTE, P. G., 1956. The microscopic diagnosis
Lisbon. of human malaria. II. A morphological study of the
CELLI, A. and SANFELICE, F., 1891. Sui parassiti del globulo erythrocytic parasites. Inst. Med. Res., Fed. Malaya,
rosso nell'uomo et negli animali. Quar. 1st. Igene Sper. No.24. pp. 251.
Univ. Roma, 1 (N.S.) 33-63 and Ueber die Parasiten GARNHAM, P. C. C., 1966. Malaria parasites and other
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Hyg. 27 : 619-622. chimpanzee, with particular reference to the presistence
CIUCA, M., CHELARESCU, M., SOFLETEA, A., of Plasmodium reichenowi and Plasmodium vivax.
CONSTANTINESCU, P., TERITEANU, E., CORTEZ, Ann. Soc. Beige de Med. Trop. 36 : 811-821.
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Experimentale a Petude de Pimmunite dans le new world monkey to Plasmodium malariae from man.
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Populaire Roumaine (Bucharest), 61-108. GENDLE'MAN, C. A., 1936. Sull'infezione sperimentale dell'
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ATANASIU, M., ISFAN, T., CONSTANTINESCU, Inst. Narkomzdrava Assr. Abhhazii 2 : 70. (NS).
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Plasmodium vivax, Plasmodium falciparum si 33 : 734-83.
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564. febre terzana; diagnosi differenziale tra i parassiti
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CONSTANTINESCU, P., 1964. Recherches sur la quartana. Arch. Sc. Med. 13 : 173-195.
transmission experimentale de P. malariae a Phomme. GORDON, R. M. and DAVEY, T. H., 1932. P. malariae in
Arch. Roum. Path. Exp. Microbiol. 23 : 763-776. Freetown, Sierra Leone. Ann. Trop. Med. & Parasit. 26
COATNEY, G. R. and YOUNG, M. D., 1941. The taxonomy of : 65-84.
the human malaria parasites with notes on the principal GRASSI, B. and FELETTI, R., 1890. Parasites malariques chez les
American strains. Am. Assoc. Adv. Sci. (No.15), 19-24. oiseaux. Arch. Ital. de Biologie 13 : 297-300.
COLLINS, W. E. and CONTACOS, P. G., 1969. Infectivityof GRASSI, B. and FELETTI, R., 1892. Contribuzione allo studio die
Plasmodium malariae in the Aotus trivirgatus monkey parassiti malarici. Atti Accad. Gioenia. Series 4,5 : 1-
to Anopheles freeborni mosquitoes. J. Parasit. 55 : 81.
1253-1257. GRASSI, B., BIGNAMI, A., and BASTIANELLI, G., 1899.
COLLINS, W. E., JEFFERY, G. M., GUINN, E., and SKINNER, Risoconto degli studi fatti sulla malaria durante il mese
J. C., 1966. Fluorescent antibody studies in human di Gennaio. Rend. Atti Accad. Lincei 8: 100-104.
malaria. IV. Cross-reactions between human and simian GREEN, R., 1929. Observations on some factors influencing the
malaria. Am. J. Trop. Med. & Hyg. 15 : 11-15. infectivity of malarial gamete carriers in Malaya to
CONSTANTINESCU, P. and NEGULICI, E., 1967. The Anopheles maculatus. Bull. Inst. Med. Res. Fed. Malay
experimental transmission of Plasmodium malariae to States 5 : 1-41.
Anopheles labranchiae atroparvus. Trans. Roy. Soc. GUAZZI, M. and GRAZI, S., 1963. Considerazioni su un caso di
Trop. Med. & Hyg. 61 : 182-188.
CONTACOS, P. G. and COLLINS, W. E., 1969. Plasmodium
malariae: Transmission from monkey to man by
mosquito bite. Science, 165 : 918-919.
228 PRIMATE MALARIAS

REFERENCES—Continued
malaria quartana recidivnte dopo 53 anni di latenza. MACKERRAS, M. J. and ERCOLE, Q. N ., 1948. Observations
Riv. di Malariol. 42 : 55-59. on the life-cycle of Plasmodium malariae. Australian J.
HEMMING, F., (Ed.), 1954. Opinions and declarations rendered Exper. Bioi. & Med. Sci. 26 : 515-519.
by the International Commission on Zoological MACKERRAS, M. J. and ROBERTS, F. H. S., 1947.
Nomenclature. Opinion 283, Vol. 7, London, pp. 225. Experimental malarial infections in Australasian
HYLKEMA, B., 1920. The development of the parasites of anophelines. Ann. Trop. Med. Parasit. 41 : 329-356.
quartan malaria in the Myzomyia ludlowi and their MAROTTA, G. and SANDICCHI, G., 1939. Contributo
trans- mission to man. Meded. v.d. Burg. Geneesk. d. all'infezione sperimentale di anofeli con Plasmodium
Nederl.- Indie 6 : 51. (NS). malariae c inoculazione della malattia all'uomo. Riv. di
IYENGAR, M. O. T., 1933. Experimental infection of anopheline Malariol. 18 : 89-94. (NS).
mosquitoes. Ind. J. Med. Res. 20 : 841-861. MAYNE, B., 1932. Note on experimental infection of Anopheles
JAMES, S. P., 1931. Some general results of a study of induced punctipennis with quartan malaria. Pub. Hlth. Rep. 47 :
malaria in England. Trans. Roy. Sac. Trop. Med. & 1771-1773.
Hyg. 24 : 477-525. MER, G., 1933. Observations on the development of Plasmodium
JANCSO, N., 1921. Experimentelle Untersuchungen über die malariae Lav. in Anopheles elutus Edw. Ann. Trop.
Malaria-infection des Anopheles und des Med. Parasit. 27 : 483-488.
Menschenbeeinin£Iussenden Umstiinde. Arch. f. MEUWISSEN, J. H. E. TH., 1968. Antibody responses of patients
Schiffs-u. Trop. -Hyg. 25 : 5. (NS). with natural malaria to human and simian Plasmodium
KHAW, O. K. and KAN, N. C., 1934. Some observations on the antigens measured by the fluorescent antibody test.
prevalence of malaria in Nanking and its vicinity. Trop. geogr. Med. 20 : 137-140.
Chinese Med. J. 48 : 109-123. (NS). MUIRHEAD-THOMSON, R. C., 1957. Notes on the characters of
KHODUKIN, N. I. and LISOVA, A. I., 1930. Zur Frage über die P. malariae oocysts of possible value in mixed
Moglichkeit von Erkrankungen an Malaria im Winter infections. Am. J. Trop. Med. & Hyg. 6 : 980-986.
(In Russian). Meditz. Muisl' Uzbekitana 1 : 76. (NS). RODHAIN, J., 1948. Contribution a I'etude des Plasmodiums des
KITCHEN, S. F., 1949. Quartan malaria. Malariology, Vol. II, anthropoides Africains. Ann. Soc. Beige de Med. Trop.
edited by Mark F. Boyd. W. B. Saunders Co.. Phila. 28 : 39-49.
KNOWLES, R. and BASU, B. C., 1943. Laboratory studies on the RUSSELL, P. F. and MOHAN, B. N., 1939. On experimental
infectivity of Anopheles stephensi. J. Malar. Inst. Ind. 5 malaria infections in certain Anopheles of south-eastern
: 1-29. (NS). Madras. J. Mal. Inst. Ind. 2 : 425-431.
KNOWLES, R., SENIOR-WHITE, R., and DAS GUPTA, B. M., SCHUURMAN, C. J. and HUININK, A. S. B., 1929. A malaria-
1930. Studies in the parasitology of malaria. Ind. J . problem in Java's South-coast. Meded. Dienst d.
Med. Res. Memoir 18 : 1-436. Volksgezondh. in Mederl.-Indie 18 : 120-142. (NS).
KRUSE, W., 1892. Der Gegenwartige Stand Unserer Kenntnisse SHUTE, P. G., 1951. Mosquito infection in artificially induced
von den Parasiten Protozoen. Hyg. Rdschr. 2: 357-380 malaria. Brit. Med. Bull. 8 : 56-63.
and 453-500. SHUTE, P. G. and MARYON, M., 1951. Studies in the
LABBE, A., 1894. Recherches zoologiques et biologiques sur les transmission of Plasmodium malariae by Anopheles
parasites endoglobulaires du sang des vertebrates. Arch. mosquitoes. Parasitology 41 : 292-300.
Zool. exp. 2 : 55-258. SHUTE, P. G. and MARYON, M., 1952. A study of human
LAVERAN, A., 1880. Note sur un nouveau parasite trouve dans le malaria oocysts as an aid to species diagnosis. Trans.
sang de plusieurs malades atteints de fievre palustre. Roy. Soc. Trop. Med. & Hyg. 46 : 275-292.
Bull. Acad. Med. 9 : 1235-1236. SHUTE, P. G. and MARYON, M., 1955. Transmission of
LENTINI, D. and TECCE, T., 1955. Recidiva a lunga scadenza di Plasmodium malariae by laboratory-bred Anopheles
infezione malarica quartana. Riv. di Malariol. 34 : 259- maculipennis var. atroparvus Meigen. Ann. Trop. Med.
265. Parasit. 49: 451-454.
LEWKOWICZ, X., 1897. Ueber den Entwickelungsgang und die SIDDONS, L. B., 1944. The experimental transmission of quartan
Einteilung der Malariaparasiten. Zentralbl. Bakt. 1 Abt. malaria by Anopheles culicifacies Giles. J. Malar. Inst.
Orig. 21 : 130-133. Ind. 5: 361-373.
LUPASCU, G., CONSTANTINESCU, P., NEGULICI, E., STEPHENS, J. W. W. and CHRISTOPHERS, S. R., 1902. The
GARNHAM, P. C. C., BRAY, R. S., KILLICK- relation of species of anopheles to malarial endemicity.
KENDRICK, R., SHUTE, P. G., and MARYON, M., Rep. Malar. Comm. Roy. Soc. 7th series, p. 20.
1967. The late primary exoerythrocytic stages of STRICKLAND, C., ROY, D. N., and SEN GUPTA, S. C., 1940.
Plasmodium malariae. Trans. Roy. Soc. Trop. Med. & Anopheles maculatus and malaria. Trans. Roy. Soc.
Hyg. 61 : 482-489. Trop. Med. & Hyg. 33 : 639-652.
LUPASCU, G., CONSTANTINESCU, P., NEGULICI, E., SULZER, A. J., WILSON, M., and HALL, E. C., 1969. Indirect
SHUTE, P. G., and MARYON. M. E., 1968. fluorescent-antibody tests for parasitic diseases. V. An
Parasitological and clinical investigations on infections evaluation of a thick-smear antigen in the IF A test for
with the VS Romanian strain of Plasmodium malariae malaria antibodies. Am. J. Trop. Med. & Hyg. 18 : 199-
transmitted by Anopheles labranchiae atroparvus. Bull. 205.
Wid. Hlth. Org. 38 : 61-67. SUR, S. N., SARKAR, H. P., and BANERJI, K. M., 1932.
Plasmochin as a malarial gametocide. Ind. Med. Gaz.
 PLASMODIUM MALARIAE 229

REFERENCES—Continued
67 : 490-493. YOUNG, M. D., STUBBS, T. H., and COATNEY, G. R., 1940.
SWELLENGREBEL, N. H., SCHUFFNER, W., and Studies on induced quartan malaria in Negro paretics. I.
SWELLENGREBEL-DE GRAAF, J. M. H., 1919. The Periodic phenomena of the asexual cycle. Am. J. Hyg.
susceptibility of anophelines to malarial-infections in 31 : 51-59.
Netherlands India. Meded. Burgerlijk. Geneesk. Dienst. YOUNG, M. D., COATNEY, G. R., and STUBBS, T. H., 1940a.
Ned. Indie. 3 : 1-64. (NS). Studies on induced quartan malaria in Negro paretics.
YOUNG, M. D. and BURGESS, R. W., 1947. The transmission of II. The effect of modifying the external conditions. Am.
Plasmodium malariae by Anopheles maculipennis J. Hyg. 32 : 63-70.
freeborni. Am. J. Trop. Med. 27 : 39-40. YOUNG, M. D., HARDMAN, N. F., BURGESS, R. W.,
YOUNG, M. D. and BURGESS, R. W., 1961. The infectivity to FROHNE, W. C., and SABROSKY, C. W., 1948. The
mosquitoes of Plasmodium malariae. Am. J. Hyg. 73 : infectivity of native malarias in South Carolina to
182-192. Anopheles quadrimaculatus. Am. J. Trop. Med. 28 :
YOUNG, M. D., COATNEY, G. R., and MCLENDON, S. B., 303-311.
1941. Studies on induced quartan malaria in Negro
paretics. III. Measurements of the paroxysmal phases. (NS) = Not seen.
Southern Med. Jour. 34 : 709-712.
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 19
Plasmodium brasilianum Gonder and von
Berenberg-Gossler , 1908

Porter, et al, (1966) summarized the records

IN 1908, Gonder and involving 1994 primates from Panama, collected
von Berenberg-Gossler had the opportunity to from 1931 through 1957, among which 4 species
examine the blood of a Cacajao monkey, in 3 genera exhibited the parasite.
Brachyurus calvus (= Cacajao calvus) imported The parasite is fairly common in
to Hamburg, Germany, from the Amazon region northwestern Brazil. In 1969, Deane and
of Brazil. In it, they encountered a quartan-like Ferreira Neto reported P. brasilianum from the
malaria parasite to which they gave the name territory of Amapa, also known as Brazilian
Plasmodium brasilianum. The following year Guyana, in eastern Amazonia. There is only one
(1909), von Berenberg-Gossler made a careful report of a natural infection in Venezuela
study of the parasite and compared it with P. (Serrano, 1967) but numerous surveys in
malariae, which it closely resembles, and other Colombia, the most recent was that of
malarias then known from monkeys. Marinkelle and Grose (1968), have shown the
Seidelin (1912) had kept a spider monkey parasite to be widespread. The work of Dunn
(Ateles sp.) in his laboratory in Merida, Yucatan and Lambrecht (1963) extended the distribution
for some time and in its blood, he found delicate of this parasite into the lowland forests of
rings which some authors, including Wenyon Colombia and Peru.
(1926), considered to be a plasmodium. Following the work of the Taliaferros in
Repeated examination of the blood showed only Panama, little attention was given to the
rings with one or two chromatin masses which is malarias of the New World monkeys until after
emphasized on his colored plate, and, therefore, the National Institute of Allergy and Infectious
as suggested by Garnham (1966), it was Disease's group launched an extensive program
probably a piroplasm. following accidental and purposeful infections in
Plasmodium brasilianum is relatively man with P. cynomolgi in 1960. Beginning in
common in the monkeys of Panama where Clark 1964, Deane and his co-workers entered upon an
(1930, 1931) first encountered it. Then, the overall study of the simian malarias of Brazil
Taliaferros (1934, a, b, and 1944) carried out an which has been extremely fruitful.
exhaustive experimental study of the parasite.

231
 PLASMODIUM BRASILIANUM 233

Cycle in the Blood stains a median blue, harbors a compact pink-

staining nucleus with a darker reddish area and
PLATE XXXVI occupies an eccentric position. The dark
The initial forms in the peripheral blood are
pigment, in the form of short rods, is scattered in
small rings with a prominent dark-staining
the cytoplasm (Fig. 29). The microgametocyte
nucleus. As growth proceeds, a small vacuole is
exhibits a large diffuse nucleus which stains
formed which eventually disappears in the older
dark pink. The cytoplasm takes a very pale blue
forms. The rings often exhibit accessory dots.
to purplish stain, with the pigment in large
The nucleus may elongate to form a part of the
granules more or less scattered in the matrix.
periphery of the older rings (Figs. 6, 7). With the
The asexual cycle requires 72 hours which
disappearance of the vacuole, the compact
the Taliaferros described and illustrated in
parasite displays irregular protrusions (Figs. 10,
precise detail. They went on to show (1934) that
11). These loitering amoeboid forms are not
the decidedly synchronous cycle could be
unlike similar ones seen in P. malariae. At about
reversed in a short time if the infected animals
this time black granular pigment appears
were subjected to a reversal of night and day
scattered in the blue-staining cytoplasm. Band
conditions. Under normal conditions sporulation
forms appear during the late trophozoite stage
took place around 8 a.m. but after the conditions
(Figs. 16, 19) and although these forms are
were reversed the cycle slowly became modified
found in the blood of all simian species
so that it occurred at 8 p.m. Some years later
susceptible to the infection, they occur with
(1940) Young, Coatney and Stubbs produced the
greater frequency in Alouatta. The host cell is
same results with P. malariae in man.
not enlarged and does not appear
inconvenienced by the parasite. At about 50
hours, the parasite occupies a large part of the Sporogonic Cycle
erythrocyte, at which time, it begins to divide. PLATE XXXVII
The process of schizogony moves toward Clark and Dunn (1931) reported
completion generally with decided development of P. brasilianum to the sporozoite
synchronicity. At maturity, each schizont level in Anopheles tarsimaculatus (= aguasalis),
harbors 8 to 12 merozoites but there may be only and A. albimanus. Garnham et al (1963) infected
4 or up to 16 arranged more or less as a rosette A. aztecus, A. atroparvus, and A. albimanus. At
or, what is sometimes described, as a "daisy- an incubation temperature of 26 to 28° C, the
head" (Fig. 28). The mature schizont may oocysts grew slowly and were characterized by a
produce a slight increase in the size of the host fragile cyst wall. On the 8th day oocysts
cell; a condition not found with P. inui. With measured 20 to 24 µ and contained pigment
appropriate staining, Ziemann's stippling can be arranged along irregular threads, in circles, or in
demonstrated during the late ring stage. clusters. By the 12th day, the oocysts
The young gametocytes are difficult to approached maturity with diameters of 38 to 45
recognize and, as is common in all quartan µ. Sporozoites were present in the salivary
infections, sexual forms are scarce at best. The glands on day 13. The sporozoites were reported
mature forms fill the host cell and cause some to be sickle-shaped with a central nucleus. In the
enlargement of it. The macrogametocyte, which

PLATE XXXVI.—Plasmodium brasilianum.

Fig. 1. Normal red cell. Figs. 24-26. Developing schizonts.
Figs. 2-5. Young trophozoites. Figs. 27-28. Mature schizonts.
Figs. 6-13. Growing trophozoites. Fig. 29. Mature macrogametocyte.
Figs. 14-23. Nearly mature and mature trophozoites Fig. 30. Mature microgametocyte.
(Note band-forms Figs. 16, 19)
234 PRIMATE MALARIAS

PLATE XXXVII.—Developing oocysts of Plasmodium brasilianum in Anopheles freeborni mosquitoes. X 580.

Fig. 1. 8-day oocysts with small grains of pigment. Fig. 6. 13-day oocyst showing small number of vacuoles.
Fig. 2. 9-day oocysts showing pigment near periphery. Fig. 7. 1-day oocyst showing earliest stage of differentiation.
Fig. 3. 10-day oocyst. Fig. 8. 15-day differentiating oocyst.
Fig. 4. 11-day oocysts. Fig. 9. 18-day fully differentiated oocyst.
Fig. 5. 12-day oocyst.
 PLASMODIUM BRASILIANUM 235

fresh state, they measured about 14 to 16 µ in

length. TABLE 29.—Oocyst diameters of Plasmodium brasilianum in
Anopheles freeborni and A. stephensi.
Garnham et al (1963) studied the fine
structure of the sporozoites of P. brasilianum. Days after A. freeborni A. stephensi
Infection
Their material was not of the best quality and
No. Range Mean* No. Range Mean
therefore they were unable to give a complete
and precise description. They found that the 6 43 9-13 11
pellicle was about 30 Mµ in width and appeared 7
8
176
148
8-18
11-20
13
15
as two layers separated by a less dense area. An 9 181 12-25 18 24 14-24 19
10 181 12-32 21 23 15-28 23
apical cup was not demonstrated but a well- 11 183 14-39 25 13 28-37 30
defined micropyle was seen. Peripheral fibers 12 123 14-42 30
13 142 17-53 34
were hollow, but their exact number could not 14 196 17-60 40† 16 28-51 38†
be determined. The "paired organelle" was an 15 175 20-63 41†
16 150 17-70 46†
obvious structure suggesting a secretory 17 112 26-70 53†**
function.
In our studies, A. freeborni has proved to be Totals 1810 9-70 76 14-51

the most suitable mosquito for observing the * Measurements expressed in microns; incubation temperature of
25º C.
sporogonic cycle of P. brasilianum (Table 29). † Oocyst differentiation.
At an incubation temperature of 25° C, on day 6, ** Sporozoites present in the salivary glands.
the mean oocyst diameter was 11 µ, with a range
of 9 to 13 µ. The oocysts continued to grow so sporozoites were not present in the salivary
that by day 14, the mean size was 40 µ, with a glands until 17 days after feeding.
range of 17 to 60 µ. At this time, the first signs A few measurements of oocysts developing
of differentiation were apparent. The in A. stephensi mosquitoes gave values well
development was slow, however, and within the range of the diameters seen in the

FIGURE 50.—Range in oocyst diameters and the mean oocyst diameter curves of Plasmodium cynomolgi and P. brasilianum in
Anopheles freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
236 PRIMATE MALARIAS

A. freeborni, none of the mosquitoes of this Sporozoites of P. brasilianum in A. aztecus

species survived long enough to determine if were shown to be infective (Garnham et al,
sporozoites would invade the salivary glands, 1963) in that a strain from the squirrel monkey
Other species which have developed sporozoites (S. sciureus) was passaged, by the combination
in the salivary glands were A. maculatus and of mosquito feeding and intravenous inoculation
rarely, A. quadrimaculatus (Collins et al, 1969). of dissected salivary glands, into another squirrel
If a comparison is made of the growth rate monkey and also into a Cebus capucinus
of P. brasilianum and P. cynomolgi (Fig. 50), monkey. In our studies, one Ateles paniscus
one sees a marked difference between the two monkey was infected through bites of A.
parasites. The mean oocyst diameter of P. freeborni mosquitoes, and six monkeys (2 A.
cynomolgi at day 8 is about the same as that of paniscus, 3 S. sciureus, and 1 Aotus trivirgatus)
P. brasilianum at day 15. The maximum oocyst were infected by the intravenous or intrahepatic
diameters of P. cynomolgi far exceed those of P. inoculation of infected salivary glands. The
brasilianum and the time required for prepatent periods in these animals ranged from
completion of the life cycle, i. e., the appearance 18 to 43 days with a mean of 33.1 days.
of sporozoites in the salivary glands, was shorter
by 6 days. A comparison of the oocyst growth Cycle in the Tissue
rates of the two quartan parasites, P. malariae
and P. brasilianum, (Fig. 51) indicates that the
PLATE XXXVIII
The first workers to study the EE bodies of
mean oocyst diameters of these two parasites in
P. brasilianum were Garnham et al (1963). They
A. freeborni mosquitoes are almost identical.
made two unsuccessful attempts to find the
Such a close similarity in oocyst growth rate
tissue stages but were rewarded on the third try
curves, was not found between any of the other
when they located a single 11-day old EE body
human and simian malaria parasites. From a
after examining some 500 sections. They
study of the sporogonic growth rate, it is
described the parasite as oval, 20 by 38 µ, and
strongly suggestive, that these parasites, P.
located in a parenchyma cell of the liver
brasilianum and P. malariae, are almost
possessing two enlarged nuclei. The parasite was
identical.

FIGURE 51.—Range in oocyst diameters and the mean oocyst diameter curves of Plasmodium malariae and P. brasilianum in
Anopheles freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
 PLASMODIUM BRASILIANUM 237

enclosed within a definite membrane which was other simian malarias and in P. malariae of man.
decidedly convoluted or scalloped. Small Lupascu et al (1967) reported them in the late
peripheral vacuoles were present, filled with a stages of P. malariae. In P. brasilianum, the
pink-staining substance. The cytoplasm was clefts were not as prominent as represented in P.
granular and tenuous, and exhibited vague malariae.
cleavage lines. The nuclei of the schizont were Lupascu et al also reported strands in their
large and arranged in a cluster of irregular dots 15-day material of P. malariae which stained
measuring 1 to 1.5 µ. They estimated that the red and were limited to that one stage. In P.
brasilianum, on the other hand, the strands were
schizont was a day or so from maturity
basophilic and present in the 14-day and 21-day
indicating a primary cycle of some twelve days.
sections; such strands have not been described
In 1969, Sodeman et al described the EE for the other simian malarias. A prominent
stages of a strain of P. brasilianum which, like feature of the 21-day stage was the retraction of
Garnham's strain, had been isolated from a the cytoplasm from the limiting membrane,
naturally infected squirrel monkey, Saimiri sometimes associated with delicate vacuoles.
sciureus. They were unable to locate the Garnham et al described a similar feature in his
parasites in biopsy material taken at 7 days, but 11-day stage, and Lupascu et al for the late stage
found them in tissue taken at 14, 18, and 21 days of P. malariae. Bray (1957) found the same
after sporozoites were introduced. These were condition in his P. ovale material.
illustrated in a series of four well-executed Greatly enlarged host cell nuclei were also
plates. found in P. brasilianum by Garnham and in liver
The 14-day EE bodies (Figs. 1, 2) were
cells housing P. malariae and P. ovale. This
elliptical while those of the 18th (Fig. 3) and
finding has not been described for any EE
21st days (Figs. 4-6) showed a tendency toward
bodies of the other simian malarias.
lobulation. A distinct cell membrane was evident
Considering the slow rate of growth of the
surrounding the EE bodies. The edges of the
parasite, the size of the P. brasilianum tissue
bodies were smooth except in situations where
schizont is remarkable. The 21-day stage is only
shrinkage, probably due to fixation, produced a
6 µ larger than the average size of the 11-day
scalloped effect. The nuclei of the EE bodies
parasite of P. cynomolgi. Plasmodium malariae
were irregular in shape, stained magenta, varied
in man at 14 days averaged 47 µ as compared
from 0.5 to 1.5 µ in diameter, and were
with 30.6 µ for P. brasilianum.
distributed evenly through the cytoplasm. The
Further comparisons were made by
cytoplasm stained pale blue and was granular in
texture. Irregular-shaped, dark blue, aggregated Sodeman et al, but suffice it to say here that
flocculi (Fig. 3) were present in all the stages, those authors were of the opinion that P.
though not found in every EE body; they were brasilianum had certain features which might
least prominent in the 21-day bodies. The distinguish it from the other simian malarias,
parasitized liver cells commonly exhibited including P. inui, and that there was close
enlarged double nuclei (Fig. 5). morphologic resemblance to the human
malarias, especially P. malariae. The presence
Among the special morphological features
of enlarged host cell nuclei, needle-like clefts,
of the P. brasilianum EE bodies were the
and peripheral retraction with vacuoles are
presence of needle-like clefts in the 14- and 21-
features not seen previously in simian malaria
day material. In many cases, these clefts were
material. However, they are all present in the
outlined with basophilic material, and basophilic
human malarias. They mentioned also, that there
strands. The authors pointed out that the lack of
was greater morphologic relationship between
clefts and strands in the 18-day bodies may
the EE bodies of P. brasilianum and P. malariae
indicate individual variation prominent in EE
than between P. brasilianum and P. inui.
bodies of the same species along with the rarity
Additional relationships between these species
of the forms for study. Garnham et al (loc. cit.)
will be dealt with in later sections.
mentioned clefts but described them as vague.
Clefts have been reported in the EE bodies of
238 PRIMATE MALARIAS
 PLASMODIUM BRASILIANUM 239

Course of Infection geoffroyi), provided parasites were in sufficient

numbers.
In our studies, infections have been
Taliaferro and Taliaferro (1934) showed
obtained in Saimiri sciureus, Ateles paniscus,
that blood-induced infections with P.
and Aotus trivirgatus monkeys both by blood
brasilianum were characterized by an initial rise
inoculation and by the introduction of
in the number of parasites, a marked dimunition
sporozoites. In intact A. paniscus monkeys (Fig.
in numbers, a low grade blood infection, and
52), the parasitemia rose slowly to a peak of
finally, short periods of subpatent parasitemia
approximately 10,000 per mm3 by day 16,
interspersed with spontaneous recrudescences.
followed by a fall, and then a secondary rise to a
The height to which the parasitemia rose varied
level almost equal to the initial peak by day 60.
among individuals of the same species, but
In the intact S. sciureus monkeys, the
showed a tendency to be more acute among the
parasitemia curve exhibited a saddle-back effect
white-throated (Cebus capucinus) and spider
with two peaks, on days 8 and 20, followed by a
monkeys (Ateles geoffroyi) than among the
slow decline in the parasitemia. In A. paniscus,
howlers (Alouatta palliata (= villosa). Sharp
the median parasitemia by day 60 was 10,000
peaks in temperature occurred at sporulation,
per mm3, whereas in the S. sciureus, the median
particularly in the spider, howler, and night
parasitemia, on day 60, was approximately 100.
monkeys (Aotus zonalis (= trivirgatus) and in
In splenectomized S. sciureus monkeys, the peak
the marmoset (Leontocebus (= Saguinus)

Figure 52.—Median parasitemia curves of Plasmodium brasilianum in 13 intact Ateles paniscus, 7 intact Saimiri sciureus, and 12
splenectomized S. sciureus monkeys inoculated with parasitized blood.

PLATE XXXVIII.—Exoerythrocytic bodies of Plasmodium brasilianum in liver tissue of the squirrel monkey, Saimiri sciureus.
Fig. 1. 14-day body showing elliptical shape. X 580. Fig. 5. 21-day body showing two enlarged host cell nuclei.
Fig. 2. 14-day body. X 580. X 1450.
Fig. 3. 18-day body showing dark, irregular-shaped, Fig. 6. 21-day body showing irregular-shaped parasite
aggregated flocculi. X 1450 nuclei. X 1450.
Fig. 4. 21-day body. X 580.
240 PRIMATE MALARIAS

parasitemia was reached after about 35 days. At the infection could produce thereby preventing
60 days, the median count was approximately any visible reoccurrence of the infection.
70,000 per mm3. None of the animals, whether The first investigators to carry out
intact or splenectomized, died or required experiments toward infecting man with P.
chemotherapy for survival. One splenectomized brasilianum were Clark and Dunn (1931,
A. trivirgatus monkey, infected by the 1931a). These investigators selected 8
inoculation of sporozoites, had a peak volunteers who had recently arrived in Panama
parasitemia of 35,600 per mm3 on day 35 after from their home states in the U. S. where
which, the parasitemia slowly declined to a malaria was not endemic. Five of the volunteers
subpatent level by day 90. No further parasites and a control monkey were given parasitized
were seen in the blood of the animal during the blood intravenously from a black spider
next 57 days, when it died. monkey. Two other volunteers plus each of the
In our experience, infections with P. original five and a control animal received
brasilianum in Ateles and Saimiri monkeys parasitized blood subcutaneously also from a
usually persist for an extended period of time, black spider monkey. The control monkeys
during which the presence of gametocytes developed the infection but only one of the
allows for the infection of mosquitoes, and such volunteers gave any evidence of infection which
infectivity can continue for at least 249 days. On was recorded as a few doubtful intracorpuscular
the other hand, daily feedings on an A. bodies on an occasional blood film. After two
trivirgatus monkey, infected with P. weeks of observation the authors considered the
brasilianum, resulted in no infections; an trial a failure.
unexpected occurrance in view of our other At that juncture, they decided to attempt
successes. infection by mosquito bite. They were able to
During the course of an infection, the infect A. albimanus, and A. tarsimaculatus
parasitemia may rise and fall in what, at times, which were allowed to bite seven volunteers.
appears to be a predetermined pattern. This was The men were observed for varying lengths of
observed in an A. paniscus monkey (AT-36) time, but again, only one exhibited any evidence
inoculated with parasitized blood. The animal of infection which consisted of several
had a low-grade infection for approximately 7 elevations of temperature at or near 100° F. and,
weeks at which time it was splenectomized. The on two occasions, "one or two forms which we
parasitemia rose to a maximum of 120,500 per consider to be malarial parasites but there was
mm3 one month later and then slowly dropped to nothing satisfactory to report." It is unlikely, but
a near negative level after six weeks. This was possible, that a P. brasilianum infection was
followed by a subsequent rise to a maximum initiated in any of the volunteers.
count of 52,000 per mm3 followed by a Our own studies toward infecting man with
subsequent drop to zero. Following the initial P. brasilianum actually began in London. The
parasitemia, 6 additional periods of high senior author (GRC) had stopped there in 1962
parasitemia occurred, followed by drops to to visit Professor P. C. C. Garnham at the
negative or near negative levels. The intervals London School of Hygiene and Tropical
between these peaks were 3.5, 3, 4, 5, 4, and 4 Medicine. In the course of the conversations, he
months, respectively, after which the animal mentioned our good fortune in getting P.
failed to exhibit parasites during an observation cynomolgi to grow in man and offered the
period of 18 months. Because the animal was prediction that it probably would be extremely
blood-inoculated, the appearance of pronounced difficult, if not impossible, to get other species
waves of parasitemia following periods of to infect man. I thought otherwise, and offered a
latency were true recrudescences. The wager that we could infect man with P.
observations in this animal suggest an almost set brasilianum in a matter of weeks. Upon my
time interval for the development of new return to Washington, the problem was--where
antigenic variants--about four months. It would to obtain P. brasilianum? Dr. Carl Johnson, then
appear that the animal had then been able to Director of the Gorgas Memorial Laboratory in
produce an immunity to all the variants which
 PLASMODIUM BRASILIANUM 241

Panama, had some 8 months before agreed to be infected after feeding on a squirrel monkey (S.
on the lookout for a brasilianum infected sciureus) naturally infected with P. brasilianum.
monkey, but we had had no word from him. This strain or isolate was subsequently
Knowing Dr. Carl's penchant for not writing transmitted by their bites to one of 3 volunteers
letters, a young staff member was dispatched to whose prepatent period was 63 days. All
Panama with instructions to remain there until together, we have been able to study brasilianum
he had obtained a monkey infected with P. infections in 25 volunteers, of which 18 were
brasilianum. He departed for Panama and four induced by the intravenous inoculation of
days later called one of us (PGC) from Miami, parasitized homologous blood. Because there
Florida, with the query "what do I do with the was little difference between the parasitologic
infected monkey". The secret of the young man's and clinical picture in these infections, their
phenomenal success in obtaining an infected parasite counts and median parasitemia curve
monkey so readily was that Dr. Johnson had have been combined and are shown graphically
been holding the animal for some time and in figure 53. The parasite counts rarely exceeded
welcomed the opportunity to get it to us. Once 50 per mm3. The maximum count was 200 per
the monkey, a spider (A. paniscus) (originally mm3. The duration of parasitemia did not exceed
reported incorrectly as A. geoffroyi) was 27 days. The clinical manifestations were
ensconced in the laboratory in Chamblee, generally milder than seen in P. cynomolgi or P.
Georgia, A. freeborni mosquitoes were allowed knowlesi infections; symptoms consisted mainly
to feed on it; they became infected and were of headache and loss of appetite. Fevers were
allowed to bite 9 volunteers. Five of the nine present, the maximum being 103.8° F. The
became infected (3 Caucasians and 2 Negroes) quartan fever pattern was hardly the rule, but
with prepatent periods of 29 to 64 days appeared more consistently in the sporozoite
(Contacos et al, 1963). Later, two additional induced infections than in those induced by
men were infected via mosquito bites with this blood inoculation. It did not appear, in this small
same isolate of P. brasilianum. In another number of cases, that the parasitologic or
experiment, A. freeborni mosquitoes were also clinical picture was enhanced by blood passage

Figure 53.—Parasite counts and median parasitemia curve of 25 infections of Plasmodium brasilianum in man (7 sporozoite-
and 18 blood-induced).
242 PRIMATE MALARIAS

from volunteer to volunteer. When the parasite TABLE 30.—Natural infections of Plasmodium brasilianum
reported in Neotropical Primates from Panama,
was blood-passaged back to the monkey, typical Colombia, Venezuela, Peru, and Brazil.
infections resulted.
This was the second simian malaria, in HOST SPECIES REFERENCE
Alouatta fusca Deane et al, 1969
point of time, experimentally transmitted to man
by mosquito bite and the ease with which human Alouatta palliata Clark, 1931
infections were obtained points up its zoonotic Alouatta seniculus straminea Serrano, 1967
and/or anthroponotic potential when man Deane et al, 1969

introduces himself into a simian environment. Alouatta villosa Porter et al, 1966
Galindo*
Also, there is another point which bears
discussion and that is the true identity of the Ateles fusciceps Clark,1931
Porter et al, 1966
brasilianum parasite. In the preceeding sections
we have called attention to the close parallelism Ateles geoffroyi Clark, 1931
Porter et al, 1966
between P. malariae and P. brasilianum to the Marinkelle and Grose, 1968
point where only an expert might presume to tell
Ateles g. geoffroyi Galindo*
the difference either in the blood, fixed tissue, or
Ateles g. grisestens Galindo*
the sporogonic cycle. This being the case, one
wonders if P. brasilianum isn't actually a strain Ateles panistus (includes “A Dunn and Lambrecht, 1963
variegates”)
of P. malariae which became adapted to New
World monkeys sometime after the early sixteen Ateles p. paniscus Deane et al, 1969
hundreds. Ateles p. chamek Deane et al, 1969

Brathyteles arachnoides Deane et al, 1969

Host Specificity Callicebus moloch ornatus Renjifo and Peidrahita, 1949**
As mentioned earlier, P. brasilianum has
Callicebus torquatus Deane et al, 1969
been found in Panama, Venezuela, Colombia,
Peru, and Brazil. A list of the animals found Cebus albifrons Dunn and Lambrecht, 1963
Marinkelle and Grose, 1968
infected in nature through 1969, is given in
Cebus apella (probable Dunn and Lambrecht, 1963
Table 30. infection)
Although marmosets have not been found
Cebus apella Marinkelle and Grose, 1968
infected in nature Deane and his coworkers Deane et al, 1969
(1969) were able to infect Callithrix jacchus by
Cebus tapucinus Porter et al, 1966,
transfer of parasitized blood from Ateles Marinkelle and Grose, 1968
paniscus. They also transferred the infection
Cebus c. tapucinus Clark, 1931
from A. fusca to Lagothrix lagotricha and to
Cebus c. imitator Clark, 1931
Saimiri sciureus. The Taliaferros (1934) Galindo*
transferred the infection from Aotus zonalis (=
Chiropotes chiropotes Deane et al, 1969
trivirgatus), the night monkey, to Leontocebus
(= Saguinus) geoffroyi, a marmoset, as well as Lagothrix cana Deane et at, 1969
from Ateles darisensis (= paniscus) , the black Lagothrix infumata Dunn and Lambrecht, 1963
spider monkey, to Alouatta p. palliata (=
Lagothrix lagotricha Deane et al, 1969
villosa), the mantled howler . Garnham et al, 1963
Marinkelle and Grose, 1968
Clark and Dunn (1931) showed that
Anopheles tarsimaculatus (= aguasalis) and, A. Saimiri boliviense Dunn and Lambrecht, 1963
albimanus were susceptible to infection with P. Saimiri sciureus Renjifo et al, 1952
brasilianum. Garnham et al (1963) were able to Garnham et al, 1963
Dunn and Lambrecht, 1963
infect A. aztecus and A. atroparvus. In our Roca-Garcia (not published)
studies, A. freeborni, A. stephensi, A. sundaicus, Marinkelle and Grose, 1968
Deane et al, 1969
A. quadrimaculatus, A. b. balabacensis, and A. Groot†
maculatus have been found infectible. The * According to Dunn and Lambrecht, 1963
** According to Marinkelle and Grose, 1968
relative susceptibility to infection varied from † According to Garnham, 1966
 PLASMODIUM BRASILIANUM 243

one species to another (Table 31). By far, the reproduction and, 2) acquired immunity in
most susceptible species was A. freeborni. No conjunction with natural immunity which results
infections were obtained with A. albimanus. in a heightened death rate of the parasites. The
marked lowering of the asexual reproduction is
Immunity and Antigenic due to a derangement of the asexual
reproduction during crisis, a lengthening of the
Relationships cycle in some parasites, and a decrease in the
number of merozoites in the segmenters.
Taliaferro and Taliaferro (1934a) found that Defense against P. brasilianum thus involves a
immunity to superinfection was demonstrated by suppression of the infection, a replacement of
a rapid decrease and disappearance of parasites the erythrocytes lost by schizogony and
from the blood of monkeys whose infection was erythrophagocytosis. Acquired immunity takes
latent, when injected intravenously with large time to develop, but once acquired, responds
numbers of the same strain or combination of immediately (Taliaferro and Cannon, 1936).
strains. This immunity was effective After superinfection with large numbers of
immediately after the initial infection had abated parasites, active concentration and phagocytosis
and lasted for more than a year. They also of the parasites begin within one hour instead of
showed, that there was no significant passive waiting for from one to several weeks.
immunity, i.e., that serum from monkeys with Antisera to P. brasilianum gave only a low
latent infections with P. brasilianum, was level fluorescent antibody cross-reaction to P.
without protective action when injected into fieldi antigen (mean reciprocal titer ratio of
uninfected monkeys, subsequently exposed to 100:24) and much lower levels to other primate
infection. malaria antigens (Collins et al, 1966a). In the
Taliaferro and Cannon (1936) stated that reverse procedure, P. brasilianum antigen
immunity to P. brasilianum, whether as a reacted highest to P. inui antisera and at a lower
manifestation of recovery from initial infection level to P. cynomolgi and P. knowlesi (mean
or as immunity to superinfection, depended reciprocal titer ratios of 100:57, 100:31, and
primarily upon the greatly increased rate of 100:24, respectively). In many cases, antisera of
phagocytosis. The process of phagocytosis is P. malariae reacted with the P. brasilianum
accomplished by the macrophages of the spleen, antigen at a higher level than to the homologous
and in a descending extent, by those in the liver antigen (Collins et al, 1966); the reaction to
and bone marrow. The Taliaferros (1944) antisera of P. falciparum was at a much lower
interpreted the host's response in terms of level to the P. brasilianum than to the
immunity as 1) natural immunity associated with homologous antigen.
marked death of the parasites which may be
associated with a sporadic variation in asexual
TABLE 31.—Comparative infectivity of Plasmodium brasilianum to seven species of Anopheles.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

F-1 100
F-1 : St-1 14 110 148 60.9 16.2 12.9
F-1 : Sund 3 32 44 71.9 9.1 6.0
F-1 : Q-1 18 237 274 51.9 22.6 4.2
F-1 : Bal 14 263 201 51.0 6.0 3.2
F-1 : Mac 25 221 342 61.1 7.0 2.1
F-1 : Alb 10 182 192 50.0 0.0 0.0

* F-1 = Anopheles freeborni, St-1 = A. stephensi, Sund = A. sundaicus, Q-1 = A. quadrimaculatus, Bal = A. b. balabacensis, Mac = A.
maculatus, Alb = A. albimanus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. freeborni to another
species where the GII of A. freeborni = 100.
244 PRIMATE MALARIAS

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BERENBERG-GOSSLER, VON H. V., 1909. Beitrage zur LUPASCU, G., CONSTANTINESCU, P., NEGULICI, E.,
naturgeschichte der malariaplasmodien. Arch. f. Protist. GARNHAM, P. C. C., BRAY, R. S., KILLICK-
16 : 245-280. KENDRICK, R., SHUTE, P. G. and MARYON, M.,
BRAY, R. S., 1957. Studies on malaria in chimpanzees. IV 1967. The late primary exoerythrocytic stages of
Plasmodium ovale. Am. J. Trop. Med. & Hyg. 6 : 638- Plasmodium malariae. Trans. Roy. Soc. Trop. Med. &
645. Hyg. 61 : 482-489.
CLARK, H. C., 1930. A preliminary report on some parasites in MARINKELLE, C. J. and GROSE, E. S., 1968. Plasmodium
the blood of wild monkeys of Panama. Am. J. Trop. brasilianum in Colombian monkeys. Trop. geogr .Med.
Med. 10: 25-31. 20 : 276-280.
CLARK, H. C., 1931. Progress in the survey for blood parasites of PORTER, J. A., JR., JOHNSON, C. M., and DE SOUSA, L.,
the wild monkeys of Panama. Am. J. Trop. Med. 11 : 1966. Prevalence of malaria in Panamanian primates. J.
11-20. Parasit. 52 : 669-670.
CLARK, H. C., and DUNN, L. H., 1931. Experimental efforts to RENJIFO, S., SANMARTIN, C., and DE ZULUETA, J ., 1952. A
transfer monkey malaria to man. Am. J. Trop. Med. 11 : survey of the blood parasites of vertebrates in Eastern
1-10. Colombia. Acta Tropica 9 : 151-169.
CLARK, H. C., and DUNN, L. H., 1931a. Experimental efforts to SEIDELIN, H., 1912. Notes on some blood-parasites in man and
transfer monkey malaria to man. Surgery, Gyn. & mammals. Ann. Trop. Med. & Parasit. V : 501-508.
Obst., 52: 428-429. SERRANO, J. A., 1967. Infeccion natural de un araguato, Alouatta
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, seniculus straminea, por Plasmodium brasilianum en
J. R., 1969. Infectivity of Plasmodium brasilianum for Venezuela. Acta Cientif. Venezolana 18 : 13-15.
six species of Anopheles. J. Parasit. 55 : 685-686. SODEMAN, T. M., HELD, J. R., CONTACOS, P. G., JUMPER,
COLLINS, W. E., JEFFERY, G. M., GUINN, E., and SKINNER, J. R., and SMITH, C. S., 1969. Studies of the
.J. C., 1966. Fluorescent antibody studies in human exoerythrocytic stages of simian malaria. IV.
malaria. IV. Cross-reactions between human and simian Plasmodium brasilianum. J. Parasit. 55 : 963-970.
malaria. Am. J. Trop. Med. & Hyg. 15 : 11-15. TALIAFERRO, W. H. and TALIAFERRO, L. G., 1934.
COLLINS, W. E., SKINNER, J. C., and GUINN, E. G., 1966a. Morphology, periodicity and course of infection of
Antigenic variations in the plasmodia of lower primates Plasmodium brasilianum in Panamanian monkeys. Am.
as detected by immuno-fluorescence. Am. J. Trop. J. Hyg. 20 : 1-49.
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KILPATRICK, .J. W., and JONES, F. E., 1963. brasilianum in monkeys by reversal of light and dark.
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 20
Plasmodium inui Halberstaedter and von Prowazek, 1907
probability, the above authors, especially Leger
AT the same time that and Bouilliez (1913), were dealing with a mixed
Halberstaedter and von Prowazek were studying infection because they demonstrated a 48-hour
the parasite of the orangutan, they came across cycle, which, of course, could not have been P.
parasites of malaria in Macacus cynomolgus (= inui.
Macaca fascicularis) from Java and in M. Noguchi (1928) reported finding P. inui in
nemestrina from Sumatra and Borneo. Because an M. cynomolgus (= M. fascicularis) which he
the new parasite was different from the one in had splenectomized in connection with work on
the orangutan, they wrote a brief description of Bartonella. Green (1932) working in Malaya
it, accompanied by illustrations. They named it saw P. inui in a pig-tailed macaque, M.
Plasmodium inui, taking the species name from nemestrina.
the old generic name of the host, Inuus. Sinton and Mulligan (1933) discussed the
It is not unlikely that Mathis and Leger ‘P. inui group' of monkey parasites and the
(1911) saw the same parasite in some macaques following year Sinton (1934) redescribed the
from Indochina listed as M. mulatta and M. parasite in a paper which was, as Eyles wrote in
lasiotis tcheliensis. Bray (1963) believed the 1963, "so meticulously prepared that it should
hosts were actually M. assamensis. serve as a model for those of us continuing to
Leger and Bouilliez (1912) found a work on monkey malaria."
plasmodium in M. cynomolgus (= M. It appears that after the early thirties natural
fascicularis) which they described briefly. infections with P. inui were seen only rarely
Bouilliez later (1913) published a short paper on (Singh et al, 1951; and Sezen, 1956) until the
the same material. In 1913, Leger and Bouilliez Eyles group went to Malaya in 1960. Their
gave a detailed description of the parasite and studies and those of other investigators will be
identified it as P. inui Halberstaedter and von dealt with later.
Prowazek. They believed it was the same
parasite as P. cynomolgi Mayer, 1907. In all

245
 PLASMODIUM INUI 247

Cycle in the Blood abundant than in the asexual forms, and they are
without a vacuole.
PLATE XXXIX The cytoplasm of the adult
The earliest ring forms are 2 to 3 µ in
macrogametocyte stains a delicate steel blue
diameter, usually with a single, fairly large,
with scattered pigment, some of which appears
nucleus. Less commonly, the nucleus may be
to be embedded. The nucleus is eccentric and
double. As the trophozoite grows, the rings
appears light red with a darker stained irregular
increase in size exhibiting a large vacuole and
area and a small vacuole. The parasite fills the
limited cytoplasm (Fig. 4). As growth continues,
host cell which is slightly enlarged (Fig. 24).
the parasite becomes amoeboid, stippling
The microgametocyte is distinctive for the way
appears, and some pigment is evident (Figs. 6-
it takes the stain. The cytoplasm is reddish-
8). Band forms, reminiscent of P. malariae, are
purple overlaid with scattered brown pigment
not uncommon (Fig. 9). The host cell may
granules. The off-center nucleus may occupy a
become somewhat enlarged. As growth
third or more of the parasite. The prominent
proceeds, the trophozoite becomes large,
reddish-purple area shows a net of thin threads
occupying about half the host cell, the nucleus
which surrounds a deeper staining bar-shaped
may be pleomorphic, the pigment becomes more
area. The parasite fills the host cell (Fig. 25).
prominent, but is still delicate, and the large
The asexual cycle in the blood occupies 72
vacuole disappears. Stippling, much like
hours.
Ziemann's stippling in P. malariae, is evident
and with some Giemsas, prominent (Figs. 13-
15). Sporogonic Cycle
Young schizonts appear after 48 hours, and PLATE XL
by the time 4 to 8 nuclei are evident, the parasite According to Bano (1959), the penetration
may almost fill the erythrocyte, or it may appear of the ookinetes of P. inui into the gut of A.
contracted with an irregular periphery. In the aztecus mosquitoes was very slow at an
early stages, the nuclei may appear as if on the incubation temperature of 28° C. The earliest
surface and in the company of a vacuole. Their recognizable stage in many 70-hour oocysts,
staining progresses from reddish-black to measuring 10 µ in diameter, was a distinct
purplish-black. Sometimes a red-staining mass uninucleate condition suggesting a resting stage.
(Fig. 20) is evident which disappears before the Binucleate oocysts were observed at 72 to 79
mature schizont is formed. The pigment has a hours; their size varied from 10.5 to 11 µ in
greenish cast, stippling is evident but diminishes diameter. At 81 to 83 hours, the oocysts
as development proceeds (Figs. 16-20). measured 12 to 12.6 µ. A multinucleate
The mature schizonts may be asymmetrical condition was observed in the 96 to 106-hour
or fill the host cell. The latter, carrying the fully oocysts which varied from 13.3 to 15.5 µ in
mature schizont, appears depleted. The pigment diameter.
is coalesced and appears as a yellowish-black Garnham (1966) reported that oocysts in
mass. The merozoites stain purple and number mosquitoes incubated at 25° C. were only 8 µ in
up to 18; 12 is the usual number (Figs. 21-23). diameter on the 5th and 6th day after feeding.
The young gametocytes are compact, have
little, if any, amoeboidity, their pigment is more

PLATE XXXIX.—Plasmodium inui.

Fig. 1. Normal erythrocyte. Figs. 16-19. Developing schizonts.
Figs. 2-4. Young trophozoites. Figs. 20-23. Almost mature and mature schizonts.
Figs. 5-10. Growing trophozoites. Fig. 24. Mature macrogametocyte.
Figs. 11-15. Older and mature trophozoites. Fig. 25. Mature microgametocyte.
248 PRIMATE MALARIAS
 PLASMODIUM INUI 249

On the 7th day, the oocysts measured 13 µ and In our studies (Table 32), the oocyst growth
the pigment was concentrated in crossed lines at rate was observed in four species of mosquitoes
the center. On the 14th day, the oocysts incubated at 25° C. In. A. b. balabacensis, after
measured 22 µ but maturity was not attained 6 days of extrinsic incubation, the oocyst
until the 21st day. At 28° C, the oocysts grew diameters ranged from 9 to 17 µ with a mean of
more quickly, and after 12 days, reached 40 µ 13 µ. The oocysts grew slowly, so that by day
and after 14 days, a size of 50 µ. Just before the 14, the mean size was 50 µ with a range of 15 to
final differentiation, Garnham noted a number of 73 µ. At this time, differentiation within the
darkly staining spheres, 7 µ in diameter. These oocyst was apparent. On the l5th day,
seemed to give rise to vesicles, 10 µ in diameter, sporozoites were present in the salivary glands.
in which sporozoites were discerned. Malaria In A. freeborni and A. maculatus
pigment was usually visible throughout the mosquitoes, the mean oocyst diameters were
growth of the oocyst. noticeably smaller than in the A. b.
balabacensis. This was probably due to the

TABLE 32.—Oocyst diameters of Plasmodium inui in Anopheles b. balabacensis, A. freeborni, A. maculatus, and
A. quadrimaculatus.

Days after A. b. balabacensis A. freeborni A. maculatus A. quadrimaculatus

Infection
No. Range* Mean No. Range Mean No. Range Mean No. Range Mean

6 100 9-17 13 114 7-14 10 23 9-13 12

7 131 8-21 15 216 8-18 13 267 8-18 13 211 8-20 14
8 150 12-27 21 308 9-27 16 293 8-20 15 215 8-21 16
9 100 15-34 27 164 8-30 15 315 9-30 19 203 12-30 21
10 100 21-38 32 163 9-28 17 193 9-30 19 200 12-32 22
11 148 15-51 36 229 11-40 24 207 11-45 21 123 12-45 27
12 150 17-44 31 245 9-50 29 199 9-50 25 223 12-55 37
13 50 22-50 38 175 9-68 30 226 11-55 26† 244 14-71 40†
14 150 15-73 50† 132 11-68 42† 366 12-63 35† 291 15-68 48†
15 50 22-53 37†** 88 11-53 37† 359 13-78 41† 225 21-77 52†
16 132 13-65 34†** 130 15-79 49†

Totals 1129 8-73 1836 7-68 2580 8-78 2065 8-79

* Measurements expressed in microns; incubation temperature 25º C

† Oocyst differentiation.
** Sporozoites present in the salivary glands.

Plate XL.—Developing oocysts and sporozoites of Plasmodium inui (Mulligan strain) in Anopheles b. balabacensis mosquitoes.
X 580 (Except Figures 1 and 2).
Fig. 1. 6-day oocysts showing a few pigment granules. Fig. 10. 14-day oocyst.
near periphery. X 740. Fig. 11. 15-day oocyst.
Fig. 2. 7-day oocysts. X 740. Fig. 12. 15-day oocyst.
Fig. 3. 8-day oocyst showing clumping of pigment. Fig. 13. 16-day oocyst showing early signs of
Fig. 4. 9-day oocysts showing clumped pigment. differentiation.
Fig. 5 10-day oocyst. Fig. 14. 16-day differentiating oocyst.
Fig. 6. 11-day oocysts showing pigment and small Fig. 15. 17-day oocysts.
vacuoles. Fig. 16. 17-day oocyst.
Fig. 7. 12-day oocysts showing one of normal size and Fig. 17. 17-day oocyst showing sporoblast formation.
one much smaller. Fig. 18. Sporozoites emerging from salivary gland tissue
Fig. 8. 12-day oocyst. 17 days after feeding.
Fig. 9. 13-day oocysts.
250 PRIMATE MALARIAS

presence of a large number of small oocysts in Garnham (1966) reported that sporozoites
each of these two species. Oocyst differentiation may be found in the salivary glands 15 to 17
was apparent in the A. freeborni mosquitoes by days after feeding when the mosquitoes are
day 14. However, sporozoites did not appear in incubated at 28° C. At a temperature of 25° C,
the salivary glands until 18 or 19 days after the development requires 3 weeks. His
feeding, and then, at a low level. In A. experimental mosquito hosts were not ideal (A.
maculatus, oocyst differentiation was apparent aztecus and A. atroparvus) and sporozoites
by day 13 and sporozoites were present in the disappeared or became scarce in the salivary
salivary glands after 16 days of extrinsic glands in a few days. In dried preparations, the
incubation. Fewer of the A. maculatus (99 out of sporozoites measured 12 to 13 µ in length.
313) had infected salivary glands than did the A. Transmission of P. inui was first obtained
b. balabacensis (95 out of 144) after 15 days of by Garnham (1951) through the bites of infected
incubation. The intensity of gland infections was A. atroparvus mosquitoes coupled with the
high in both species. intravenous inoculation of infected salivary
In A. quadrimaculatus, the oocyst growth glands. Mohiuddin (1957) passaged the parasite
rate was similar to that in A. b balabacensis. to M. mulatta by the inoculation of dissected
Differentiation was apparent after 13 days, but glands from infected A. aztecus. He also allowed
no positive salivary glands were found until day infected A. atroparvus and A. aztecus
20, and then, at a very low level. mosquitoes to feed on a monkey which later
A comparison of the oocyst growth rate of developed a patent infection. It is impossible, in
P. inui with that of P. cynomolgi in A. b. this instance, to say which of the species
balabacensis mosquitoes (Fig. 54) shows that transmitted the infection, maybe both. Eyles
the P. inui are smaller and that sporozoites (1960) obtained infections by the intravenous
appear in the salivary glands 5 days later than in inoculation of salivary glands from infected A.
P. cynomolgi. quadrimaculatus. Shortt et al (1963) transferred

FIGURE 54.—Median oocyst diameter curves and ranges in oocyst diameters of Plasmodium cynomolgi and Plasmodium inui in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
 PLASMODIUM INUI 251

the parasite by the intravenous inoculation of Held et al (1968) were well aware of the
salivary glands containing sporozoites from A. uncertainty regarding the taxonomy of certain
aztecus and from A. stephensi. quartan parasites of simians and elected to study
In our studies (Collins et al, 1966, 1968), the exoerythrocytic stages of the parasite in 4
we obtained transmission of 12 different isolates different strains: 1) The L strain isolated from an
of P. inui by the bites of A. b. balabacensis, A. A. leucosphyrus mosquito by our unit in Kuala
maculatus, or A. stephensi mosquitoes. Among a Lumpur, Malaysia. Its blood stages are
total of 18 transmissions, the prepatent periods indistinguishable from the original strain
ranged from 10 to 26 days with a mean of 16.4 isolated by Sinton; 2) The OS strain, employed
days. Twelve additional passages have been by Coatney et al (1966) in their successful
obtained by the intravenous and/or intrahepatic attempt to infect man. The origin and the
inoculation of sporozoites from A. freeborni designation of the OS strain are described in the
(twice), A. quadrimaculatus (twice), and A. section dealing with Course of Infection; 3) The
maculatus mosquitoes (8 times). The prepatent CDC strain, isolated from a long-tailed
periods ranged from 11 to 31 days with a mean macaque, M. fascicularis, received directly from
of 17 days. the Philippines, and indistinguishable from the
Transmission of the infection to man original Sinton-isolated strain; 4) The Philippine
(Coatney et al, 1966) was made via the bites of strain, isolated by Lambrecht et al (1961) and
A. maculatus and A. stephensi mosquitoes. The designated the Cebu strain.
prepatent periods in the volunteers were 31 and Infection of clean rhesus monkeys was by
56 days. intrahepatic inoculation of sporozoites and the
site of the inoculation was marked by a stainless
Cycle in the Tissue steel ligature. To make sure of the age of the EE
bodies found subsequently, each animal received
PLATE XLI infective material on a single day. EE bodies of
Garnham (1951) was the first to see EE
the L strain were found in 6-, 7-, 8-, and 9-day
stages of P. inui which he described and
biopsy material; in OS strain material at 6, 7, &
presented in a series of 15 figures. In this work,
8 days; in the CDC strain material, at 7 days;
he gave a single rhesus monkey sporozoites
and in Philippine strain material at 10 days,
from Anopheles m. atroparvus mosquitoes
following exposure.
infected with the Mulligan strain on 2
In each of the four strains, the EE, bodies
consecutive days. Liver biopsies were taken on
were round or slightly elliptical. The edges were
days 8 and 12, after the first exposure to
smooth. The nuclei stained magenta, were
infection, and in that material he found EE
generally round, although other shapes were
stages of variable size. Garnham assumed that
seen, and measured 0.5 to 1.0 µ in diameter. The
the smaller forms in each of the biopsies,
cytoplasm stained a pale blue. Dark blue
resulted from sporozoites injected on the second
cytoplasmic aggregates up to 6 µ in diameter
day and the larger forms from those of the first
were seen in some older forms. Some EE bodies
day. On that basis, he described 7-, 8-, 11-, and
took a deeper stain than others giving the
12-day forms. It is not impossible that the same
impression of compactness. Small pink staining
variation in size might have resulted had the
vacuoles, less than 3 µ in diameter, were seen in
animal been exposed on only a single day. In
a few sections. Some sections exhibited a
1966, Garnham described 9- and 10-day forms
separation of 1 to 5 µ between the parasite and
of the parasite but gave no details regarding the
the host tissue. Each of the EE bodies was
strain of P. inui employed or the method used in
measured and the results compared with the
obtaining the material for the descriptions.
measurements given by Garnham (1951, 1966)
Shortt et al (1963) described 8- and 9-day
and by Shortt et al (1963).
EE forms of P. osmaniae, later named P. shortti
The EE bodies studied by Held et al were
Bray, 1963; we consider the parasite to be P.
considerably larger than corresponding ones
inui for reasons explained later.
described by Garnham and by Shortt et al. The
latter authors (1963) in their description of the
252 PRIMATE MALARIAS

PLATE XLI—Exoerythrocytic bodies of Plasmodium inui in liver tissue of Macaca mulatta. X 580 (Except Figs. 2 and 8). Figures
1, 2, 4, 6, 7, and 8 are examples of the Leucophyrus strain; Figures 3 and 5 of the OS strain; Figure 9 of the Philippine strain.
Fig. 1. 6-day body. Fig. 6. 8-day body showing individual nuclei.
Fig. 2. 6-day body. X 1450. Fig. 7. 9-day body showing clumped flocculi.
Fig. 3. 7-day body. Fig. 8. 9-day body showing flocculi and host cell
Fig. 4. 7-day body. nucleus. X 742.
Fig. 5. 8-day body showing prominent flocculi. Fig. 9. 10-day body.
 PLASMODIUM INUI 253

EE bodies of P. osmaniae stressed their size, will show (Fig. 55) a peak parasitemia of
they were larger than P. inui, as an argument for approximately 35,000 per mm3 on the 14th day
species status and Garnham (1967) supported of patent parasitemia. The parasitemia then
that view. If that criterion were accepted, then dropped and fluctuated between 4,000 and 6,000
each of the strains studied by the Held group per mm3 for the remainder of the 60-day
would represent separate species. We hold that observation period. Such levels were commonly
this is untenable, for the present at least, because maintained for months and even years. In some
all other essential characters of these strains are individual monkeys, parasite levels of 100,000
so similar. And, too, observations on a few EE per mm3 or greater were maintained for many
bodies which lack consistent specific months. Sporozoite induced infections (20
morphologic differences, with the possible monkeys) rose to a peak of approximately
exception of size at a given time after infection, 15,000 per mm3 by day 14 and thereafter
can hardly supplant the results of prolonged dropped to a level of approximately 2,500 per
study of the blood stages by competent mm3 after 60 days. In splenectomized monkeys
investigators over many years. (27 animals), the peak parasitemia of
approximately 500,000 per mm3 was obtained
Course of Infection after 16 days. This dropped rapidly to a level of
70,000 per mm3 after 30 days of patent
Although P. inui infects many species of parasitemia. Some of the animals continued to
monkeys, most of the studies have been con- maintain a high level of parasitemia for many
fined to the rhesus monkey, M. mulatta. Eyles months. Garnham (1966) reports that M. mulatta
(1963) stated that infections produced in this monkeys which have severe infections are apt to
animal were moderate when compared to P. develop a sterilizing immunity and throw off the
cynomolgi and remarkably long lasting. In our infection entirely. He also reported a similar
own experience, many animals have maintained occurrence of splenectomized monkeys. We
infections for many years. have not had this experience. Some of our
A perusal of the median parasitemia curve animals have maintained fairly high parasitemias
for 66 intact M. mulatta monkeys infected with for 3 to 4 years after splenectomy.
P. inui by the inoculation of parasitized blood There is no evidence that P. inui possesses

FIGURE 55.—Median parasitemia curves for 113 infections of Plasmodium inui in Macaca mulatta monkeys (66 blood-induced
and 20 sporozoite-induced infections in intact animals, and 27 blood-induced infections in splenectomized animals).
254 PRIMATE MALARIAS

the mechanism for true relapse. Several of our received bites from 19 A. maculatus. Infection of
monkeys, infected by sporozoite inoculation, the mosquitoes was proved by postprandial
had their blood infection cured by the ad- dissection. Each man became infected. The
ministration of schizontocidal drugs. They failed prepatent periods were 31 and 56 days; and
to develop secondary infections during a 6- parasitemia continued for 21 days in one and for
month observation period. Later, they were 24 days in the other. The maximum parasite
shown to be susceptible to reinfection when count was 2,520 per mm3 of blood. Each of the 5
inoculated with parasitized blood of the same volunteers who received parasitized blood
strain. developed infections which were patent for 10 to
Infections in the M. fascicularis and M. 26 days with a maximum parasite count of 450
radiata monkeys are usually less severe than per mm3 (Fig. 56).
those in the rhesus monkey (Garnham, 1966). The quartan pattern was well marked in two
Plasmodium inui is infectious to man as of the volunteers with maximum fever of 103.2°
shown by Das Gupta (1938). The volunteer was F. The others exhibited low-grade, remittent-
given blood directly from an infected S. irus (= type fever or, in the case of the Negro patient, no
M. fascicularis) monkey by intramuscular fever at all. No morphological changes were
injection. Twenty-three days later the patient observed in the parasites as a result of their
developed a patent parasitemia and febrile sojourn in man. Blood from 4 of the volunteers
symptoms on the 28th day, which continued for was inoculated into clean rhesus monkeys which
a total of 3 days. The maximum temperature produced typical P. inui infections.
observed was 102° F. The strain of P. inui The major complaints voiced by the
employed by Das Gupta was obtained directly volunteers were headache, malaise, muscular
from Col. Sinton which leaves little doubt as to and joint pains, and loss of appetite. When chills
the identity of the parasite. occurred, they were mild and of short duration.
Our own studies in man (Coatney et al, In no case was anti-malarial therapy necessary.
1966) were carried out with the OS strain of P. Some interesting biological facts emerged
inui. The name originally suggested for the as a result of this study. Blood was drawn from
parasite (Shortt et al, 1961) was P. osmaniae. one volunteer on day 16, following his exposure
Because that name was only a suggested one, to infection by mosquito bite, and given to
Bray (1963) proposed the name P. shortti. Eyles another, who developed a patent infection 47
obtained the parasite through the courtesy of days later. At the time blood was drawn, the
Professor Garnham and after carefully donor had mild symptomatic complaints
comparing it with the original strain of P. inui although parasites could not be demonstrated in
was unable, on morphological grounds, to his blood until day 56. The fact that the recipient
separate it from the original strain. Nevertheless, became infected shows that tissue schizogony
he elected to consider it a subspecies, P. inui had taken place by day 16 and, further, that only
shortti. The parasite was sent to us from our a small number of parasites is needed to
installation in Kuala Lumpur, Malaysia, and establish a patent infection in man.
after careful study, we agreed with Eyles that it It is well known that Negroes are
was an inui-type parasite; until further studies universally susceptible to P. malariae, the
convince us of its taxonomic status, we prefer to human quartan parasite, and we had shown that
designate the osmaniae-shortti parasite as the OS the quartan parasite of New World monkeys, P.
strain of P. inui. brasilianum (see Chapter 19) and P. inui of Old
Two Caucasian male volunteers were World monkeys are infective to Negroes. In
exposed to the bites of infected mosquitoes and contrast to these successes, we have not been
5 other volunteers, including one Negro, able to infect Negroes with tertian parasites, P.
received their infections by the intravenous cynomolgi (see Chapter 6) and P. schwetzi (see
inoculation of parasitized blood from one of the Chapter 12). Likewise, it is sometimes difficult
previously infected volunteers. to infect Negroes with the human tertian
One volunteer was bitten by 5 Anopheles parasite, P. vivax (see Chapter 5). So far we
maculatus and by 7 A. stephensi. The other, cannot account for this resistance phenomenon.
 PLASMODIUM INUI 255

FIGURE 56.—Parasitemia and median parasitemia curve for 7 infections of Plasmodium inui (OS strain) in human volunteers.

It probably should be mentioned, too, that Host Specificity

prior to, and subsequent to, our successful
transmission of this strain of P. inui to man, by Plasmodium inui naturally infects many monkeys as
mosquito bite, we had made several attempts, listed below:
using other strains, each time without success. SPECIES REFERENCES
To some this might indicate that true P. inui Cercopithecus mitis Garnham, 1966
would not grow in man. Since the osmaniae- (Experimentally)
shortti parasite did infect man, some might Cynopithecus niger Eyles and Warren, 1962
consider that reason enough for accepting it as Macaca cyclopis Hsieh, 1960
an entity outside the inui group. However, as M. fascicularis Many references
mentioned earlier, Das Gupta was able to infect M. mulatta Schmidt et al, 1961;
man with the original strain of P. inui following Notananda et al, 1961
inoculation of parasitized blood. Why our earlier M. nemestrina Halberstaedter and yon
attempts failed and this one succeeded, we are Prowazek, 1907
not prepared to say. M. radiate Mulligan and Swaminath,
1940
Presbytis cristatus Collins et al, 1970
P. obscurus Eyles et al, 1962
256 PRIMATE MALARIAS

Plasmodium inui has been isolated from or P. shortti (= P. inui) developed low
Anopheles leucosphyrus (Wharton et al, 1962) parasitemias of P. inui upon challenge. Voller
and A. b. introlatus (Warren and Wharton, and Rossan (1969) confirmed this observation
1963), in Malaya. Experimentally, P. inui is by finding that in monkeys with chronic
infectious to a large number of mosquitoes as infections of P. knowlesi, infections with P. inui
listed below: developed more slowly and had lower peak
SPECIES REFERENCES parasitemias than did animals infected with P.
Anopheles albimanus Eyles, 1960a inui alone.
A. atroparvus Weyer, 1937; Garnham, Antisera to P. inui gave a fluorescent anti-
1951; Mohiuddin, 1957; body cross-reaction at a very high level to P.
Shortt et al, 1963 fieldi and P. shortti (= OS strain P. inui) (mean
reciprocal titer ratios of 100:107 and 100:88)
A. aztecus Mohiuddin, 1957; Bano,
1959; Shortt et al, 1963;
and reacted at somewhat lower levels to P.
Garnham, 1966 brasilianum, P. coatneyi, and P. cynomolgi
(mean reciprocal titer ratios of 100:57, 100:57,
A. b. balabacensis Collins et al, 1968 and 100:46, respectively). In the reverse
procedure, P. inui antigen cross-reacted to none
A. freeborni Eyles, 1960
of the heterologous antisera at a high level, the
A. gambiae Garnham, 1951 highest response was to the OS strain with a
mean reciprocal titer ratio of 100:35 (Collins et
A. letifer Warren and Wharton, 1963 al, 1966). Antigen to the OS strain gave
A. maculatus Warren and Wharton,
fluorescent antibody cross-reactions of a much
1963;Collins et al, 1966, lower magnitude with P. fieldi and P. inui giving
1968 the highest levels of response (mean reciprocal
titer ratios of 100:47 and 100:35, respectively).
A. philippinensis Warren and Wharton, 1963 In the reverse procedure, the OS strain antigen
A. quadrimaculatus Eyles, 1960 gave a high level cross-reaction to the P. inui
antigen only (mean reciprocal titer ratio of
A. stephensi Garnham, 1951; Shortt et al, 100:88),
1963; Collins et al, 1966, In a further study of antigenic relationships
1968
(Collins et al, 1970), antisera from monkeys
In addition to the above, we have obtained
each infected with one of 15 different isolates of
infections in A. kochi, A. vagus, A. sinensis, and
P. inui from different geographical and
A. riparis. Thirteen species of mosquitoes are
ecological areas of South Central and Southeast
listed (Table 33) for comparison as to their
Asia were allowed to react with their
susceptibility to infection with P. inui. The most
homologous and heterologous antigens using the
readily infected was A. b. balabacensis and the
fluorescent antibody technique. It was shown,
least was A. letifer.
using the test of relatedness statistic, that there
Immunity and Antigenic were significant antigenic differences between
Relationships each pair of isolates of P. inui included in the
study. The OS strain parasite was, on the
Singh and Singh (1940) observed that
average, more distantly related to the remainder
homologous superinfection with P. inui resulted
of the strains than was any other strain. The next
in a very mild and transient parasitemia. Chronic
most distantly related was a parasite from the
infections with P. inui afforded no protection
Celebes.
against either P. cynomolgi or P. knowlesi.
El-Nahal (1967) reported that antisera to P.
Monkeys with chronic infections of P.
inui and P. shortti (= OS strain P. inui) failed to
cynomolgi or P. knowlesi showed no resistance
respond to exoerythrocytic antigens of P.
to typical infections with P. inui.
cynomolgi and P. malariae using a fluorescent
In contrast, Voller et al. (1966) found that
antibody procedure.
monkeys immune to infection with P. knowlesi
 PLASMODIUM INUI 257

TABLE 33.—Comparative infectivity of Plasmodium inui to thirteen species of Anopheles.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other
Bal 100
Bal : F-1 63 492 629 83.9 65.5 59.9
Bal : Kochi 5 131 88 93.9 87.5 52.1
Bal : St-1 109 991 759 66.1 69.2 44.9
Bal : Mac 23 123 208 80.5 68.3 41.4
Bal : Q-1 83 1223 1176 47.9 38.6 17.8
Bal : Vagus 6 74 29 83.8 62.1 9.6
Bal : Sin 2 31 17 83.9 17.6 9.2
Bal : Atro 29 583 570 34.6 15.3 7.1
Bal : Phil 1 16 2 87.5 100 4.2
Bal : Rip 2 61 3 88.5 100 3.8
Bal : Alb 46 696 835 39.4 5.1 1.5
Bal : Let 5 66 50 80.3 10.0 1.3

* Bal = Anopheles b. balabacensis; F-1 = A.freeborni; Kochi = A. kochi; St-1 = A. stephensi; Mac = A. maculatus; Q-1 = A. quadrimaculatus;
Vagus = A. vagus; Sin = A. sinensis; Atro = A. atroparvus; Phil = A. philippinensis; Rip = A. riparis; Alb = A. albimanus; Let = A. letifer.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. b. balabacensis to
another species where the GII of A. b. balabacensis = 100.

REFERENCES
BANO, L., 1959. A cytological study of the early oocysts of seven EYLES, D. E., LAING, A. B. G., WARREN, McW. and
species of Plasmodium and the occurrence of SANDOSHAM, A. A., 1962. Malaria parasites of
postzygotic meiosis. Parasitology 49 : 559-585. Malayan leaf monkeys of the genus Presbytis. Med. J.
BOUILLIEZ, M., 1913. Nouvelles recherches experimentales sur Malaya 17 : 85-86.
un Plasmodium des singes. C. R. Soc. BioI. (Paris) 74: EYLES, D. E. and WARREN, McW., 1962. Plasmodium inui in
1070-1072. Sulawesi. J. Parasit. 48: 739.
BRAY, R. S., 1963. Malaria infections in primates and their EYLES, D. E., 1963. The species of simian malaria: taxonomy,
importance to man. Ergeb. Mikrob. Immunit. Experim. morphology, life cycle, and geographical distribution of
Therap. 36 : 168-213. the monkey species. J. Parasit. 49 : 866-887.
COATNEY, G. R., CHIN, W., CONTACOS, P. G., and KING, H. GARNHAM, P. C. C., 1951. The mosquito transmission of
K., 1966. Plasmodium inui, a quartan-type malaria Plasmodium inui Halberstaedter and von Prowazek, and
parasite of Old World monkeys transmissible to man. J. its pre-erythrocytic development in the liver of the
Parasit. 52 : 660-663. rhesus monkey. Trans. Roy. Soc. Trop. Med. & Hyg. 45
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, : 45-52.
J. R., 1966. Studies on the transmission of simian GARNHAM, P. C. C., 1966. Malaria parasites and other
malarias. I. Transmission of two strains of Plasmodium haemosporidia. Blackwell Scientific Publications.
inui by Anopheles maculatus and A. stephensi. J. Oxford. pp.1114.
Parasit. 52 : 664-668. GARNHAM, P. C. C., 1967. Abstract of a paper by Coatney et al,
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, 1969. Trop. Dis. Bull. 64 : 464.
J. R., 1968. Some observations on the transmission of GREEN, R., 1932. A malarial parasite of Malayan monkeys and its
Plasmodium inui. J. Parasit. 54 : 846-847. development in anopheline mosquitoes. Trans. Roy.
COLLINS, W. E., SKINNER, J. C., and GUINN, E. G., 1966. Soc. Trop. Med. & Hyg; 25 : 455-477.
Antigenic variations in the plasmodia of certain HALBERSTAEDTER, L. and VON PROWAZEK, S., 1907.
primates as detected by immuno-fluorescence. Am. J. Untersuchunger über die malariaparasiten der affen.
Trop. Med. & Hyg. 15 : 483-485. Arb. K. Gesundh.-Amte (Berl.) 26 : 37-43.
COLLINS, W. E., WARREN, McW., SKINNER, J. C., and HELD, J. R., CONTACOS, P. G., JUMPER, J. R., and SMITH, C.
ALLING, D. W ., 1970. Plasmodium inui: Serologic S., 1968. Studies of the exoerythrocytic stages of
relationships of Asian isolates. Exp. Parasit. 27 : 507- simian malaria. III. Plasmodium inui. J. Parasit. 54 :
515. 249-254.
DAS GUPTA, B. M., 1938. Transmission of P. inui to man. Proc. HSIEH, H. C., 1960. Malaria parasites of the Tiawan monkey.
Natl. Inst. Sci. India 4: 241-244. Formosan Science. 14: 477-487.
EL-NAHAL, H. M. S., 1967. Study of serological cross-reactions LAMBRECHT, F. L., DUNN, F. L., and EYLES, D. E., 1961.
of exo-erythrocytic schizonts of avian, rodent and Isolation of Plasmodium knowlesi from Philippine
primate malaria parasites by the fluorescent antibody macaques. Nature. 191 : 1117-1118.
technique. Bull. WId. Hlth. Org. 37 : 154-158. LEGER, M. and BOUILLIEZ, M., 1912. Sur un Plasmodium des
EYLES, D. E., 1960. Anopheles freeborni and A. quadrimaculatus singes. Passages par especes variees. Action pathogene.
as experimental vectors of Plasmodium cynomolgi and C. R. Soc. BioI. 73 : 310-313.
P. inui. J. Parasit. 46 : 540. LEGER, M. and BOUILLIEZ, M., 1913. Recherches
EYLES, D. E., 1960a. Susceptibility of Anopheles albimanus to experimentales sur "Plasmodium inui" Halberstadter et
primate and avian malarias. Mosq. News 20 : 368-371.
258 PRIMATE MALARIAS

REFERENCES—Continued
Prowazek d'un "Macacus cynomolgus". Ann. Inst. SINGH, J., RAY, A. P., NAIR, a. P., and BASU, P. a., 1951.
Pasteur. 27 : 955-985. Isolation of a strain of P. inui from mixed infection in
MATHIS, C. and LEGER, M., 1911. Plasmodium des macaques Malayan monkey. Ind. Jour. Malariol. 5 : 433-445.
du tonkin. Ann. Inst. Pasteur. 25 : 593-601. SINTON, J. A. and MULLIGAN, H. W., 1933. A critical review
MAYER, M., 1907. Ueber malaria beim affen. Med. Klin. 3 : 579- of the literature relating to the identification of the
580. malarial parasites recorded from monkeys of the
MOHIUDDIN, A., 1957. Notes on a new strain of "Plasmodium families aercopithecidae and aolobidae. Rec. Mal. Surv.
inui". Riv. di. Malariol. 36 : 203-208. India III: 381-443.
MULLIGAN, H. W. and SWAMINATH, C. S., 1940. Natural SINTON, J. A., 1934. A quartan malaria parasite of the lower
infection with Plasmodium inui in Silenus sinicus from oriental monkey, Silenus irus (Macacus cynomolgus).
South India. J. Malar. Inst. Ind. 3 : 603-604. Rec. Mal. Surv. India 4 : 379-410.
NOGUCHI, H., 1928. Etiology of oroya fever. XII. Influence of VOLLER, A., GARNHAM, P. C. C. and TARGETT, G. A. T.,
malarial infection (Plasmodium inui), splenectomy, or 1966. Cross immunity in monkey malaria. Jour .Trop.
both, upon experimental carrion's disease in monkeys. Med. & Hyg. 69 : 121-123.
J. Exp. Med. 47 : 812-827. VOLLER, A. and ROSSAN, R. N., 1969. Immunological studies
NOTANANDA, V., NILUBOL, S., and SWASDIWONGHORN, on simian malaria parasites. IV. Heterologous
P., 1961. A preliminary note on the discovery of simian superinfection of monkeys with chronic Plasmodium
malaria in Chingmai. Minuten Nadeln. 1 : 27. knowlesi infections. Trans. Roy. Soc. Trop. Med. &
SCHMIDT, L. H., GREENLAND, R., ROSSAN, R. and Hyg. 63 : 837-845.
GENTHER, C., 1961. The occurrence of malaria in WARREN, McW., and WHARTON, R. H., 1963. The vectors of
wild-caught rhesus monkeys. Science 133 : 753. simian malaria: identity, biology, and geographical
SEZEN, N., 1956. Studies on the life-cycle of two strains of distribution. J. Parasit. 49 : 892-904.
Plasmodium inui and the development of immunity. WHARTON, R. H., EYLES, D. E., WARREN, McW. and
Turk. Ij. tecr. Biyol. Dreg. 16 : 240-242. (NS). MOORHOUSE, D. E., 1962. Anopheles leucosphyrus
SHORTT, H. E., RAO, G., OADRI, S. S., and ABRAHAM, R., identified as a vector of monkey malaria in Malaya.
1961. Plasmodium osmaniae, a malaria parasite of an Science 137 : 758.
Indian monkey Macaca radiata. Jour. Trop. Med. & WEYER, F., 1937. Versuche zur ubertragung der affen-malaria
Hyg. 64 : 140-143. durch stechmiicken. Arch. Schiff. f. Tropenhyg 41:
SHORTT, H. E., BAKER, J. R. and NESBITT, P. E., 1963. The 167-172.
pre-erythrocytic stage of Plasmodium osmaniae. Jour.
Trop. Med. & Hyg. 66 : 127-129. (NS) = not seen
SINGH, J. and SINGH, H., 1940. Observations on immunity in
monkey malaria as evidenced by the results of super-
infection. J. Malaria Inst. India 3 : 99-114.
 21
Plasmodium rodhaini Brumpt, 1939

from a chimpanzee to each of two people by the

IN 1920, Reichenow same route (1940, 1940a, 1941). Other human
studied the malarias of chimpanzees and gorillas infections resulted under similar circumstances,
in the Cameroons and found three species which as detailed in the above papers, with moderate to
he considered identical to P. vivax, P. low parasitemias and fever up to 40° C. The
falciparum, and P. malariae in man. Blacklock above results convinced him that the P. rodhaini
and Adler (1922) in Sierra Leone and Schwetz parasite of Brumpt was in fact P. malariae. In
(1933, 1933a, 1934) in the Belgian Congo saw 1943, Rodhain and Dellaert reported the transfer
the same three forms. The first authors did not of P. rodhaini from a chimpanzee to a paretic in
accept the Reichenow view of their being human the Hospital Stuyvenberg. The patient became
malaria counterparts because they proposed a infected and from him twenty-three other
new name P. reichenowi for the falciparum-like patients were given the infection in tandem.
parasite. Schwetz, on the other hand, considered It was clear that P. rodhaini of the
them identical to those of man. Rodhain and chimpanzee when blood passaged to man would
Muylle (1938) did not accept the Schwetz grow and produce disease. However, there was
opinion because they were unable to infect no evidence that the reciprocal would be equally
human subjects with falciparum- and vivax-type true. This was answered by Rodhain in 1948
parasites from the chimpanzee. Brumpt (1939), when he reported the successful transfer of P.
after considering the morphological similiarity malariae to each of three young chimpanzees.
between the ape and human forms, the failures Following this experiment, he again declared
in cross infection experiments, and the lack of that the quartan parasite in man and the
success in infecting known human vectors with chimpanzee was P. malariae.
the chimpanzee parasites, decided that the forms In 1956, Garnham et al transferred P.
were different. In that paper, he named the malariae to an intact chimpanzee which
vivax- like parasite Plasmodium schwetzi (see exhibited a light infection. When the spleen was
chapter 12); to the quartan parasite he gave the removed, the parasitemia increased to a peak of
name Plasmodium rodhaini in honor of the 160,000 per mm3. They were not able to infect
Belgian investigator Dr. Jerome Rodhain. At mosquitoes. Bray (1960) was able to infect
that point, each of the three human-like malarias Anopheles gambiae mosquitoes when fed on
of apes had received names. man with P. malariae, and on chimpanzees
Rodhain was not satisfied. His interest was carrying P. rodhaini. Plasmodium malariae
more biological than taxonomic and, to that end, infections were obtained in the chimpanzees by
he entered upon a susceptibility study which mosquito bites, but he was not able to carry out
continued intermittently during the rest of his the reverse procedure. Nevertheless, he was
life. In 1940, he succeeded in transferring P. willing to accept the name P. malariae for the
malariae from man to the chimpanzee by the quartan parasite of man and the higher apes.
inoculation of parasitized blood and P. rodhaini Garnham (1966) concurred in that opinion,

259
260 PRIMATE MALARIAS

which is accepted generally, when, in speaking we were unable to obtain an infected animal
of Rodhain's work of 1940 (loc. cit.) he wrote from which we might obtain material, we
"This was the first step in the sinking of P. elected not to include a plate depicting the blood
rodhaini into synonomy with P. malariae." At forms of the parasite; reference should be made
present, we cannot take issue with the view that to our plate of P. malariae.
the quartan parasite of man and the chimpanzee Plasmodium rodhaini is a quartan malaria
are the same parasite. of the chimpanzee (Pan stayrus verus). The
Granted that P. rodhaini is synonomous infection extends along the coast of West Africa
with P. malariae, then, one must conclude that from Sierra Leone to Angola and thence east to
the human parasite became adapted to the some point deep in the Congolese Republic. The
chimpanzee, or that it was a simian parasite infection rate appears to be low. Reichenow
originally which became adapted to early man. It (1920) found it in only two of eight
seems likely, in the light of recent work chimpanzees, Rodhain and Dellaert (loc. cit.)
involving other simian malarias (Coatney, reported finding it in some of the young
1968), that in relatively recent times it went chimpanzees available to them, and Bray,
from man to the apes, and, therefore, qualifies as according to Garnham (1966) examined seventy-
an anthroponosis. If that is not the case, then it is eight chimpanzees in Liberia and found it in
a zoonosis of long standing. only eight percent of them.
For reasons stated above, plus the fact that

REFERENCES
BLACKLOCK, B., and ADLER, S., 1922. A parasite resembling plasmodiums humains. Receptivite de l'homme au
Plasmodium falciparum in a chimpanzee. Ann. Trop. Plasmodium malariae. (Plasmodium rodhaini Brumpt)
Med. & Parasit. 16 : 99-106. du chimpanze. C. R. Soc. BioI. 133 : 276-277.
BRAY, R. S., 1960. Studies on malaria in chimpanzees. VIII. The RODHAIN, J., 1941. Les plasmodiums des anthropoides de
experimental transmission and pre-erythrocytic phase I'Mrique centrale et leurs relations avec les
of Plasmodium malariae, with a note on the host-range plasmodiums humains. Bull. Acad. Roy. Med. Belgique
of the parasite. Am. J. Trop. Med. & Hyg. 9 : 455-465. 6 : 21-60.
BRUMPT, E., 1939. Les parasites du paludisme des chimpanzés. RODHAIN, J., 1948. Susceptibility of the chimpanzee to P.
C. R. Soc. Bioi. 130 : 837-840. malariae of human origin. Am. J. Trop. Med. & Hyg.
COATNEY, G. R., 1968. Simian malarias in man: facts, 28 : 629-631.
implications, and predictions. Am. J. Trop. Med. & RODHAIN, J., and DELLAERT, R., 1943. L'infection a
Hyg. 17 : 147-155. Plasmodium malariae du chimpanze chez l'homme.
GARNHAM, P. C. C., LAINSON, R., and GUNDERS, A. E., Etude d'une premiere souche isolee de l'anthropoide
1956. Some observations on malaria parasites in a Pan satyrus verus. Ann. Soc. Belge Med. Trop. 23 : 19-
chimpanzee, with particular reference to the persistence 46.
of Plasmodium reichenowi and Plasmodium vivax. RODHAIN, J ., and MUYLLE, G., 1938. Sur la specificite des
Ann. Soc. Beige Med. Trop. 36 : 811-821. plasmodium des anthropoides de I'Afrique centrale. C.
GARNHAM, P. C. C., 1966. Malaria parasites and other R. Soc. BioI. 127 : 1467-1468.
haemosporidia. Blackwell Scientific Publications, SCHWETZ, J ., 1933. Sur les parasites malariens (Plasmodium)
Oxford. des singes superieurs (Anthropoides) africains. C. R.
REICHENOW, E., 1920. Ueber das vorkommen der Soc. Biol. 112 :710-711.
malariaparasiten des menschen bei den afrikanischen SCHWETZ, J ., 1933a. Sur une infection malarienne triple d'un
menschenaffen. Centralbl. f. Bakt. I. Abt. Orig. 85 : chimpanze. Zentrabl. f. Bakt. Parasit. Infektion-
207-216. skrankheiten I. Abt. Orig. 130 : I0s-II0.
RODHAIN, J ., 1940. Les plasmodiums des anthropoides de l' SCHWETZ, J ., 1934. Sur le paludisme des pygmees. C. R. Soc.
Afrique centrale et leurs relations avec les plasmodiums Biol. 115 : 1228-1229.
humains. Ann. Sac. BeIge Med. Trop. 20 : 489-505.
RODHAIN, J., 1940a. Les Plasmodiums des anthropoides de
I'Mrique centrale et leurs relations avec les
 SECTION 5
Falciparum-Type Parasites
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 22
Plasmodium falciparum (Welch, 1897)

resolved some of the mysteries of the parasite, as

SYNONYMS: Oscillaria seen in the peripheral blood, by setting forth its
malariae Laveran, 1881; Plasmodium malariae 48-hour cycle, and showing that the small
Marchiafava and Celli, 1885; Laverania parasites and the crescents were parts of the
malariae Feletti and Grassi, 1890; Haemamoeba same cycle. On clinical grounds, they pointed
praecox Grassi and Feletti, 1890, partim; out its perniciousness. Mannaberg (1893), in his
Ematozoo falciforme Antolisei and Angelini, concisely written little book, pointed out the
1890; Haemamoeba immaculata Grassi, 1891; error of ascribing triple etiology, as Feletti and
Haemamoeba laverani Labbe, 1894; Grassi (1890) had done, to the malignant tertian
Haematozoon falciforme Thayer and Hewetson, parasite and his illustrations, colored Plate IV,
1895; Haematozoon falciparum Welch, 1897; leave little doubt that he was familiar with the
Haemosporidium sedecimanae Lewkowicz, circulating blood forms of the parasite.
1897; Haemosporidium undecimanae Several different names were proposed for
Lewkowicz, 1897; Haemosporidium the parasite between 1885 and Welch's
vigesimotertianae Lewkowicz, 1897. Haematozoon falciparum of 1897. The latter
As mentioned in an earlier chapter, Laveran name called attention to the sickle-shaped
in his early studies (1880,1881) of the parasites parasites and, probably for that reason, was
of human malaria saw each of the principal widely accepted by the scientific community,
species and in 1881 used the name Oscillaria even though taxonomically incorrect. Such a
malariae for these parasites, including the situation is intolerable because of the priority
crescent-shaped bodies; then, and later, he rule and a long struggle began among
steadfastly held to the belief that only one taxonomists to resolve the dilemma.
species was involved. Garnham (1966) gives an In 1929, Sergent et al made an exhaustive
interesting account of Laveran's first study of the situation and came to the conclusion
observations of the falciparum parasite in 1880 that the correct name for the malignant tertian
in the blood of a young soldier who had been in parasite should be Plasmodium praecox Grassi
Algeria for about a year. In 1892, Grassi and and Feletti, 1890. In 1935, Giovannola
Feletti, as an honor to Laveran, proposed the reexamined the problem and decided that the
genus name Laverania which was zoologically correct name should be Plasmodium
correct, providing two genera are recognized. immaculatum (Grassi and Feletti, 1892); but, in
However, since most authors recognize only the 1938, Christophers and Sinton pointed out that if
genus Plasmodium Marchiafava and Celli, 1885, that name were accepted, it must be credited to
the latter took precedence under Opinion No.104 Grassi, 1891. The latter authors went on to point
of the International Commission on Zoological out that malariae was the name applied
Nomenclature, 1928. originally by Laveran and is, therefore, the de
The confusion which surrounded the jure name.
naming of this parasite was linked to its masked It was abundantly clear that strict adherence
periodicity and to the presence of crescent- to the rules of zoological nomenclature would
shaped bodies. Marchiafava and Bignami (1892) create intolerable confusion and when Sergent et

263
264 PRIMATE MALARIAS

al (1939) withdrew their proposal of 1929, there Polumordvinov (1945) reported infections in
appeared a united front, joined by Coatney and southern Tadjikistan at 2750 and 2850 meters
Young in 1941, for the general adoption of the which is the limit of human settlements in that
commonly used de facto name. However, a region. Garnham (1948) described an epidemic
ruling was necessary to give status to the in the highlands of Kenya, as high as 2600
concensus. At the 1954 meeting of the meters, where at Kericho, for example, the
International Commission on Zoological parasite rate was 8 percent prior to the epidemic,
Nomenclature, the trivial name falciparum of but rose to 36 percent by the end of it. Hackett
Welch (1897) was validated but privilege was (1945), working in Bolivia, demonstrated
given for its use with either the genus transmission at 2600 meters.
Plasmodium or with Laverania (see Hemming, There are many accounts of havoc among
1954). The ruling settled the specific name, and, early civilizations which is attributed to
it was hoped, at least in some quarters, that in malignant malaria, but proof that malaria was
spite of privilege, Plasmodium would be the the actual culprit was lacking until early in this
accepted name for the genus. However, Bray century. Examples from this era include the
(1958) redefined the genus Laverania and report by Raffaele and Coluzzi (1949) for the
consigned two species to it: falciparum and area around Cassino in Italy. Prior to 1943,
reichenowi. Although he, and certain others, felt malaria, although present, was of little
strongly about the designation, he was willing to importance. In 1945, following the bloody
concede "the use of the genus is not obligatory." fighting between the German and the Allied
There was a great deal of heated discussion Armies, with destruction of dykes and other
regarding Bray's proposal, most of it without the control measures in 1943-44, about 100 percent
benefit of printers ink, with the result that, in of the people were infected; 43 percent of the
1963, he relegated Laverania to subgeneric rank. infections were P. falciparum. The mortality in
During the years since 1963, the use of some villages of the area was 10 percent. In this
Laverania as a genus name has continued to lose hemisphere, the classic example of introducing
favor. In fact, Garnham (1966), although he an efficient vector into a susceptible P.
supported Bray's proposal in 1958, fails to falciparum population is that of Anopheles
mention its revival after the 1954 decision. We gambiae in northeast Brazil as recounted by
feel, that in the interest of uniformity and Soper and Wilson (1943). The first A. gambiae
convenience, the name of the malignant tertian probably arrived in the area from Africa in 1929;
parasite should be Plasmodium falciparum its transmission potential was recognized in
(Welch, 1897). 1930, but scant attention was paid to it until it
Plasmodium falciparum has a worldwide reached the Assu and Apode valleys in Rio
distribution and is concentrated in the tropics Grande do Norte in 1938. It spread over some
and subtropics. It invades the temperate zone 12,000 square miles leaving illness, death, and
and, as a consequence, it used to be common in desolation until finally eradicated in 1940 by an
southeastern United States, the littoral areas of encircling technique which, at its inception,
the Mediterranean, and in the Balkans. It has many malariologists called an "audacious
since disappeared from those areas generally as experiment."
a result of better economic conditions, good Among the human malarias, P. falciparum
control, and/or eradication programs. is considered the youngest evolutionarily and the
Altitude has an important bearing on the least efficient as a parasite because its malignant
transmission of P. falciparum, and other species, nature tends to eliminate its host.
too, but the height at which it disappears is We have studied many different strains of
variable depending on the temperature at which P. falciparum, some of which will be discussed
the vector can maintain itself. Under ordinary later in this chapter.
circumstances, transmission fails above 1500
meters. Exceptions are not uncommon, however.
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 PLASMODIUM FALCIPARUM 267

Cycle in the Blood parasite count of about 5,000 per mm3, yet he
continued to show a high proportion of
PLATE XLII segmenters for several days.
The youngest ring forms of Plasmodium
During schizognoy, the nucleus divides
falciparum are smaller than those of other
repeatedly, the parasite increases in size until it
human malarias and are commonly referred to as
may occupy a large part of the host cell. At first
tiny, hair-like rings, with a vacuole, and a
the pigment comes together in small aggregates,
prominent nucleus. Sometimes, there is an
but, as the parasite nears maturity, it collects in a
accessory chromatin dot (Fig. 2-5). Multiple
single yellowish-brown mass. The mature
invasion of the host cell by equal-aged parasites
schizont is less symmetrical than those of other
is more common in this species than in the other
human malarias and its merozoites number 8 to
human malarias (Figs. 6, 10, 11). Field and
20; the usual number is about 16 (Figs. 20-25).
Shute (1956) illustrate 7 ring-stage parasites in a
One of the striking features of erythrocytes
single cell and state that "eight rings in a cell has
infected with the asexual parasites is the early
been recorded." Appliqué or accollé forms are
development of Maurer's dots, or clefts, which
common and, hence, have some diagnostic
make their appearance shortly after the hair-like
value. As development proceeds, the overall size
ring stage. As the development of the parasite
of the parasite is increased, the vacuole and the
proceeds, these abnormalities become more
nucleus of the parasite become more prominent
pronounced. They are demonstrable in the
(Fig. 14), and tenue forms (Fig. 15) may appear;
parasitized red cells only under certain staining
aside from these abnormal forms (see Field and
procedures, not ordinarily applied, and therefore
Shute, 1956), there is no appreciable
are not shown in our plate.
amoeboidity. The parasite now becomes smaller
The mature gametocytes are unique among
and more compact, the cytoplasm stains a deep
human malarias because of their sickle or
blue, it loses its vacuole, the nucleus ceases to be
crescent shape, a feature well appreciated by
circular, and dark pigment grains appear in the
Laveran. In most malarias, the gametocytes
cytoplasm (Figs. 16-19).
appear about the same time as the asexual forms,
At this juncture, the number of parasites in
but in falciparum malaria, it is about 10 days
the peripheral blood decreases due to their
after the first appearance of the asexual, forms
penchant for retreating into the deeper
that they appear as a wave of full grown
circulation—a practice common to P. coatneyi,
parasites. Preceding their appearance, the young
too—so that in cases of high synchronicity, it is
gametocytes have been growing in the blood
sometimes difficult to find late developing
spaces of the spleen and bone marrow.
forms. In general, however, the phenomenon of
The macrogametocyte is relatively slender,
asynchronicity produces enough tardy forms to
has pointed ends, and is generally longer than
permit following the remainder of the cycle. As
the microgametocyte. The cytoplasm stains a
a rule, the presence of appreciable numbers of
decided blue. The nucleus is compact and may
segmenters in the peripheral blood is an
be masked by pigment granules which appear to
indicator of grave consequences, but this is not
cover it. The red cell may be seen as stretching
always the case. In our own experience, we have
across the curvature of the gametocyte (Figs. 27,
seen a case in which the patient was not
particularly ill, was ambulatory, and had a

PLATE XLII.—Plasmodium falciparum.

Fig. 1. Normal red cell. Figs. 19-22. Developing schizonts.
Figs. 2-11. Young trophozoites. Figs. 23-26. Nearly mature and mature schizonts.
Figs. 12-15. Growing trophozoites. Figs. 27, 28. Mature macrogametocytes.
Figs. 16-18. Mature trophozoites. Figs. 29, 30. Mature microgametocytes.
268 PRIMATE MALARIAS

28). The microgametocyte is sausage-shaped and, in the same year, Grassi et al (1898)
with blunt-rounded ends. The cytoplasm stains observed complete development of P.
light blue to purplish-blue. The nucleus occupies falciparum in Anopheles claviger (= A.
about half the total length of the parasite. It is maculipennis). In 1899, Bastianelli and Bignami
diffuse, and generally shows some dark red dots not only described the development of the
scattered in a pale pink area. Lying well within parasite in mosquitoes, but, also, demonstrated
the periphery of the nuclear area are clustered its transmission to man.
dark brown to black pigment granules. The host Shute and Maryon (1952) observed the
cell generally hugs the body of the parasite, but development of oocysts of P. falciparum in A.
may show as a bulb-like area in the slight atroparvus mosquitoes incubated at a
curvature of the gametocyte (Figs. 29, 30). temperature of 25° C. The black pigment
The asexual cycle is 48 hours. granules (between 10 and 20 in number) were
usually arranged (between days 3 and 7) in a
Sporogonic Cycle double semicircle around the periphery of the
oocyst. By the 8th day, the pigment was
PLATE XLIII obscure; the oocysts measured from 8 to 60 µ in
There have been many studies on the
diameter. The sporogonic cycle was completed
sporogonic cycle of P. falciparum since Ross
in 11 to 12 days. From the 3rd to the 5th day, the
(1897) described finding oocysts on the gut of a
daily increase in oocyst diameter was
mosquito which had fed on a gametocyte carrier.
approximately 4 µ. From the 6th to the 10th day,
Bastianelli et al (1898) observed pigmented
the daily increase was about 10 µ.
oocysts in anopheline mosquitoes which had fed
Our studies of the sporogonic cycle of this
on an individual infected with P. falciparum,

PLATE XLIII.—Developing oocysts and sporozoites of Plasmodium falciparum in Anopheles freeborni mosquitoes. X 580.
Fig. 1. 7-day oocyst. Fig. 5. 14-day oocyst showing numeous small vacuoles.
Fig. 2. 7-day oocyst showing line of pigment. Fig. 6. 14-day differentiated oocyst.
Fig. 3. 8-day oocyst. Fig. 7. Sporozoites present near salivary gland tissue
Fig. 4. 10-day oocyst. 14 days after feeding.
 PLASMODIUM FALCIPARUM 269

parasite have been limited, but we have followed Experimentally, P. falciparum has been
its development in A. freeborni infected with the transmitted to man via the bites of many species
Malayan IV and the McLendon strains of P. of mosquitoes on numerous occasions. In that
falciparum, and in A. quadrimaculatus infected connection, Garnham (1966) lists 66 species of
with the McLendon strain only (Table 34). In A. anophelines which will serve as hosts of P.
freeborni, with the Malayan IV strain, on day 5, falciparum.
oocysts had mean diameters of 12 µ, with a
range of 8 to 15 µ; on day 12, the mean size was Cycle in the Tissue
50 µ, with a range of 21 to 78 µ. There are some
differences in the development of the 2 strains in
The tissue stages of Plasmodium,
A. freeborni. The Malayan IV had slightly larger
falciparum have been demonstrated in
mean oocyst diameters, but, more significantly,
experimental infections of man as well as
sporozoites were present in the salivary glands
chimpanzees. This species of human malaria
on day 12 whereas the McLendon strain required
differs from P. vivax and P. ovale in that the
14 days. The development in the A.
exoerythrocytic cycle is restricted to a single
quadrimaculatus was similar to that seen in A.
generation; in other words, there is no secondary
freeborni infected with the McLendon strain.
exoerythrocytic or other continuing fixed tissue
Sporozoites were present in the salivary glands
stage.
on day 14.
The tissue cycle of Plasmodium falciparum
A comparison of the oocyst growth rate of
was first demonstrated by Shortt et al (1949,
the Malayan IV strain of P. falciparum with that
1951) in liver biopsy material from a human
of P. cynomolgi (Fig. 57) shows a marked
volunteer who had been bitten by 770 Anopheles
difference between the 2 parasites. The P.
mosquitoes (93 percent infection rate) over a
cynomolgi was much larger both with regard to
period of 3 days. The strain of falciparum
mean and maximum oocyst diameters.
malaria used by these authors was of Roumanian
Sporozoites were present in the salivary glands
origin. The liver biopsy was taken 5% days after
of the mosquitoes infected with P. cynomolgi
mosquitoes had first bitten the volunteer. The
one day sooner than in those infected with P.
exoerythrocytic schizonts described by these
falciparum.
authors were considered to be 4-, 5-, and 6-day
stages. The 4-day schizonts were described as

TABLE 34.—Oocyst diameters of Plasmodium falciparum (Malayan IV and McLendon strains) in Anopheles freeborni and A. quadrimaculatus
mosquitoes.

Malayan IV strain McLendon strain

Days after
A. freeborni A. freeborni A. quadrimaculatus
Infection
No. Range Mean* No. Range Mean No. Range Mean

4 118 5-11 10
5 106 8-15 12 193 7-14 10 5 8-14 11
6 122 9-21 15 115 11-20 15 116 8-20 14
7 163 9-28 19 152 8-20 15 134 12-23 18
8 255 12-38 23 120 11-31 20 187 11-32 19
9 199 14-45 32 169 12-41 24 101 18-38 28
10 111 26-64 41 150 17-53 35 168 18-50 33
11 112 20-73 45† 132 19-61 40 126 20-58 40
12 154 21-78 50†** 150 20-72 47† 110 18-60 42
13 36 31-71 56†** 139 19-70 49† 132 27-68 48†
14 164 20-67 45†** 109 30-68 54†**

Totals 1258 8-78 1602 5-72 1188 8-68

* Measurements expressed in microns.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
270 PRIMATE MALARIAS

FIGURE 57.—Mean oocyst diameter curves and ranges in oocyst diameter of Plasmodium cynomolgi and P. falciparum (Malayan
IV strain) in Anopheles freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

ovoid or spherical in shape, with some tendency The 6-day stages showed the parasite had
toward the production of lobose projections. undergone very rapid growth, expanding in an
They measured about 30 µ in diameter and were irregular fashion in many directions. Six-day
surrounded by a thin membrane. The cytoplasm forms measured 50 to 60 µ in length and were
was fairly dense and there were no vacuoles. described as being more "misshapen" than P.
Nuclei were rather sparsely distributed, slightly vivax because of the pronounced tendency to
irregular masses measuring approximately 1.5 µ lobosity. The number of nuclei had increased
in diameter. Cytoplasm tended to condense tremendously, the cytoplasm being thickly
around each nucleus; and, according to the strewn with them. Near maturity, the parasite
authors, this possibly represented the first step in appeared to break up into small islands of
the formation of the so-called pesudocytomeres. cytoplasm, measuring approximately 2 µ in
Five-day old schizonts measured roughly diameter, each island contained two very small
50 µ in their longest dimension. The tendency to nuclei.
produce the lobose character was more With a final division of the nuclei, one has
pronounced. There was no local tissue reaction. a mature schizont, measuring 60 µ or more in its
There was a tendency for the cytoplasm to greatest dimension, containing an enormous
separate into areas resembling cytomeres. These number of merozoites. Each merozoite, about
pseudocytomeres varied in shape from spherical 0.7 µ in diameter, consists of a nucleus with a
to elongate; each contained a large number of trace of cytoplasm. The number of merozoites in
nuclei. The size of the individual nuclei, in a large mature schizont was estimated as roughly
active division, was roughly 0.8 µ. The 40,000.
membrane surrounding the parasites was less Jeffery et al. (1952) carried out a study,
apparent. involving 14 patients, designed to demonstrate
 PLASMODIUM FALCIPARUM 271

the exoerythrocytic stages of Plasmodium The parasites described by Jeffery et al

falciparum in the human liver. Inoculation was (1952) were considered similar to those
either by the bites of heavily infected described by Shortt et al in 1951. However, a
mosquitoes, the intravenous inoculation of few differences, which may be due to
dissected salivary glands, or both. Each interpretation, were noted. For example, very
volunteer received from 2 to 6 inoculations. little, if any, compression of host tissues
Biopsies were performed 3 to 8 days after the surrounding the parasite was observed by these
first inoculation. Material from 13 of the 14 authors. Shortt et al described the tendency
patients was negative. The 14th patient, who toward lobation in the parasite. Jeffery et al
received a total of 8,516 mosquito bites along noted the same thing, but in addition, reported
with the intravenous inoculation of 1,403 pairs that it was not unusual to find parasites,
of salivary glands, yielded material positive for including mature schizonts, with smooth
falciparum tissue schizonts. The authors found outlines, although in many others, the lobose
125 parasites; 100 were complete. character was extremely pronounced. Jeffery et
The smallest parasites were 15 µ in their al felt it was not possible to designate a
greatest diameter. However, the authors stated particular parasite as being of a specific age
that due to shrinkage, the living parasites were since inoculations were carried out over a period
undoubtedly larger than 15 µ. The biopsy was of 4 days which was some 3 to 6 days before
done on day 6 and inoculations occurred on day biopsy was taken. Shortt et al (1951) also had
0, 1, 2, and 3, and consequently, the tissue stages inoculated their patient on 3 consecutive days
could have been anything from 3 to 6 days old. and correlated their parasites with time,
The cytoplasm was described as being granular, designating them as being 4, 5, and 6 days of
sometimes containing small vacuoles. The age on the basis of size. On this basis, then, the
outline of the parasite appeared to be wavy smallest parasite found by Jeffery et al, which
which was probably due to shrinkage. The was 15 µ, could have been 3 days old, whereas
smallest number of nuclei observed was about the smallest parasites found by Shortt et al were
40. about twice that size and were designated as
Parasites approximately 25 µ in their being 4 days old.
greatest diameter were also observed. The Shortt et al (1951), in comparing the pre-
cytoplasm was homogeneous, contained a few erythrocytic stages described for cynomolgi and
vacuoles, and a larger number of nuclei. The vivax malaria with those of falciparum malaria,
largest parasite, about 40 µ, was considered to indicated several points of difference; namely, 1)
be nearly mature. The cytoplasm was the larger size of the mature schizont, 2) the
vacuolated, the outline of the parasite was smaller size of the merozoites, 3) the greater
smooth, and, occasionally, small lobes were number of merozoites, and 4) the rapidity of
observed. The stage considered nearest maturity development of the tissue stages to full maturity,
was described as having an increase in the i.e., 6 days for P. falciparum compared with 8 or
complexity of the vacuoles and an apparent more for P. vivax and P. cynomolgi.
formation of cords and islands of cytoplasm Bray (1958, 1960) and Bray and Gunders
(pseudocytomeres) from which the merozoites (1962, 1963) studied the tissue stages of P.
arise. Most parasites at this stage were 55 to 60 falciparum in chimpanzees. The youngest forms
µ in their greatest diameter. were 2-day stages. These were described as
The parasites were found in the lying within a vacuole in the liver parenchymal
parenchymal tissue of the liver lying within the cell and measuring about 4 µ in diameter. The 2-
liver cords. The host cells were enlarged but no day parasite was enclosed in a well-defined
changes were observed in the host cell nuclei. envelope and contained 2 nuclei which were
There were no detectable morphologic reactions triangular in shape. At 2 days and 15 hours, the
to the presence of the parasite. In addition, there tissue stages measured 7 µ and contained up to
was no leukocytic infiltration into areas around 20 nuclei. Three-day forms, still within a
the parasite. vacuole in the liver cell cytoplasm, averaged 9 µ
272 PRIMATE MALARIAS

and contained 25 nuclei. At 4 days, the parasite merozoites become differentiated from the 2
measured 18 µ and contained flocculated nucleate cytomeres.
cytoplasm studded with about 145 large nuclei. There is direct and indirect evidence to
Bray (1958) considered the 5-day forms in suggest that the exoerythrocytic cycle of
the chimpanzee liver similar in all respects to the falciparum malaria is limited to a single
4-day stages described by Shortt et al and the generation--the first. In other words, no true
smaller schizont described by Jeffery et al. The relapse occurs because no tissue stages remain
average greatest dimension was 32 µ, with a after the development of the primary falciparum
range of 41 to 28 µ. The outline of the parasite malaria infection. Bray (1958) found no tissue
was generally regular without lobations. The schizonts in biopsies of liver taken 12 days after
cytoplasm was rarely vacuolated. However, infection. This contrasts greatly with the ease
when vacuoles were present, they were small with which he found P. vivax and P. ovale,
and rarely numbered more than 2. The contour under similar experimental conditions, in liver
of the parasite was indented by the host cell biopsies from chimpanzees; P. vivax and P.
nucleus. ovale are relapsing malarias.
Three different types of nuclei could be Sodeman et al (1969) described 6- and 7-
seen in the chimpanzee material: 1) parasites day stages of P. falciparum in the liver tissue of
with tiny dots of chromatin measuring 0.5 µ in the owl monkey, Aotus trivirgatus. The parasites
diameter and usually associated with a smooth resembled the 2.5 to 5-day stages observed in
homogeneous appearance of the cytoplasm, 2) the chimpanzee and in man. The owl monkey
parasites with round pieces of chromatin EE bodies were considered non-viable because
approximately 1 µ in diameter, and 3) parasites the host failed to develop a patent infection.
with relatively large pieces of chromatin Fairley et al (1947), by subinoculating a
measuring 1.0 to 1.7 µ which were circular, large volume of blood from volunteers exposed
oblong, or even square in shape, and these were to falciparum infection by bites of 7 to 20
associated with a cytoplasm which tended to infected mosquitoes, concluded that the tissue
display aggregates of basophilic material. cycle ended at 6 ½ days. When blood taken 160
The exoerythrocytic cycle of falciparum hours, or later, after exposure to infection, was
malaria appears to be slightly longer in the subinoculated into volunteers, the subinoculees
chimpanzee than it is in man. Bray (1958) developed patent falciparum infections. As
considered the 8-day parasites in his chimpanzee stated earlier, Shortt et al found that, blood taken
material to be nearly mature and resembled the from their one volunteer at 135 hours after
6-day schizonts of Shortt et al and the large exposure to infection with approximately 716
schizonts of Jeffery et al. infected mosquitoes was infectious when
The final stages of schizogony were inoculated into another volunteer. It appears
described as very complex, and defined by Bray obvious that the difference of 25 hours was
(1960) as aposchizogony. Garnham (1966) probably due to the difference in the number of
stated that the common appearance of the tissue infective bites (maximum of 20 versus
schizont on the 6th day is that of an ovoid approximately 700). Ciuca et al (1937) showed
structure, about 40 µ in its greatest dimension, that the tissue cycle of a Roumanian strain of
containing hundreds of cytomeres, each falciparum malaria was completed during the
cytomere measuring 3 to 5 µ in diameter. The 6th day after exposure to infection.
cytomeres are considered as hollow spheres, the Garnham (1966) states that the prepatent
surface studded with small nuclei. At first there period for falciparum malaria is 5½ days since
are only a few of these cytoplasmic islands or the tissue schizont ruptures at that time releasing
cytomeres, but by the process of aposchizogony merozoites into the blood stream as indicated by
(Bray, 1960) a large number of cytomeres are experiments similar to those of Fairley, i.e., that
eventually produced, each becoming smaller and a large volume of blood taken from the
with progressively fewer nuclei (i.e., 8, 4, 2 volunteer at 135 hours after exposure was
nuclei) with each division. At maturity, injected into another volunteer and the
subinoculee developed malaria.
 PLASMODIUM FALCIPARUM 273

Course of Infection of 11 days for 220 naturally induced falciparum

infections involving 6 different strains, which
included the Costa and the Long strain. The
The course of infection is inaugurated with
mean incubation period for this same group was
the entry of merozoites into red cells of the
13.1 days. The range for the prepatent periods
circulating blood, and according to Garnham
was 6 to 25 days; for the incubation period, 7 to
(1966), Shute demonstrated a parasite in a thick
27 days. Seventy-five percent of the prepatent
blood film from a patient who had received
periods ranged from 9 to 11 days. Among the
500,000 sporozoites of a Roumanian strain of
220 infections, only 2, 1, and 1 patients had
Plasmodium falciparum intravenously 5 days
prepatent periods of 6, 7, or 8 days, respectively.
earlier. Shortt et al (1951) showed that the
Eyles and Jeffery (1949) transmitted
prepatent period could be as early as 5½ days
Santee-Cooper strain falciparum and Panama
when exposure to infection was massive and
strain falciparum by bites of Anopheles
blood was subinoculated into volunteers. We
albimanus. With the former the prepatent period
prefer to define prepatent period as the interval
was 13 days, and with the latter, 10 to 13 days
from the time of exposure to the demonstration
(median of 11.5 days). Later, Eyles and Young
of parasites in the blood of the host by more
(1951), working with mosquito inoculated
conventional methods; namely, the thick blood
Santee-Cooper falciparum, reported prepatent
film. On this basis, the prepatent period
periods from 7 to 13 days. Jeffery et al (1952),
observed by Shortt et al, in the volunteer from
in their studies on the fixed tissue stages of
whom liver biopsy material was obtained, was 7
falciparum malaria, observed prepatent periods
days. In addition, Shortt et al observed a
ranging from 7 to 13 days after massive
prepatent and incubation period of 8 days in a
exposures to infection, either by mosquito bite
patient who served as a control for mosquito
or by inoculation of suspensions of sporozoites.
infectivity and who was bitten by a total of 370
The total number of mosquitoes biting ranged up
mosquitoes on 4 consecutive days.
to 8,516, with an 86.8 percent infection rate. The
Ciuca et al (1937a), in their description of
median was 9 days. Jeffery et al (1963) in
12 falciparum malaria infections, induced by the
studies with a Thailand strain of resistant
bites of infected mosquitoes and/or the
falciparum malaria reported prepatent periods of
intravenous inoculation of sporozoite
11 days in 2 control patients who received no
suspensions, reported prepatent periods ranging
medication; and, interestingly enough, prepatent
from 11 to 20 days and incubation periods from
periods as short as 10 days in patients receiving
11 to 21 days, with medians of 12 days.
drug suppressively to which this strain was
Burgess and Young (1946) experimentally
resistant. Of interest, also, was the fact that these
transmitted the McLendon strain of falciparum
infections were in patients all of whom had
malaria by bites of Anopheles quadrimaculatus
experienced previous malaria infections. The
and A. freeborni and obtained prepatent periods
incubation periods ranged from 15 to 19 days.
of 15 days and incubation periods of 12 and 18
Lunn et al (1964) and Contacos et al
days in non-immunes. Coatney et al (1947)
(1964), working with a Southern Rhodesian
reported that prepatent periods for 31 mosquito-
strain of P. falciparum, reported prepatent
induced infections of falciparum malaria
periods of 10 and 11 days and 9 to 19 days,
(McLendon strain) ranged from 9 to 13 days
respectively. Powell et al (1965) records
with a mean of 11 days; the incubation periods,
prepatent periods of 9 days for Thailand and
based on the first temperature of 101° F or
Malayan Camp strains of falciparum. In fact,
higher, ranged from 10 to 15 days with a mean
they observed a single 8-day prepatent period in
of 12.2 days.
an individual who had received ineffective
Fairley et al (1947) reported prepatent
antimalarial suppression and/or prophylaxis.
periods ranging from 7 to 12 days (mean 9.5
Chin et al (1967) reported prepatent periods with
days) with New Guinea strains of P. falciparum.
3 chloroquine resistant, or multi-resistant, strains
Kitchen (1949) reported a mean prepatent period
of falciparum malaria, which ranged from 9 to
274 PRIMATE MALARIAS

11 days. Contacos and Collins (1968) and for a week or even longer. Its presence suggests
Collins et al (1968) reported prepatent periods of a high degree of susceptibility by the patient
11 and 12 days for Malayan IV strain infections. and/or a greater invasiveness on the part of the
The studies of Boyd and Kitchen (1937) parasite. Patients showing a remittent course of
seemed to indicate that increases in dosages of fever throughout are more likely to be over-
sporozoites (principally the number of infected whelmed by the infection unless the fever
mosquitoes biting) 1) did not materially change pattern changes over to an intermittent pattern.
the proportion of takes, 2) did not appreciably Most commonly the infection begins and
shorten the prepatent period, and 3) did not continues as an intermittent fever which is more
shorten the incubation period (i.e., elevation of often tertian than quotidian in type. The duration
temperature to 100° F or more). In 60 cases with of the paroxysms tends to be longer than
5 strains, prepatent periods ranged from 6 to 25 observed for vivax and the peak of the
days with a median of 11 days. The incubation paroxysms is usually broken into several
periods ranged from 7 to 27 days with a median secondary peaks. In their experience, patients
of 12 days. They did observe, however, that the whose clinical onset preceded the appearance of
duration of the incubation period tended to vary parasites in the peripheral blood may have an
with the season of the year; being shortest in the abrupt onset, with cerebral symptoms.
4th quarter (mean of 10.1 days) and longest in Jeffery et al (1959) saw that the mean
the 2nd quarter (mean of 13 days). maximum fever varied very little between
As was stated earlier, it has been the intent Panama, McLendon, and Santee-Cooper strain
in this monograph to stress the biologic rather falciparum infections. The mean maximum
than clinical aspects of primate malaria and, temperature recorded for McLendon strain was
especially, the human malarias. Therefore, for a 105.5, for the Panama strain 105.1, and the
description of the various clinical "pernicious Santee-Cooper strain 105.0° F. The maximum
forms" (cerebral, algid, gastrointestinal, etc.) and fever, usually occurred some time between the
blackwater fever (hemoglobinuria), the reader is 5th and 7th day of patent parasitemia. In their
referred to Boyd's Malariology (1949) and experience, the initial fever patterns for each of
James et al (1932) for excellent descriptions of the 3 strains were quotidian, followed by a
these features of falciparum malaria. tertian, and then a remittent type of fever for the
In contrast to P. vivax, P. falciparum Santee-Cooper and McLendon strains, but a
infections are considered to be more malignant remittent and then tertian pattern for the Panama
than the benign vivax. Kitchen (1949) stated that strain. In many cases of Panama strain
most persons who have had experience with falciparum, the fever might more accurately be
falciparum infections "will attest the capacity of described as continuous, rather than remittent.
P. falciparum both to assume a malignant role These authors stated "it is not surprising that
and to evoke protean clinical manifestations." Of there seems to be some confusion in the
all the human malarias, this one is potentially the literature describing the P. falciparum febrile
most dangerous. attack. It is almost impossible to describe a
Boyd and Kitchen (1937) found that 'typical' fever curve for P. falciparum."
falciparum malaria infections exhibit the same Quotidian periodicity prevailed as the initial
general types of febrile reactions that are pattern in their series of infections. Some of
observed in vivax infections, although them remained quotidian until the termination of
pronounced differences did occur. In 60 cases of the infection whereas others converted to tertian
falciparum malaria, the succession of the periodicity. Jeffery et al (1959) emphasized that
different febrile types was not observed as often the most prominent characteristic of the fever
as in vivax and a larger proportion of the patterns, in the 3 strains of falciparum studied by
intermittent fevers were tertian. The onset was them, was extreme variability; in other words,
described, usually, as a remittent fever with a no typical periodicity or pattern could be
tendency to higher fever peaks. Remittent fever, determined.
when present, appears at the onset and may last Boyd and Kitchen (1937) reported that
 PLASMODIUM FALCIPARUM 275

there is no apparent limit to the parasite density a situation where 35 percent of the erythrocytes
which may obtain in P. falciparum infections; were parasitized in a Negro patient who became
the potentialities of this species for comatose and developed renal failure but
multiplication being so great they regarded daily survived this malignant experience.
smears and counts to be essential in following The duration of the primary attack in
the infections. In our studies, falciparum falciparum malaria, according to Boyd and
infections are followed even more closely; Kitchen (1937), is shorter than that of vivax
namely, blood smears are made, stained, and malaria, the mean being 10.8 days and the range,
read every 8 hours to preclude infections getting 2 to 36 days. However, the data of other workers
out of hand. show that the time of such attacks is extremely
Boyd and Kitchen (1945) reported that the variable. Ciuca et al (1955) reported parasitemia
mean interval from the first day of parasitemia continuing for up to 27 months, and African
to the day of maximum parasitemia ranged from strains persist for up to approximately 18 months
4.2 to 11 days for 8 different strains of according to Covell (1960). Some of the
falciparum malaria; the mean for the exotic American strains have persisted for 503 days.
strains was 6.7 and the indigenous strains, 8.9 Verdrager (1964) reported falciparum infections
days. The mean maximum parasite counts for of 3 years duration.
these various strains ranged from 11,140 to Eyles and Young (1951), in summarizing
369,200 per mm3. The mean for the exotic their observations on the duration of
strains was 83,870 and the indigenous strains Plasmodium falciparum infections induced by
103,950 per mm3. sporozoites or parasitized blood, reported that
Coatney et al (1947) reported, in 7 patients, following and including one or more clinical
that parasite densities went above 100,000 per attacks, parasites were present continuously in
mm3 of blood. The maximum parasite densities the blood stream for varying periods of time and
in the primary attacks ranged from 10 to 250,000 that the height of parasitemia in these successive
per mm3, the latter corresponding to 5 to 10 waves tended to become lower and lower as
percent of the red cells. They described 10-fold time went by. The length of this period of
increases or decreases in the parasite count continuous remittent parasitemia had a mean of
within a 12 hour period even in the absence of 121 days with extremes of 32 and 224 days after
treatment. This calls attention to the fact that in the beginning of patent parasitemia. They
developed infections, low parasite counts are no reported further, that the period of continuous
guarantee of a favorable prognosis. In a parasitemia was followed by a period of
Thailand strain (Jeffery et al, 1963), maximum intermittent parasitemia which averaged 100
parasite counts ranged from 3,394 up to 35,022 days with a range of 0 to 283 days. The duration
per mm3 of blood. of infection appeared to be similar, whether
According to Kitchen (1949), a count of infections were induced by mosquito bites
500,000 parasites per mm3 (12.5 percent) (sporozoites) or by parasitized blood. Three of
probably gives a patient about a 50-50 chance of the infections persisted for more than one year,
surviving provided treatment is started the longest being 480 days.
immediately and pernicious symptoms do not Eyles and Young (1951), in studying a
appear. Field and Niven (1937) showed, by group of 13 sporozoite-induced cases, Santee-
analysis of some 750 cases, that the mortality Cooper strain, observed 9 of them throughout
rate increased greatly as parasite counts their infections and for 6 months after the last
increased above 100,000 per mm3 (less than 0.5 parasites were seen. All 13 infections were
percent mortality with counts less than 100,000; observed through the long primary attack with
7 to 20 percent mortality with counts ranging continuous parasitemia. Apparently, 4 had to be
from 100,000 to 500,000; and 63 percent treated because of dangerously high
mortality with counts over 500,000). Chopra et parasitemias. They described the general pattern
al (1932) reported a patient with more than 50 of the infections as characterized by a clinical
percent of the erythrocytes parasitized who died attack which varied from practically
within 12 hours of admission. We have observed
276 PRIMATE MALARIAS

asymptomatic to one of severe dimensions. The (8 weeks) of the termination of clinical activity
clinical period was followed by an but did not observe any clinical reactivation after
asymptomatic period during which patients 24 weeks; whereas James et al (1932) reported
carried parasites in their blood continuously. secondary clinical attacks in 3.3 percent of their
This was then followed by a period of varying infections after 24 weeks. In our studies with
duration during which parasites were only drug resistant strains of falciparum malaria, we
intermittently observed in peripheral smears. have found that recurrences (recrudescences)
The mean length of the initial clinical episode could occur up to 80 days after subcurative or
was 9 days and the mean number of clinical ineffective antimalarial therapy. For this reason,
episodes was 1.4. The mean total hours of fever, we require a 90-day period of follow-up before
over 101.0° F, orally, was 90.4. The median recording a cure.
maximum parasitemia was 65,000 per mm3 of Since 1960, a large number of strains of
blood. The fact that only one clinical episode falciparum malaria have been found resistant to
was observed in most cases and the fact that antimalarial drugs. They are resistant not only to
two-thirds of their original 13 cases were able to chloroquine and other quinolines, but also to
terminate their clinical symptoms spontaneously one, or more, or all, of the synthetic
indicated to them that they were working with a antimalarials, including chlorguanide,
strain of low virulence. pyrimethamine, and mepacrine. In addition,
Jeffery and Eyles (1954) carried out studies some strains from Southeast Asia have shown
on the Panama strain of P. falciparum. Of the 24 varying degrees of resistance to quinine.
sporozoite-induced infections, 12 had attacks The susceptibility of the chimpanzee to
which had to be partially suppressed. The mean falciparum malaria has been studied by several
length of the initial clinical episode was 13.1 workers. Mesnil and Roubaud (1920) failed to
days; the mean number of clinical episodes was infect these animals when exposure was by the
3.3. The mean total hours of fever, over 101.0° bites of infected mosquitoes. Lefrou and
F, was 124.8. The median maximum parasite Martignoles (1954) demonstrated the persistence
count was 49,121 per mm3; the mean maximum of P. falciparum parasites for as long as 3 weeks
parasite count was 73,741 per mm3. The general in 3 of 4 chimpanzees inoculated with blood
pattern of this strain of malaria was very similar containing the parasite. However, Bray (1958,
to that of the Santee-Cooper strain (Eyles and 1960) studied the susceptibility of the
Young, 1951). Probably the most significant chimpanzee to falciparum malaria in detail and
difference was the greater severity of the showed that the sporozoite was able to develop
primary clinical period observed with the in the parenchymal cells of the liver. Although
Panama strain; its clinical attack was described patent parasitemia obtained in the intact
as being quite severe. The mean length of the chimpanzee, it did not persist for more than one
period of continuous remittent parasitemia for or, at the most, 2 cycles. However, if a
the Panama strain was 115.7 days with a range splenectomized chimpanzee was exposed to
of 36 to 220 days. The terminal period of infection by the bites of infected mosquitoes, the
intermittent parasitemia had a mean duration of blood stages appeared, developed, and
168.3 days. The mean total duration of infection multiplied rapidly; the gametocytes failed to
was 279 days with a range of 114 to 503 days. reach maturity (Bray, loc. cit.; Rodhain and
Boyd and Kitchen (1937) observed renewal Jadin, 1964).
of clinical activity after spontaneous cessation of In the Aotus trivirgatus monkey, P.
the primary attack or subcurative therapy of the falciparum often produces very high
primary attack in, roughly, 58 percent of their parasitemias. Geiman and Meagher (1967)
cases. Renewal of clinical activity was observed reported a peak parasitemia, in a splenectomized
as many as 4 times in some patients. Most of the monkey, on the first passage from man, of
recurrences were observed within a period of 8 180,000 per mm3. Subsequent passage into
weeks following the primary attack. Kitchen splenectomized A. trivirgatus monkeys produced
(1949) reported recrudescences within 2 months even higher peak parasitemias, some reaching
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 PLASMODIUM FALCIPARUM 279

FIGURE 58.—Median parasitemia curves of Plasmodium falciparum infections in Aotus trivirgatus monkeys infected by the
intravenous inoculation of parasitized blood from man.

levels as high as 87 percent of the red cells being passage from man to intact monkeys, peak
infected (Geiman et al, 1969). parasitemias of approximately 70,000 per mm3
In our studies, P. falciparum has been were obtained after 14 days of patent
passed from man to the A. trivirgatus monkey on parasitemia. Secondary infections, initiated by a
5 occasions, in intact animals 3 times, and in small number of parasites given to intact
splenectomized animals, twice (see Plate XLIV). monkeys, resulted in peak parasitemias of
We have elected to term sub-passages in approximately 300,000 per mm3 on patent
monkeys as secondary infections. The primary parasitemia day 14 and subsequently declined.
infections had similar courses of parasitemia in However, if a large number of parasites were
both the splenectomized and the intact monkeys given, the parasitemia reached a peak of
(Fig. 58). On subsequent passage (Fig. 59), peak approximately 1,000,000 per mm3 by day 10
parasitemias of approximately 300,000 per mm3 after which the animal, in most cases, died;
obtained in the splenectomized monkeys by day some were saved from death by administering
8 and in intact animals, by day 14. The number antimalarial drugs.
of parasites in the inoculum greatly affected The gametocytes in A. trivirgatus monkeys
subsequent parasitemia in intact A. trivirgatus were readily infectious to mosquitoes (Contacos
monkeys (Fig. 60). During the first, or primary,

PLATE XLIV.—Plasmodium falciparum in the owl monkey, Aotus trivirgatus.

Fig. 1. Normal red cell. Figs. 13-18. Developing schizonts.
Figs. 2-5. Young trophozoites. Figs. 19-21. Nearly mature and mature schizonts.
Figs. 6-10. Growing trophozoites. Figs. 22, 23. Mature macrogametocytes.
Figs. 11, 12. Mature trophozoites. Figs. 24, 25. Mature microgametocytes.
280 PRIMATE MALARIAS

FIGURE 59.—Median parasitemia curves of Plasmodium falciparum infections in Aotus trivirgatus monkeys infected by the
intravenous inoculation of parasitized blood from other A. trivirgatus monkeys.

and Collins, 1968; Collins et al, 1968). This is monkey, Cebus capucinus. The animals were
illustrated in Figure 61 where Anopheles infected by the inoculation of parasitized blood
freeborni mosquitoes were allowed to feed on a from infected A. trivirgatus and C. capucinus
monkey infected with a Malaysian strain of P. monkeys. The prepatent periods ranged up to 30
falciparum. This animal (AO-23) had been days; the periods of patent parasitemia up to 72
infected by blood-passage from man. After a days. Infections reached high parasitemia levels
low, transient course of parasitemia, the animal in 3 of 8 monkeys; the highest was 662,700 per
was able to eliminate the infection. Following mm3. In all cases but one, the infection was self-
splenectomy, the monkey was re-inoculated with limiting.
the same strain of parasite. Eight days after In the squirrel monkey (Saimiri sciureus),
inoculation, the first mosquito infections were P. falciparum infections are remarkably
obtained and these continued for the next 40 transient. Young and Rossan (1969) reported an
days. On 16 of the days, the infection rate was infection in an intact monkey which persisted at
100 percent and, in many instances, the number a detectable level for 21 days with a maximum
of oocysts per gut exceeded 500. parasitemia of 2,210 per mm3. After 49 days,
Young and Baerg (1969) observed parasites were again demonstrable for 3
infections of P. falciparum in the white faced consecutive days, but were not seen again. On
 PLASMODIUM FALCIPARUM 281

FIGURE 60.—Median parasitemia curves of Plasmodium falciparum infections in Aotus trivirgatus monkeys infected by the
inoculation of parasitized blood.

FIGURE 61.—Parasitemia and infectivity of Anopheles freeborni mosquitoes to a strain of Plasmodium falciparum from Malaysia
(Malayan-IV in an Aotus trivirgatus monkey.

one occasion we gave a heavy inoculum to a days after inoculation. Upon reinoculation, the
splenectomized S. sciureus monkey; 3 days animal experienced a low, transient parasitemia.
later, the parasite count was approximately When mosquitoes were allowed to feed, they
120,000 per mm3. This high level was became infected but the infection was low.
maintained for approximately 2 weeks; 20 days Porter and Young (1967) recorded
after infection, the parasite count had dropped to infections of P. falciparum in the marmoset,
15,400 per mm3. Thereafter, the parasitemia Saguinus geoffroyi. In 4 intact animals,
declined rapidly; parasites were last seen 29 inoculated with parasitized blood, patent
282 PRIMATE MALARIAS

infections were seen in from 1 to 2 days; the parasitemia was transient. In splenectomized
periods of patent parasitemia ranged from 4 to animals, however, the parasitemia not only
15 days. The maximum parasite count was persisted but exhibited all forms of the
22,660 per mm3. schizogonic cycle in the peripheral blood.
Gibbons, Hylobates lar, have also been Rodhain and Jadin (1964) also reported the
shown susceptible to infections with P. infection of splenectomized chimpanzees but the
falciparum (Ward et al, 1965; Ward and gametocytes were unable to develop to maturity.
Cadigan, 1966; Gould et al, 1966; Cadigan et al, Ward et al (1965), Ward and Cadigan (1966),
1969). In studies on a large number of animals, Gould et al (1966), and Cadigan et al (1969)
these workers found that once the infection found splenectomized gibbons (Hylobates lar)
became patent, the parasite levels rose rapidly to susceptible to falciparum infection either by
one percent of red cells infected or higher; after sporozoites or by the inoculation of parasitized
about 2 weeks, the parasite counts began to fall. blood.
Subsequent rises occurred, but the parasite Probably the most exciting results relating
counts tended to diminish with succeeding to P. falciparum in non-human primates, in
waves. The median duration of detectable terms of practical laboratory studies, are those
parasitemia was approximately 31 weeks. In involving South American monkeys. Taliaferro
individual animals, this varied from 6 weeks to and Taliaferro (1934) and Taliaferro and Cannon
as long as 72 weeks. The infections did not (1934) were able to infect brown howler
produce overt disease. Experimentally, monkeys, Aloutta palliata (= fusca) by the
splenectomized gibbons can be infected by the inoculation of parasitized blood from man.
inoculation of sporozoites (Gould et al, 1966). However, we had to wait for over 3 decades
Attempts to infect mosquitoes were before Geiman and Meagher (1967), Contacos
unsuccessful. and Collins (1968), Collins et al (1968), Geiman
Cadigan et al (1966) reported the successful et al (1969), and Voller et al (1969) showed that
infection of splenectomized M. mulatta siamica, splenectomized and intact owl monkeys, A.
M. nemestrina, and M. irus (= fascicularis) trivirgatus, were highly susceptible to infection
monkeys with P. falciparum which had been with P. falciparum. In 1969, Sodeman et al
adapted to the gibbon, H. lar. After periods of demonstrated that EE bodies would develop
14, 6, and 10 days, detectable parasitemias only partially in the owl monkey and that blood
persisted for 18, 19, and 16 weeks, respectively. infections did not result from sporozoite
Peak parasitemias ranged from 850 to 2,420 per inoculation. Infections have also been obtained
mm3. In general, the parasitemias were very low. in the white faced monkey, Cebus capucinus,
Passages from man to M. fascicularis monkeys (Young and Baerg, 1969), the squirrel monkey,
by the inoculation of parasitized blood produced Saimiri sciureus, (Young and Rossan, 1969),
infections in 4 of 5 animals. The parasitemias and the marmoset, Saguinus geoffroyi, (Porter
were of low grade but persisted for as long as 19 and Young, 1967),
weeks. Cadigan et al (1966) reported low-order
infections in splenectomized M. mulatta
Host Specificity siamica, M. nemestrina, and M. irus (=
fascicularis).
Plasmodium falciparum naturally infects
A great number of anopheline mosquitoes
man only. Experimentally, however, infections
have been shown to be natural or experimental
have been obtained in a number of primates.
hosts for P. falciparum. Garnham (1966) lists 66
Early attempts to infect chimpanzees with P.
species as hosts for this parasite, and no doubt,
falciparum resulted in failure (Mesnil and
many more are susceptible to infection. We have
Roubaud, 1920; Blacklock and Adler, 1922;
made only limited studies in this area. Among
Rodhain, 1939). Bray (1958), however, obtained
those we have examined (Table 35), A. freeborni
infections in chimpanzees (Pan troglodytes
was the most susceptible with either the
versus) by the intravenous inoculation of
McLendon or Malayan IV strains. These results
infected salivary glands. In intact animals, the
and those of our previous studies (Collins, 1962;
 PLASMODIUM FALCIPARUM 283

Collins et al, 1963, 1964) have confirmed the analyzed according to race, suggested that race
opinion of many investigators that the infectivity did not play a significant role in regulating
of isolates of P. falciparum to different and/or modifying the prepatent or incubation
anophelines is dependent to some extent on the periods. However, the Negro does have a
geographical origin of either the parasite or the tendency to be able to clinically tolerate
mosquito. This is well illustrated in Table 35 in falciparum malaria infections better than the
which it is shown that with the McLendon strain Caucasian.
from southern United States, the gut infection The presence of genetic traits which inhibit
index ratio between A. freeborni and A. parasite multiplication or survival, such as sickle
quadrimaculatus was 100:38, whereas with the cell hemoglobin and enzyme deficiencies, have
Malayan IV strain from southeast Asia, the ratio been reported but are not universally accepted. It
was 100:4.3. The results of comparative has been suggested, that possession of the sickle
infectivity studies are so variable between cell trait or sickle cell character has a "tolerating
different isolates of P. falciparum that it is often effect" on the course of falciparum malaria in
necessary to feed a number of species on the individuals with these hemoglobin types; in
host of a "new" isolate in order to determine other words, individuals with sickle cell type
which of the species available will serve as hemoglobin are rather poor hosts for
suitable experimental vectors. For example, with Plasmodium falciparum.
the Panama strain of P. falciparum, A. Allison (1954) and Mackey and Vivarelli
albimanus mosquitoes are very good hosts (1954) suggested that sickling hemoglobin,
(Jeffery et al, 1950), but with the Thailand present in erythrocytes of carriers of sickle cell
strain, it is almost refractory (Collins et al, trait, was not as favorable for the development
1963). of malaria parasites as normal hemoglobin.
Immunity and Antigenic Allison (1954a, 1961, 1963) obtained good
correlation between the incidence of sickle cell
Relationships trait and the degree of malarial infection.
There appears to be no racial or innate Regions in East Africa, where malaria is
immunity among Negroes against falciparum hyperendemic, had a higher incidence of sickle
malaria as has been observed for vivax malaria. cell trait than did regions where malaria was
The results obtained by Kitchen (1949), when

TABLE 35.—Comparative infectivity of two strains of Plasmodium falciparum to different species of Anopheles.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other
McLENDON STRAIN
F-1 100
F-1 : Q-1 8 100 131 86.0 78.6 38.0
F-1 : Alb 12 300 175 42.7 29.1 32.5
F-1 : Mac 3 45 57 100.0 79.2 23.8

MALAYAN IV STRAIN
F-1 100
F-1 : St-1 6 81 127 35.8 19.7 28.8
F-1 : Mac 6 56 111 60.7 9.0 13.4
F-1 : Q-1 10 147 223 37.4 5.4 4.3
F-1 : Bal 8 147 136 37.4 11.8 2.0

* F-1 = Anopheles freeborni; Q-1 = A. quadrimaculatus; Alb = A. albimanus; Mac = A. maculatus; St-1 = A. stephensi;
Bal = A. b. balabacensis.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. freeborni to another
species where the GII of A. freeborni = 100.
284 PRIMATE MALARIAS

epidemic or absent. Allison suggests that the falciparum parasites induce "a highly effective
sickle cell trait may confer a certain degree of degree of premunition in the indigenous host in
resistance against malaria and, for that reason, the sense that the children look thoroughly
the trait survives more successfully where the healthy, although 80 percent or more have a
malaria infection rates are more severe. heavy load of parasites in the blood, while the
It is thought, in some quarters, that the adults appear strong, well grown, and energetic,
parasite cannot effectively utilize the abnormal with a relatively low parasitemia." However, if
hemoglobin in the sickled cell. Mackay and residents of the endemic areas move to other
Vivarelli (1954) reported that sickle cells were endemic areas, and are exposed to new strains,
seldom parasitized in a blood film from an they develop very heavy infections.
individual with sickle-cell trait even when the Most studies have indicated that immunity
sickle cells predominated. Miller et al (1956), on to malaria is a residual immunity acquired
the other hand, reported results contrary to those through infection and that this immunity is not
of Mackay and Vivarelli; namely, that life-long. Rather, it is usually of short duration
falciparum malaria parasites can and do, enter unless the individual is infected repeatedly.
and develop readily in cells which undergo Boyd et al (1936) concluded that homologous
sickling. In 2 of their 3 cases, parasites were but not heterologous immunity is acquired
found as frequently in sickled cells as in through infections of P. falciparum. This is
nonsickled cells. contrary to what they observed with vivax
Miller et al (1956) suggested that it was not malaria. In falciparum malaria, they found that
necessarily the sickle cell hemoglobin alone reinoculation with a different strain resulted in
which was detrimental to the development of the an infection oftentimes as severe as the first. The
falciparum parasites, but other factors were homologous immunity lasted approximately 4
functioning, too. When the parasitized red cells, months. The latter is generally characterized as a
containing sickle cell hemoglobin, adhere to the clinical immunity; namely, no clinical attack
walls of blood vessels, as falciparum infected with or without low patent parasitemia--if you
red cells have a tendency to do, the oxygen will--an asymptomatic attack. The degree of
supply to these parasitized cells would be very immunity, acquired through infection, is
much decreased; and, under such circumstances, probably dependent on the parasite densities and
the loss of oxygen would bring about a relative the duration of the parasitemia. Ciuca et al
anoxia which could conceivably induce sickling (1934) observed that repeated inoculations with
of the parasitized erythrocyte, thereby falciparum malaria progressively increased the
interfering with the multiplication of the degree and/or the duration of immunity.
parasite. Boyd et al (1939) recorded absence of
Another genetic trait which acts adversely cross-immunity between vivax and falciparum
is the deficiency of the enzyme glucose-6- malarias whether the reinoculations were
phosphate dehydrogenase (G6PD). High effected during the incubation period, the acute
frequencies of this deficiency are found in primary attack, or shortly after termination of
malarious areas, and especially, in those that attack.
endemic for falciparum malaria. It has been However, Boyd and Kitchen (1945)
suggested that such a deficiency may limit reexamined and modified their opinions
parasite multiplication. Allison and Clyde concerning heterologous immunity between
(1961) found a significant lowering of the falciparum malarias. They stressed the fact that
parasite rate in males deficient in G6PD and the the primary attacks of falciparum malaria were
proportion of falciparum infections with parasite more severe in Caucasian than in Negro patients.
counts above 1,000 per mm3 was significantly In Caucasians, the primary infections usually
lower in G6PD deficient children than in normal consisted of 2 or 3 successive waves of patent
children. parasitemia whereas in Negro patients only one
Acquired immunity to falciparum malaria, wave of patent parasitemia was commonly
as with the other human malarias, is strain observed. Generally, the heterologous immunity
specific. Garnham (1966) stated that P. was characterized by a shortened period of
 PLASMODIUM FALCIPARUM 285

clinical activity. In addition, reinoculation of barrier when they treated falciparum infections
Caucasian patients with heterologous strains with gamma globulin prepared from cord blood.
resulted in infections similar to the primary If one turns to serology for insight into
infections in Negro patients; namely, one wave relationships between various primate malarias,
of patent parasitemia with parasite densities some interesting facts emerge. In this regard,
lower in the Negro patients. Their final Kuvin and Voller (1963) studied the differences
conclusion was that immunity acquired from a in response of sera from 26 individuals from
falciparum malaria infection has "appreciable West Africa, previous malarial histories
heterologous value." unknown, against P. falciparum and B strain P.
That humoral immunity exists in cynomolgi antigens. The mean titers of the
falciparum malaria was shown by Cohen et al former were 1:24 versus 1:18 for P. cynomolgi
(1961) and Cohen and McGregor (1963). They indicating a high level of cross-reactivity. Diggs
demonstrated that malarial immunity can be and Sadun (1965), using sera from known
transferred passively in the 7S fraction of infections of P. falciparum and P. vivax,
gamma globulin of hyperimmune serum from quantitated the levels of cross-reactivity. Their
adults in hyperendemic areas of the Gambia. P. falciparum antisera gave a homologous
When they administered such gamma globulin response, as measured by the geometrical mean
preparations in large doses to acutely ill of the reciprocal titers, of 1:28.2 and a
Gambian children with heavy infections, a heterologous response of 1:6.3. Using the P.
consistent pattern of response was observed; vivax antisera, the homologous geometrical
namely, rapid clinical recovery and a highly mean of the reciprocal titers was 1:17.2 and the
significant reduction in parasitemia. This was heterologous mean titer was 1:9.3. Collins et al
confirmed by Edozien et al (1962) in Nigeria. (1966) were able to show that antiserum to P.
McGregor (1964) was of the opinion that falciparum would react with a number of simian
immune 7S gamma globulin acted against the malaria antigens but that the highest
late asexual forms (schizonts) or the liberated heterologous response was to P. fieldi.
(extracellular) merozoites. Meuwissen (1968) demonstrated that although
That humoral malarial immunity could be the antisera to P. falciparum would react at a
passively transferred from mother to offspring high level to both the B strain P. cynomolgi and
had been postulated for years. This was P. fieldi antigens, the highest heterologous
confirmed by Edozien et al (1962) when they response was to the latter.
demonstrated that antimalarial antibodies
(gamma globulins) can cross the placental
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COATNEY, G. R. and YOUNG, M. D., 1941. The taxonomy of relation to parasite counts in acute subtertian malaria.
the human malaria parasites with notes on the principal Trans. Roy. Soc. Trop. Med. & Hyg. 30 : 569-574.
American strains. Am. Assoc. Adv. Sci. (No.15), 19-24. FIELD, J. W. and SHUTE, P. G., 1956. The microscopic diagnosis
COATNEY, G. R., COOPER, W. C., YOUNG, M. D., and of human malaria. II. A morphological study of the
McLENDON, S. B., 1947. Studies in human malaria. I. erythrocytic parasites. Inst. Med. Res., Fed. Malaya,
The protective action of sulfadiazine and sulfapyrazine No.24. pp. 251.
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GARNHAM, P. C. C., 1966. Malaria parasites and other Zool. exp. 2 : 55-258.
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GEIMAN, Q. M. and MEAGHER, M.J., 1967. Susceptibility of a Bull. Acad. Med. 9 : 1235-1236.
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GEIMAN, Q. M., SIDDIQUI, W. A., and SCHNELL, J. V., 1969. trouve dans le sang des malades atteints de fievre
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Feletti 1892) Schaudinn 1902. The correct name for the Exot. 47 : 895-903.
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Soc. Wash. 2 : 90-91. Einteilung der Malariaparasiten. Zentralbl. Bakt. 1 Abt.
GOULD, D. J., CADIGAN, F. C., JR. and WARD, R. A., 1966. Orig. 21 : 130-133.
Falciparum malaria: transmission to the gibbon by LUNN, J. S., CHIN, W., CONTACOS, P. G., and COATNEY, G.
Anopheles balabacensis. Science 153 : 1384. R., 1964. Cycloguanil pamoate (CI-501) as a causal
GRASSI, B., 1891. Weiteres zur Malariafrage. II. Verschiedene prophylactic against a Southern Rhodesian strain of
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Antimalarial drug trials on a multiresistant strain of plasmodiums humains. Ann. Soc. Beige de Med. Trop.
Plasmodium falciparum. Am. J. Trop. Med. & Hyg. 12 19 : 563-572.
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KITCHEN, S. F., 1949. Falciparum malaria. Malariology, Vol. II, Plasmodium falciparum au chimpanzé splénectomise.
edited by Mark F. Boyd. W. B. Saunders Co., Phila. Ann. Sac. Beige Med. Trop. 44 : 531-536.
KUVIN, S. F. and VOLLER, A., 1963. Malarial antibody titres of ROSS, R., 1897. On some peculiar pigmented cells found in two
West Africans in Britain. Brit. Med. Jour. 2 : 477-479. mosquitoes fed on malarial blood. Brit. Med. Jour. 2 :
1786-1788.
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SERGENT, ED., SERGENT, ET., and GATANEI, A., 1929. Une Alouatta sp. II. Cellular reactions. Am. J. Hyg. 19 :
question de nomenclature le nom de Plasmodium 335-342.
praecox doit-il designer un parasite du paludisme THAYER, W. S. and HEWEWTON, J., 1895. The malarial fevers
humain ou un parasite du paludisme aviare? Arch. Inst. of Baltimore. Johns Hopkins Hosp. Reports 5 : 1-218.
Pasteur Alger. 7 : 223-238. VERDRAGER, J ., 1964. Observations on the longevity of
SERGENT, ED., SERGENT, ET., PARROT, L., and GATANEI, Plasmodium falciparum: with special reference to
A., 1939. Nomenclature of the malaria parasites. Brit. findings in Mauritius. Bull. Wid. Hlth. Org. 31 : 747-
Med. Jour. 1 : 747-748 and Arch. Inst. Pasteur Alger. 751.
17 : 242-243. VOLLER, A., RICHARDS, W. H. G., HAWKEY, C. M., and
SHORTT, H. E., FAIRLEY, N. H., GOVELL, G., SHUTE, P. G., RIDLEY, D. S., 1969. Human malaria (Plasmodium
and GARNHAM, P. C. C., 1949. The pre-erythrocytic falciparum) in owl monkeys (Aotus trivirgatus). J.
stage of Plasmodium falciparum. A preliminary note. Trop. Med. & Hyg. 72 : 153-160.
Brit. Med. Jour. 2 : 1006-1008. WARD, R. A. and CADIGAN, F. C., JR., 1966. The development
SHORTT, H. E., FAIRLEY, N. H., COVELL, G., SHUTE, P. G., of erythrocytic stages of Plasmodium falciparum in the
and GARNHAM, P. C. C., 1951. The pre-erythrocytic gibbon, Hylobates lar. Mil. Med. 131 : 944-951.
stage of Plasmodium falciparum. Trans. Roy. Soc. WARD, R. A., MORRIS, J. H., GOULD, D. J., BOURKE, A. T.
Trop. Med. & Hyg. 44 : 405-419. C., and CADIGAN, F. C., JR., 1965. Susceptibility of
SHUTE, P. G, and MARYON, M., 1952. A study of human the gibbon Hylobates lar to Plasmodium falciparum.
malaria oocysts as an aid to species diagnosis. Trans. Science 150 : 1604-1605.
Roy. Soc. Trop. Med. & Hyg. 46 : 275-292. WELCH, W. H., 1897. Malaria: definition, synonyms, history, and
SODEMAN, T. M., CONTACOS, P. G., SMITH, C. S., JUMPER, parasitology. Loomis and Thompson, Syst. Practice
J. R., and COLLINS, W. E., 1969. The exoerythrocytic Med. 1 : 17-76. Lea Bros. & Co., New York.
stages of Plasmodium falciparum in Aotus trivirgatus. YOUNG, M. D. and BAERG, D. C., 1969. Experimental
J. Parasit. 55 : 682-683. infections of Plasmodium falciparum in Cebus
SOPER, F. L. and WILSON, D. B., 1943. Anopheles gambiae in capucinus (white faced capuchin) monkeys. Mil. Med.
Brazil 1930 to 1940. The Rockefeller Foundation, New 134 : 767-771.
York. YOUNG, M. D. and ROSSAN, R. N., 1969. Plasmodium
TALIAFERRO, W. H. and TALIAFERRO, L. G., 1934. The falciparum induced in the squirrel monkey, Saimiri
transmission of Plasmodium falciparum to the howler sciureus. Trans. Roy. Soc. Trop. Med. & Hyg. 63 : 686-
monkey, Alouatta sp. I. General nature of infections 687.
and morphology of the parasites. Am. J. Hyg. 19 : 318-
334. (NS) = Not seen.
TALIAFERRO, W. H. and CANNON, P. R., 1934. Transmission
of Plasmodium falciparum to the howler monkey,
 23
Plasmodium coatneyi Eyles, Fong, Warren,
Guinn, Sandosham, and Wharton, 1962

mulatta, without delay. The monkey exhibited

IN the course of studies infection after a prepatent period of 14 days. The
on simian malaria begun by the late Dr. Don parasite was first taken to be Plasmodium
Eyles in Malaya, he and his co-workers isolated knowlesi on morphological grounds but when
a new species of malaria from a wild caught the periodicity was found to be tertian, rather
anopheline mosquito, Anopheles hackeri. This than quotidian, it was obvious that the
unique experience is the first instance of finding investigators were dealing with an undescribed
a new species of malaria in the vector before it species which they named Plasmodium coatneyi
was known from the primate host. The mosquito in honor of the American malariologist Dr. G.
was taken in the nipah palm area of Kampong Robert Coatney. Later, Eyles et al (1962)
Rantau Panjang near the town of Klang in the isolated P. coatneyi from a kra monkey, M. irus
State of Selangor in November, 1961. Upon (= fascicularis) taken in the same area as the
dissection, the mosquito was found to be original infected mosquito and then again in
positive with sporozoites which were injected 1963 (Eyles et al, 1963) from the blood of an M.
into an uninfected rhesus monkey, Macaca irus (= fascicularis) from the Philippines.

289
 PLASMODIUM COATNEYI 291

Cycle in the Blood The macrogametocytes take a medium blue

stain with a red nucleus, generally eccentric,
PLATE XLV enclosing a deeper staining irregular area. The
The young ring forms of P. coatneyi closely
pigment, scattered in the cytoplasm, is
resemble those of the human parasite, P.
prominent and rice-grain shaped (Fig. 24). The
falciparum. The youngest ring forms are smaller
microgametocyte stains reddish-purple and has a
than the rings of other simian parasites (not
large circular mottled nucleus which may show a
shown on plate) except, possibly, P. knowlesi.
deeper staining bar. The pigment is dark to
The typical young trophozoite has a single or
yellowish-brown and sometimes found entirely
double chromatin body (Figs. 2-5) but they may
within a vacuole (Fig. 25).
number up to four. The position in the host cell
The parasite, as pointed out by Eyles (1963)
is varied as is true with the same age growth
has a penchant for invading reticulocytes.
forms of P. falciparum. Marginal, appliqué or
Warren et al (1966) possessed a greater amount
accolé forms are common (Fig. 6), often
of material, and employed statistical methods to
considered diagnostic of P. falciparum along
show that the parasite selectively invades mature
with displaced vesicular forms (Fig. 7) with one
erythrocytes.
or two nuclei. In heavy infections, host cells
In 1968, Rudzinska and Trager, after
may harbor two or more young parasites (Fig.
studying the fine structure of the parasite and its
8); band and tenue forms are not uncommon
host cell, were able to show that the trophozoites
(Figs. 9, 10). In some instances the host cell
do not have typical protozoan mitochondria, but
carrying the young parasites is smaller than the
they do have a double-membraned organelle
normal cell.
which, it is assumed, carries out the functions of
As the trophozoites mature, their number in
the mitochondria. The young parasite feeds on
the peripheral blood becomes less. The usual
the host cell by pinocytosis, taking in portions of
form is circular or oval, usually with a vacuole
the erythrocytes through invaginations of the
and with intense blue cytoplasm (Figs. 11-13).
plasma membrane or through the cytostome.
The youngest of these forms rarely show
Digestion of the hemoglobin takes place in small
pigment, but as the parasite grows the granules
vesicles derived from the food vacuole. The
become prominent; they do not coalesce.
macrogametocytes have two plasma membranes;
Maurer's spots or clefts are prominent and
the inner one thickened in places. The cytoplasm
characteristic of this species. These were
displays Palade's particles, has toxonemes and
originally described by Eyles et al (loc. cit.) and
vesicles of endoplasmic reticulum. The
their fine structure more recently by Rudzinska
microgametocytes have the whole inner
and Trager (1968). The bluish cast to the
membrane thickened, the cytoplasm displays
cytoplasm in Figures 11-13 probably illustrates
few Palade's particles and there are no
these spots with Giemsa stain.
toxonemes.
The early schizonts stain a deep blue, are
The host cells with trophozoites are
compact, round, and occupy at least half the host
irregularly shaped and show elevated points with
cell. The pigment remains granular with a
knob-like projections and a double membrane.
tendency to coalesce (Figs. 14-16). The older
The host erythrocyte has numerous Mauer's
and the mature schizonts fill the host cell and
clefts which, because they are sometimes
produce about 20 merozoites (Figs. 22, 23).
continuous with the membranes of the parasite,

PLATE XLV.—Plasmodium coatneyi.

Fig. 1. Normal red cell. Figs. 18-21. Developing schizonts.
Figs. 2, 3, 6, 7. Young trophozoites. Figs. 22, 23. Nearly mature and mature schizonts.
Figs. 4, 5, 8-11. Growing trophozoites. Fig. 24. Mature macrogametocyte.
Figs. 12, 13. Mature trophozoites. Fig. 25. Mature microgametocyte.
Figs. 14-17. Early schizonts.
292 PRIMATE MALARIAS

suggests that they may take their origin from animals were splenectomized, mosquito
them. infections followed almost immediately with the
The asexual cycle in the blood occupies 48 intensity of the infection in the mosquitoes
hours. usually correlated with the 48-hour asexual
periodicity (Fig. 62).
Sporogonic Cycle A comparison of the mean oocyst diameters
of P. coatneyi with P. cynomolgi (Fig. 63)
PLATE XLVI indicates that P. coatneyi is a smaller parasite
Warren and Wharton (1963) were able to
and it requires one day longer for the sporozoites
infect A. kochi, A. letifer, A. maculatus, A.
to appear in the salivary glands.
sundaicus, and A. vagus but they made no
The sporozoites in A. b. balabacensis were
comments on the development of the oocysts.
shown to be infective; infections were
Eyles (1963) in commenting on the sporogonic
transmitted to 6 M. mulatta monkeys by
cycle of P. coatneyi reported that no
mosquito bites with prepatent periods from 10 to
distinguishing characteristics were seen.
15 days with a mean of 13.2 days. Dissected
Subsequently Collins et al (1967) reported the
salivary glands and triturated bodies of A.
infection of Anopheles b. balabacensis and A.
freeborni mosquitoes infected with P. coatneyi
freeborni but only the latter consistently
were inoculated into 5 M. mulatta monkeys.
produced sporozoites in the salivary glands.
Three of the animals developed an infection with
More recently, studies were made to determine
prepatent periods of 14, 15, and 15 days,
the growth rate of the oocysts of P. coatneyi in
respectively. We do not know if A. maculatus
A. b. balabacensis, A. maculatus, and A.
will transmit this parasite although we have seen
freeborni. The results of these observations were
seemingly viable sporozoites in their glands.
presented in Table 36.
Three attempts to transmit the infection by bites
In A. b. balabacensis, the oocysts at day 6
of A. freeborni mosquitoes to rhesus monkeys
had a mean diameter of 19 µ with a range of 12
have failed.
to 26 µ. The oocysts continued to grow so that
by day 11, the mean size was 61 µ with a range
of 24 to 90 µ and sporozoites were present in the Cycle in the Tissue
salivary glands. PLATE XLVII
In A. maculatus, the oocysts appeared to Following the Held et al (1967) technique
slow down in their rate of growth after day 7. of intrahepatic inoculation of sporozoites, Held
However, oocyst differentiation was seen as and Contacos (1967) carried out a detailed study
early as day 9 and sporozoites, though scarce, of the growth stages of P. coatneyi in the rhesus
were present in the salivary glands on day 12. monkey. Liver biopsies were done on days 6, 7,
The mean diameters of the oocysts in A. 8, 9, 10, and 11 following the introduction of
maculatus were considerably smaller on days 8 sporozoites; and growth forms for each of the
through 11 than were those of A. b. days, except day 11, were described and
balabacensis. illustrated in a series of 69 figures. The 6-day
In A. freeborni, the development was forms measured 19 to 22 µ and the oldest forms,
apparently normal through day 10. After day 10, i.e., 10-day measured 40 to 48 µ. Different
there was no evidence of further development; parasites studied on the same day demonstrated
by day 12, many of the oocysts were in various the wide extent of heteromorphism in the species
stages of degeneration. No sporozoites were which served to confirm their opinion that the
found in the salivary glands although as tissue stages do not exhibit morphological
indicated earlier (Collins et al, 1967) low level characteristics which will allow for the
infections of the salivary glands of A. freeborni separation of species.
have been found. An interesting sidelight was
that fully developed infections in intact animals, Course of Infection
carrying abundant gametocytes, were rarely
The natural host of P. coatneyi is Macaca
infectious to mosquitoes. However, once the
irus (= fascicularis), the kra monkey, and in that
 PLASMODIUM COATNEYI 293

PLATE XLVI.—Developing oocysts of Plasmodium coatneyi in Anopheles b. balabacensis mosquitoes. X 580.

Fig. 1. 6-day oocyst showing clumped pigment. Fig. 6. 9-day oocyst.
Fig. 2. 6-day oocyst showing linear arrangement of Fig. 7. 10-day oocyst showing early stages of
pigment. differentiation.
Fig. 3. 7-day oocyst showing two clumps of pigment. Fig. 8. 11-day differentiated oocyst.
Fig. 4. 7-day oocyst showing large clump of pigment. Fig. 9. Fully differentiated 11-day oocyst showing
Fig. 5. 8-day oocyst. withdrawal of sporozoite mass from oocyst wall.
294 PRIMATE MALARIAS

animal, the parasite produces a mild low-grade the parasite remained unchanged and continued
infection that persists for a long time. When the to express the tertian cycle.
infection is transferred to clean, laboratory- In the rhesus monkey, M. mulatta (Fig. 65),
reared M. fascicularis by blood inoculation, the blood-induced infections may be explosive with
peak parasitemias range from 15,000 to 57,000 peak counts greater than 500,000 per mm3,
per mm3 with the older parasites retreating from resulting in death of a large proportion of the
the peripheral circulation (Fig. 64). animals (40 percent of our test animals) unless
Other monkeys, Presbytis cristatus, M. the infection is treated with schizontocidal drugs
nemestrina, and M. speciosa (= arctoides) were well ahead of the crisis. Sporozoite-induced
more resistant to infection than M. fascicularis infections in intact M. mulatta monkeys had a 33
(Fig. 64); gibbons, Hylobates lar, either refused percent mortality rate. The mortality rate in
the infection or allowed it to run a very low splenectomized M. mulatta monkeys was 100
course. In each of the hosts, the morphology of percent.

TABLE 36.—Oocyst diameters of Plasmodium coatneyi in Anopheles b. balabacensis, A. maculatus, and A. freeborni.

Days after A. b. balabacensis A. maculatus A. freeborni

Infection
No. Range* Mean No. Range Mean No. Range Mean
5 114 8-19 14
6 114 12-26 19 111 12-26 21 188 12-30 19
7 125 14-40 25 107 14-40 25 104 14-45 26
8 134 19-60 37 111 20-51 31 101 18-51 34
9 119 17-67 44 134 21-65 39† 140 12-66 41†
10 107 14-74 54† 122 20-54 38† 124 19-80 53†
11 103 24-90 61†** 124 18-70 43† 111 18-78 54†
12 131 26-63 44†** 74 21-90 49†‡
Totals 702 12-90 840 12-70 956 8-90

* Measurements expressed in microns; incubation temperature 25º C.

† Oocyst differentiation.
‡ Oocyst degeneration.
** Sporozoites present in the salivary glands.

FIGURE 62.—Infectivity of Plasmodium coatneyi to Anopheles freeborni mosquitoes when fed on a splenectomized Macaca
mulatta monkey.
 PLASMODIUM COATNEYI 295

FIGURE 63.—Range in oocyst diameters and the mean oocyst diameter curve of Plasmodium coatneyi and P. cynomolgi in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

Infections induced through the bites of A. b. Host Specificity

balabacensis in intact rhesus monkeys appear to
In nature, P. coatneyi appears to be limited
follow the general pattern of blood-induced
to the natural host, Macaca fascicularis, of
infections. Three such animals (Fig. 66)
peninsular Malaysia and the Philippines (Eyles
exhibited prepatent periods of 10, 11, and 14
et al, 1962; 1963). The best experimental host is
days after which the initial parasitemia climbed
the rhesus monkey, Macaca mulatta, which is
rapidly to peak counts of 160,000 to 800,000 per
highly susceptible to infection either by the
mm3 between the 7th and 9th days of patent
inoculation of parasitized blood or by
parasitemia only to decline, and then, exhibit a
sporozoites. Attempts to infect other simian
second rise some three weeks later. After the
hosts, the silvered leaf coloboid P. cristatus, M.
second rise, the parasitemia continued to decline
arctoides, and M. nemestrina have been
but evidenced the alternate high and low parasite
successful but the infections were all of a low
counts during an observation period of 60 days.
order. Following the discovery of this parasite
with its falciparum-like characteristics,
296 PRIMATE MALARIAS

PLATE XLVII.—Exoerythrocytic bodies of Plasmodium coatneyi in liver tissue of Macaca mulatta monkeys. X 580 (Except Fig. 6).
Fig. 1. 6-day body. Fig. 6. 9-day body. X 740.
Fig. 2. 7-day body. Fig. 7. 10-day body.
Fig. 3. 7-day body showing three prominent vacuoles. Fig. 8. 10-day body showing two prominent vacuoles.
Fig. 4. 8-day body showing abundant large flocculi. Fig. 9. 10-day body.
Fig. 5. 9-day body showing abundant, irregular-shaped
flocculi.
 PLASMODIUM COATNEYI 297

FIGURE 64.—Median parasitemia curves of infections of Plasmodium coatneyi in 8 Macaca irus ( = fascicularis), 3 M. speiosa,
( = arctoides), 3 M. nemestrina and 3 Presbytis cristatus monkeys.

FIGURE 65. Median parasitemia curves of infections of Plasmodium coatneyi in 77 intact (66 blood-induced and 11 sporozoite-
induced infections) and 6 splenectomized Macaca mulatta monkeys.

investigators were anxious to learn if the parasite that among 6 rhesus monkeys, M. mulatta,
would express the same characteristics if it exposed at the same time as the volunteers, and,
became established in man. However, to date all to bites of the same mosquitoes, five developed
attempts in that direction have failed. Garnham normal patent infections.
(1965) reported the transfer of parasitized blood Warren and Wharton (1963) were of the
from a rhesus monkey to a paretic patient and opinion, based on finding P. coatneyi in A.
we (1963 and 1967) made three unsuccessful hackeri, that the vector was zoophilic. They
attempts, over a four-year period, to infect nine were able to obtain infection, through the
volunteers with observation periods extending development of oocysts, in: A. maculatus, A.
from 90 to 180 days after biting episodes kochi, A. sundaicus, A. vagus, A. philippinensis,
utilizing A. freeborni and A. b. balabacensis and A. letifer. More recently we have infected A.
mosquitoes. It is of interest in this connection
298 PRIMATE MALARIAS

FIGURE 66.—Course of parasitemia in three Macaca mulatta monkeys infected with Plasmodium coatneyi by sporozoite
inoculation.

b. balabacensis, A. freeborni, A. stephensi, A. knowlesi, P. inui, and P. cynomolgi. The P.

albimanus, A. atroparvus, A. quadrimaculatus, knowlesi infections were severe but not fatal
and A. maculatus. Among the 13 species of indicating some degree of protection; the P. inui
mosquitoes known to be susceptible, only four: and P. cynomolgi infections were normal
A. b. balabacensis, A. hackeri, A. maculatus, and indicating no dampening of the infection due to
A. freeborni have carried the infections to the infection with P. coatneyi. Voller et al (1966)
production of sporozoites in the salivary glands. concluded there was considerable cross
The susceptibility to infection with P. coatneyi immunity between P. knowlesi, P. coatneyi, and
varies (Table 37); A. b. balabacensis was the P. fragile. However, Voller and Rossan (1969)
most susceptible followed by A. freeborni, A. demonstrated that rhesus monkeys with chronic
maculatus, A. stephensi, A. albimanus, A. P. knowlesi infections were susceptible to
atroparvus, and A. quadrimaculatus. The infection with P. coatneyi.
feedings with the other species were too limited Antisera to P. coatneyi gave a fluorescent
to permit proper evaluation. antibody cross-reaction at a very high level to P.
fieldi (mean reciprocal titer ratio of 100:107) but
Immunity and Antigenic reacted at a much lower level to other primate
malaria antigens (Collins et al, 1966), In the
Relationships reverse procedure, P. coatneyi antigen cross-
reacted highest to P. inui (mean reciprocal titer
In order to elucidate some aspects of ratio of 100:57) and at a much lower level to the
immunity, Eyles (1963), challenged rhesus P. cynomolgi and P. knowlesi antigens (mean
monkeys harboring chronic P. coatneyi reciprocal titer ratios of 100:27).
infections with superinfections. These animals
were inoculated with parasitized blood of P.
 PLASMODIUM COATNEYI 299

TABLE 37.—Comparative infectivity of Plasmodium coatneyi to Anopheles b. balabacensis, A. freeborni, A. maculatus,

A. stephensi, A. albimanus, A. atroparvus, and A. quadrimaculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other
Bal 100
Bal : F-1 20 288 389 22.6 47.6 45.7
Bal : Mac 10 93 511 67.7 46.4 15.3
Bal : St-1 8 70 133 35.7 6.0 2.9
Bal : Alb 10 163 144 53.4 1.4 0.4
Bal : Atro 5 60 166 80.0 1.2 0.15
Bal : Q-1 6 62 144 80.6 1.4 0.08

* Bal = Anopheles b. balabacensis, F-1 = A. freeborni, Mac = A. maculatus, St-1 = A. stephensi, Alb = A. albimanus, Atro = A. atroparvus, Q-
1 = A. quadrimaculatus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. b. balabacensis to
another species where the GII of A. b. balabacensis = 100.

REFERENCES
COLLINS, W. E., CONTACOS, P. G., GUINN, E. G., and HELD, the study of the exo-erythrocytic stages of simian
J. R., 1967. Studies on the transmission of simian malarias. J. Parasit. 53 : 656-657.
malaria. III. Infection and transmission of Plasmodium HELD, J. R. and CONTACOS, P. G., 1967. Studies of the
coatneyi with Anopheles freeborni and A. balabacensis exoerythrocytic stages of simian malaria. II.
balabacensis mosquitoes. J. Parasit. 53 : 1130-1134. Plasmodium coatneyi. J. Parasit. 53 : 910-918.
COLLINS, W. E., SKINNER, J. C., and GUINN, E. G., 1966. RUDZINSKA, M. A. and TRAGER, W., 1968. The fine structure
Antigenic variations in the plasmodia of certain of trophozoites and gametocytes in Plasmodium
primates as detected by immuno-fluorescence. Am. J. coatneyi. J. Protozool. 15 : 73-88.
Trop. Med. & Hyg. 15 : 483-485. VOLLER, A., GARNHAM, P. C. C., and TARCETT, G. A. T.,
EYLES, D. E., FONG, Y. L., WARREN, McW., GUINN, E., 1966. Cross immunity in monkey malaria. J. Trop.
SANDOSHAM, A. A., and WHARTON, R. H., 1962. Med. & Hyg. 69 : 121-123.
Plasmodium coatneyi, a new species of primate malaria VOLLER, A. and ROSSAN, R. N., 1969. Immunological studies
from Malaya. Am. J. Trop. Med. & Hyg. 11 : 597-604. on simian malaria parasites. IV. Heterologous
EYLES, D. E., 1963. The species of simian malaria: taxonomy, superinfection of monkeys with chronic Plasmodium
morphology, life cycle, and geographical distribution of knowlesi infections. Trans. Roy. Soc. Trop. Med. &
the monkey species. J. Parasit. 49 : 866-887. Hyg. 63 : 837-845.
EYLES, D. E., DUNN, F., WARREN, McW., and GUINN, E., WARREN, McW. and WHARTON, R. H., 1963. The vectors of
1963. Plasmodium coatneyi from the Philippines. J. simian malaria: identity, biology, and geographical
Parasit. 49 : 1038. distribution. J. Parasit. 49 : 892-904.
GARNHAM, P. C. C., 1965. The pathology of Plasmodium WARREN, McW., SKINNER, J. C., and GUINN, E., 1966.
coatneyi malaria. Omagiu Lui. Prof. Dr. M. Ciuca, Edit. Biology of the simian malarias of Southeast Asia. I.
Acad. Rep. Pop. Romane. pp. 199-203. Host cell preferences of young trophozoites of four
HELD, J. R., CONTACOS, P. G., JUMPER, J. R., and SMITH, C. species of Plasmodium. J. Parasit. 52 : 14-16.
S., 1967. Direct hepatic inoculation of sporozoites for
This page intentionally left blank.
 24
Plasmodium fragile Dissanaike, Nelson, and
Garnham, 1965

124 were infected with the cynomolgi parasite

DURING the months and 117 with the inui-like parasite. Following
of May and June, 1919, Donovan (1920) these studies, the latter parasite was sent to Dr.
examined the blood of 76 macaques, Macaca Eyles in Kuala Lumpur, Malaysia, through the
sinicus (= radiata) and 10 langurs (Presbytis kindness of Dr. Ramakrishnan.
priamus) taken in the valleys of the Nilgiri hills Eyles (1963) determined that the parasite
in southern India, but failed to find examples of actually had an asexual cycle of 48 hours and,
the genus Plasmodium. However, in an therefore, a tertian rather than quartan
addendum to the paper, he mentioned that a slide periodicity; and, that it was a new species. At the
of the blood of a M. sinicus monkey had been Washington, D. C. Symposium on Recent
sent to him and on it he had found a Advances in Simian Malaria, in 1963, as part of
plasmodium. According to Ramakrishnan and the XVI International Congress of Zoology, he
Mohan (1961), Sinton and Mulligan (1933) had outlined the salient features of the parasite: its
given a tentative identification of P. inui var. penchant for deep circulation schizogony, small
cynomolgi to this parasite. The monkey which rings resembling P. coatneyi, P. knowlesi, and P.
had supplied the blood for the smear had been falciparum, trophozoites with heavy pigment
taken at an altitude of 4,000 feet. This was the and no enlargement of the host cell. He did not
first instance of malaria not only from that area name the parasite, preferring to discuss it as the
but also from that species of monkey, and "New Nilgiri Parasite." He did mention,
Donovan suggested further study of the blood of however, that a full description with a color
monkeys from that area. plate was in preparation. His sudden untimely
In 1960, stimulated by the Eyles et al death left the text and plate unfinished.
(1960) account of P. cynomolgi in man, About this same period in time, Dissanaike
Ramakrishnan and Mohan (loc. cit.) examined had begun the study of parasites in the blood of
the blood of 13 brown monkeys (M. radiata) monkeys in Ceylon, and among the malarias
caught in the area of Kallar, Nilgiri hills. Nine which came to light was the "New Nilgiri
were found infected with a parasite tentatively Parasite" (Dissanaike et al, 1965). Because of
identified as P. inui and, following splenectomy, Dr. Eyles' connection with the parasite, the
a parasite resembling P. cynomolgi appeared Dissanaike group considered naming it for him
also. but by the time they made their decision, we had
Samples of blood, from two different already reserved his name for a new malaria
naturally infected animals, were sent from the parasite of gibbons (see chapter 7). When
Nilgiris to the Malaria Institute of India at Delhi advised of this, Dissanaike et al (1965) proposed
where Prakash and Chakrabarti (1962) studied the name Plasmodium fragile to emphasize its
infections in a total of 241 M. mulatta monkeys; effect on the host cell.

301
 PLASMODIUM FRAGILE 303

Cycle in the Blood fragmented and dispersed in the cytoplasm (Fig.

28).
PLATE XLVIII The parasite has an asexual cycle of 48
The youngest forms are delicate rings
hours.
which may occur as a multiple infection in the
host red cell. The older rings may display an
accessory chromatin dot (Fig. 3) or the Sporogonic Cycle
chromatin bodies may number up to 3 or 4 (Figs. PLATE XLIX
7, 5). As the parasite grows, it develops a
prominent vacuole, becomes amoeboid, and Eyles (loc. cit.) got oocyst development of the
exhibits numerous large pigment granules which Nilgiri parasite in Anopheles maculatus on one
are scattered throughout the cytoplasm. The occasion. Mosquitoes were fed on an intact M.
pigment is black with a prominent yellow sheen mulatta monkey infected by the inoculation of
(Figs. 11, 12) and occurs as irregular blocks or parasitized blood. On the 10th day of patent
heavy spherical bodies in marked contrast to the parasitemia, when the parasite count was 17 per
rice grain-like pigment in P. coatneyi. Just 100 red blood cells, a lot of A. maculatus
preceding schizogony, the parasite occupies a mosquitoes was allowed to feed. Each of 16
large portion of the host cell which may show mosquitoes dissected had oocysts (6.5 per gut)
some alteration marked by pallor and distortion but three salivary glands examined 13.5 days
of the periphery but without enlargement. after feeding failed to demonstrate sporozoites.
The schizonts ordinarily do not fill the host Dissanaike et al (1965) tried to infect A.
erythrocyte and generally lie to one side of it maculipennis atroparvus and A. aztecus with P.
(Figs. 14-17). The merozoites fragment from the fragile, incubating the first group of mosquitoes
main mass and, in the mature schizont, may at 27° C and the second group at the same
number up to 18 or 19 with an average number temperature for 24 hours and then at 20° C. No
of about 16. Toward the end of schizogony, development took place in either situation.
there is marked host cell distortion. The pigment In our hands, P. fragile has been a difficult
accumulates in a bulky mass but retains its black parasite with regard to mosquito infection. We
yellowish sheen (Figs. 24-26). eventually found that infections could be
Gametocytes are generally abundant in obtained if M. mulatta monkeys with chronic
developed infections. The macrogametocyte is infections were splenectomized and mosquitoes
generally oval to spherical with a prominent then allowed to feed between the 2nd and 10th
eccentrically located red nucleus. The pigment day after splenectomy. Moreover, infection of
appears shattered or disintegrating (Figs. 29-30). mosquitoes generally occurred on the days of
The tendency toward oval gametocytes is an high parasitemia when the predominant parasites
intriguing feature in the light of its close in the peripheral blood were small rings.
relationship to P. coatneyi and therefore to P. The sporogonic cycle has been studied in
falciparum. The microgametocytes may assume two species of mosquitoes, Anopheles freeborni
irregular shapes but always display a prominent, and A. b. balabacensis. Observations began 5
usually diffuse, red staining nucleus with a days after feeding and continued through day 17.
prominent 'karyosome' area. The pigment is Extrinsic incubation took place at a temperature
of 25° C.

PLATE XLVIII.—Plasmodium fragile.

Fig. 1. Normal red cell. Figs. 13-22. Developing schizonts.
Figs. 2-7. Young trophozoites. Figs. 23-26. Nearly mature and mature schizonts.
Figs. 8-10. Growing trophozoites. Figs. 27-28. Immature and mature microgametocytes.
Figs. 11-12. Mature trophozoites. Figs. 29-30. Mature macrogametocytes.
304 PRIMATE MALARIAS

PLATE XLIX.—Developing oocysts of Plasmodium fragile in Anopheles freeborni mosquitoes. X 580 (Except Figure 1.).
Fig. 1. 6-day oocyst showing peripheral arrangement Fig. 5. 11-day oocyst.
of pigment. X 1300. Fig. 6. 12-day oocyst.
Fig. 2. 8-day oocysts. Fig. 7. 13-day oocyst.
Fig. 3. 9-day oocyst. Fig. 8. 13-day oocyst.
Fig. 4. 10-day oocyst. Fig. 9. 14-day oocyst showing early differentiation.

The results of the oocyst measurements are Sporozoites were first seen in the salivary glands
presented in Table 38. In A. freeborni, at day 6, on day 16.
the mean oocyst diameter was 10.8 µ with a Measurement of the oocysts in A. b.
range of 9 to 12 µ. The oocysts continued to balabacensis at day 5 gave a mean diameter of
grow so that on day 16, they had an average 9.8 µ with a range of 8 to 12 µ. The oocysts in
diameter of 58.7 µ with a range of 35 to 77 µ. this mosquito were quite similar to those seen in
 PLASMODIUM FRAGILE 305

A. freeborni except that on day 17, oocysts A comparison of the growth rate of P.
having diameters of up to 100 µ were seen. fragile and P. cynomolgi in A. freeborni
Sporozoites were present in the salivary glands mosquitoes (Fig. 67), indicates that this parasite
on day 16. takes approximately 16 days to complete its

TABLE 38.—Oocyst diameters of Plasmodium fragile in Anopheles freeborni and in A. b. balabacensis.

Days after A. freeborni A. b. balabacensis

Infection
No. Range* Mean No. Range Mean

5 17 8-12 9.8
6 15 9-12 10.8
7 48 8-18 13.1 25 12-19 15.9
8 39 12-27 15.8 64 9-21 16.2
9 27 14-35 23.8 100 11-52 22.3
10 63 13-37 23.0 8 20-37 28.5
11 41 18-53 32.9 42 18-46 29.9
12 43 21-52 37.5
13 31 25-55 41.9
14 25 39-73 55.6† 2 50-64 57.0
15 14 41-77 61.5†
16 24 35-77 58.7†** **
17 39 30-100 60.4†**
Totals 372 8-77 297 8-100

* Measurements expressed in microns; incubation temperature was at 25° C

† Oocyst differentiation.
** Sporozoites present in the salivary glands.

FIGURE 67.—Mean oocyst growth curves and ranges in oocyst diameters of Plasmodium fragile and P. cynomolgi in Anopheles
freeborni mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).
306 PRIMATE MALARIAS

development versus 11 days for P. cynomolgi. parasitemia rose to a level of approximately

Plasmodium fragile is probably the slowest 240,000 per mm3 by day 10 and declined slowly
growing primate malaria with tertian periodicity. to a low of approximately 1,000 by day 40 and
The sporozoites in A. b. balabacensus were thereafter exhibited a secondary rise in
infective as shown by their ability to initiate parasitemia. Of the 15 animals, 5 died 8 to 12
infection in three intact M. mulatta monkeys. days after inoculation (mean 10.5 days). In
The prepatent period was 17 days in each addition, one of three animals inoculated with
animal. sporozoites did not survive the infection (Fig.
69) giving a mortality rate of 33.3 percent. The
Cycle in the Tissue two M. fascicularis monkeys had lower though
persistent parasitemias during a 60-day period of
There are no data on the exoerythrocytic
observation. The pattern of deep circulation
cycle of this parasite.
schizogony, marked by alternate days of high
and low parasitemia, is illustrated in Fig. 69.
Course of Infection
Three different groups of investigators had
studied this parasite in different hosts before it
Host Specificity
The normal hosts of Plasmodium fragile are
came into our hands through the kindness of
Macaca radiata (Ramakrishnan and Mohan,
Prof. P. C. C. Garnham. None had actually
1961) and M. sinica (Dissanaike et al, 1965). It
described the course of the infection either in the
has been shown experimentally to be infective to
normal host or in the rhesus monkey which
M. mulatta and M. fascicularis. Dissanaike, et al
supports its growth well.
(1965) inoculated three men intramuscularly
We were able to follow the course of the
with blood containing P. fragile--no infection
infection of 18 M. mulatta and 2 M. fascicularis
resulted during an observation period of one
monkeys (Fig. 68). Fifteen rhesus monkeys
month. We have made two attempts to transmit
received their infection via parasitized blood and
the infection to man by the bites of A. b.
three via the inoculation of sporozoites.
balabacensis mosquitoes but no infection
Following the passage of parasitized blood, the

FIGURE 68.—Median parasitemia curves of Plasmodium fragile in 15 Macaca mulatta and 2 M. irus ( = fascicularis) monkeys
infected by the inoculation of parasitized blood.
 PLASMODIUM FRAGILE 307

FIGURE 69.—Parasitemia of Plasmodium fragile in three Macaca mulatta monkeys infected via the bites of Anopheles b.
balabacensis mosquitoes.

resulted during an observation period of 6 Immunity and Antigenic

months. The sporozoites were proven infective
when the infection in the control monkey Relationships
became patent after 17 days. There is not much one can say about
The normal invertebrate host of P. fragile is antigenic relationships and immunity because of
not known. It is not unlikely that Anopheles the paucity of information. About all that can be
elegans is the culprit in the Nilgiris because said at present is that P. fragile can exist as a
Choudhury et al (1963) incriminated it in the mixed infection with P. cynomolgi in nature.
transmission of P. cynomolgi and P. inui, and Voller et al (1966) infected monkeys having a
probably the Nilgiri Parasite (= P. fragile). It previous history of P. cynomolgi, P. knowlesi,
will be recalled that the Nilgiri Parasite was first and P. coatneyi infections with P. fragile. In the
taken to be P. inui. However, until the natural former, a normal infection developed, but in the
vector is determined for the Nilgiris and for monkey previously infected with P. knowlesi,
Ceylon, information on its vector potential and there was a low parasitemia; in the monkey
sporogonic cycle will have to be based on infected with P. coatneyi, there was complete
information derived from experimental vectors. immunity.
We have infected A. freeborni, A. b. Serum samples from animals infected with
balabacensis, A. maculatus, and A. P. fragile gave fluorescent antibody cross-
quadrimaculatus mosquitoes. Sporozoites were reactions to P. fieldi at relatively high levels
demonstrated in the salivary glands of only the (mean reciprocal titer ratio of 100:93) and lower
first two. A comparison of the intensity of the reactions to P. gonderi and P. cynomolgi. When
infections (Table 39) shows that the most the procedure was reversed, P. fragile antigen
susceptible mosquito was A. freeborni followed failed to cross-react at high levels with any of
by A. b. balabacensis, A. maculatus, and, finally, the 9 other simian antigens (Collins et al, 1966).
A. quadrimaculatus.
308 PRIMATE MALARIAS

TABLE 39.—Comparative infectivity of Plasmodium fragile in Anopheles freeborni, A. b. balabacensis, A. maculatus, and
A. quadrimaculatus.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other

F-1 100
F-1 : Bal 8 143 152 23.1 16.4 35.8
F-1 : Mac 6 79 123 53.2 4.1 2.2
F-1 : Q-1 5 54 36 74.1 2.8 0.06

* F-1 = Anopheles freeborni, Bal = A. b. balabacensis, Mac = A. maculatus, Q-1 = A. quadrimaculatus.

** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of A. freeborni to another
species where the GII of A. freeborni = 100.

REFERENCES
CHOUDHURY, D. S., WATTL, B. L. and RAMAKRISHNAN, S. EYLES, D. E., 1963. The species of simian malaria: taxonomy,
P., 1963. Incrimination of Anopheles elegans James morphology, life cycle, and geographical distribution of
(1903) as a natural vector of simian malaria in the the monkey species. J. Parasit. 49 : 866-887.
Nilgiris. Madras State, India. Ind. J. Malariol. 17 : 243- PRAKASH, S., and CHAKRABARTI, S. C., 1962. The isolation
247. and description of Plasmodium cynomolgi (Mayer,
COLLINS, W. E., SKINNER, J. C., and GUINN, E. G., 1966. 1907) and Plasmodium inui (Halberstadter and
Antigenic variations in the plasmodia of certain Prowazek, 1907) from naturally occurring mixed
primates as detected by immuno-fluorescence. Am. J. infections in Macaca radiata radiata monkeys of the
Trop. Med. & Hyg. 15 : 483-485. Nilgiris, Madras State, India. Ind. J. Malariol. 16 : 303-
DISSANAIKE, A. S., NELSON, P. and GARNHAM, P. C. C., 311.
1965. Two new malaria parasites, Plasmodium RAMAKRISHNAN, S. P. and MOHAN, B. N., 1961. Simian
cynomolgi ceylonensis subsp. nov. and Plasmodium malaria in the Nilgiris, Madras State, India. Bull. Nat.
fragile sp. nov., from monkeys in Ceylon. Ceylon J. Soc. Ind. Mal. Mosq. Dis. 9 : 139.
Med. Sci. 14 : 1-9. SINTON, J. A. and MULLIGAN, H. W., 1933. A critical review
DONOVAN, C., 1920. Malaria of monkeys. Ind. J. Med. Res. 7 : of the literature relating to the identification of the
717-721. malarial parasites recorded from monkeys of the
EYLES, D. E., COATNEY, G. R., and GETZ, M. E., 1960. Vivax- families Cercopithecidae and Colobidae. Rec. Malar.
type malaria parasite of macaques transmissible to man. Surv. Ind. III: 381-443.
Science 131 : 1812-1813. VOLLER, A., GARNHAM, P. C. C., and TARGETT, G. A. T.,
1966. Cross immunity in monkey malaria. J. Trop.
Med. Hyg. 69 : 121-123.
 25
Plasmodium reichenowi Sluiter, Swellengrebel,
and Ihle, 1922
reichenowi for the parasite in the chimpanzee.
REICHENOW (1917, However, Sluiter, Swellengrebel and Ihle had
1917a), working in the Cameroons, saw applied the name Laverania reichenowi to the
parasites of malaria in anthropoid apes. In 1920 chimpanzee parasite in 1922, which Blacklock
he published a more detailed study of the blood acknowledged in a short note in 1926, and
parasites of the chimpanzee and the gorilla with therefore the credit for the name goes to them.
figures of falciparum-like parasites. His Later investigators have dealt with these
contention was that since these animals live in parasites as distinct species.
the vicinity of human habitations and are Schwetz (1933, 1933a, 1934) found P.
therefore exposed to bites by the same vectors, reichenowi in the blood of chimpanzees from the
their parasites are probably one and the same Stanleyville area and the upper Congo, and
with those of man. Blacklock and Adler (1922) Rodhain (1938) demonstrated the parasite from
and Adler (1923) saw parasites resembling P. the same host near Leopoldville. Ducke in 1921,
falciparum of man in chimpanzees from Sierra according to Brumpt (1939), found "crescents in
Leone. These investigators were aware that the blood of animals taken in Uganda," and Bray
Mesnil and Roubaud (1920) had had partial (1956) reported the parasite from chimpanzees
success in their attempt to infect the chimpanzee in Liberia. In 1964, van den Berghe et al
with P. falciparum via blood inoculation but examined the blood of some fifty chimpanzees
failed via sporozoites. Inasmuch as Blacklock and thirty gorillas in the eastern Congo at
and Adler doubted Reichenow's conclusion that elevations up to 2,000 meters and failed to find
the infection in the chimpanzee was actually P. P. reichenowi or any other parasite of malaria.
falciparum, they attempted (1922) to infect man More recently (1968), through the kindness of
by direct inoculation with the falciparum-like Dr. Betty June Meyers, of the Southwest
parasite from a heavily infected chimpanzee. Foundation for Research and Education, we
The attempt failed. In 1922, according to Adler were able to isolate the parasite from a
(1923) they repeated the same experiment with chimpanzee taken in the eastern part of the
P. falciparum which they injected into a three- Democratic Republic of the Congo near Lake
month-old chimpanzee. The animal did not Edward. From these reports it would appear that
become infected. They concluded, on the basis the parasite occurs in the area bounded by
of their studies, that the two parasites were 10°W--30°E and 10°N--5°S.
different and proposed the name Plasmodium

309
 PLASMODIUM REICHENOWI 311

Cycle in the Blood Very young gametocytes rarely appear in

the peripheral blood of the intact animal; but,
PLATE L with splenectomy, they make their appearance.
All investigators agree that Plasmodium
According to Bray (1957) and others, these latter
reichenowi, as seen in the peripheral blood,
forms have difficulty in maturing properly. The
closely resembles P. falciparum in the peripheral
development of the gametocytes is well
blood of man. Usually, the parasitemia is
described by Garnham et al (1956) and by Bray
marked by the presence of rings and crescents.
(1957). The young oval forms, with discrete
Other stages appear under stressful conditions or
granules or short rods of pigment, appear first.
as a result of splenectomy.
The oval body is rigid on one side and the other
The youngest parasites are small rings with
side bends around it in an arc. Cytoplasm
a prominent nucleus (Figs. 2-4) or they may
collects along the border opposite the nucleus
contain up to four small chromatin dots (Figs. 5,
and the pigment (Fig. 23). As the gametocyte
6); multiple invasion is not uncommon (Fig. 7).
grows, it swells and assumes the usual sausage
A prominent feature is the presence of
or crescent-shape first described and figured by
marginal forms which appear as clear blebs with
Reichenow (1920).
single or double nuclei (Figs. 6, 7, 9). In Bray's
The mature macrogametocyte is crescent-
experience, accolé forms accounted for 13
shaped and somewhat slender. The cytoplasm
percent of the rings. Schwetz (1934) mentioned
stains a pale blue and encloses a compact pale
the abundance of tenue-type forms but these
red-staining nucleus with greyish-black pigment
were not a prominent feature of our material. At
granules collected around it (Fig. 24). The
about 24 hours, the rings generally retreat into
microgametocyte is more robust than the distaff
the deeper circulation where development
parasite. The cytoplasm stains bluish-red and
continues. Under conditions where growth
surrounds the centrally located diffuse pale red-
continues in the peripheral blood, i.e., especially
staining nucleus and the dispersed brownish
following splenectomy, the pigment appears in
pigment (Fig. 25).
discrete clumps, the cytoplasm increases in
Although there is little difference between
amount and takes a pronounced blue stain; the
the parasites of P. reichenowi and P. falciparum,
nucleus stains bluish-purple to wine color (Figs.
one difference which appears constant is that the
11, 12). With the advent of schizogony, which
mature gametocytes of reichenowi malaria are
begins at about 42 hours and moves rapidly, the
more slender and shorter than falciparum
pigment comes together as a yellowish-black
parasites. This is easily seen if one compares
mass, vacuoles appear in the cytoplasm, and the
these forms in Plate XLII with the same forms in
host cell tends to lose color (Figs. 18, 20), but at
Plate L.
no time is it fully occupied by the parasite. The
The asexual cycle in the blood occupies 48
mature schizonts contain 10 to 12 merozoites
hours.
(Fig. 22). The asexual cycle takes approximately
48 hours. Most authors mention the prominence
of Mauer's clefts and beading along the Sporogonic Cycle
periphery of the host cell but they are not shown There are no data on the sporogonic cycle
on our plate because we did not employ special of Plasmodium reichenowi.
stains. The plate depicts what is seen ordinarily
with regular Giemsa stain.

PLATE L.—Plasmodium reichenowi.

Fig. 1. Normal red cell. Figs. 18-22. Mature schizonts.
Figs. 2-9. Young trophozoites. Figs. 23-24. Young adult and mature macrogametocytes.
Figs. 10-13. Growing and mature trophozoites. Fig. 25. Mature microgametocytes.
Figs. 14-17. Developing schizonts.
312 PRIMATE MALARIAS

Cycle in the Tissue reichenowi were not only distinct but also
nontransferrable outside the original host
species.
There are no data on this phase of the life-
cycle of reichenowi malaria. This is due to the
fact that no suitable vector has been found and Host Specificity
until that hurdle has been cleared, no progress
can be made toward elucidating the tissue phase The earliest attempt to find a vector for P.
of the parasite. reichenowi was that of Blacklock and Adler
(1922) who found no evidence of infection after
Course of Infection dissecting 40 Anopheles costalis (= gambiae)
mosquitoes which had fed on an infected
chimpanzee on two different nights. Rodhain
Nothing is known about the course of the
(1941) dissected 76 A. maculipennis atroparvus,
early natural infection. All our data are based on
out of a lot of 165 allowed to feed on a
observations on naturally infected and blood
chimpanzee with low gametocyte levels, and
inoculated captive animals; this information is
found no evidence of infection. The results led
meager indeed.
Rodhain to suggest that a "special vector" was
Except for the cursory examination of the
required. In 1957, Bray tried to infect A.
blood stages by Reichenow (1917, 1917a),
gambiae with P. reichenowi and found only 4
Blacklock and Adler (1922), Peel and Rodhain
out of 112 dissected showed oocysts and in 3 of
(1946) and others, plus the more detailed studies
them the oocysts were degenerate. No
of these stages by Bray (1956) and by Garnham
sporozoites were encountered. He concluded
et al (1956) in splenectomized animals, very
that A. gambiae "will not act as a host for P.
little is known about the overall infection. The
reichenowi ..." There appears to have been no
latter authors write of a single animal harboring
further attempts until we made our trials in 1968.
a natural infection of P. schwetzi and P.
At that time we had an infected chimpanzee
reichenowi which was treated successfully. Two
whose reichenowi infection showed many
years later the animal was splenectomized only
apparently mature gametocytes and
to have P. reichenowi parasites appear in the
consequently three species of mosquitoes were
circulating blood. The total length of the
allowed to feed: A. b. balabacensis, A. freeborni,
observed infection was in the neighborhood of
and A. stephensi. When dissected some 6 to 8
three years; the total length of the infection was
days later none showed any evidence of
undoubtedly a great deal longer.
infection. In a way, this was somewhat
There is no evidence that the parasite will
surprising because P. schwetzi, which was being
grow in the rhesus monkey. Rodhain (1938)
studied at about the same time, had infected two
gave the parasite to M. mulatta without success
of these species readily. The parasite of malaria
and that has been our experience too. In other
is a capricious animal and failure in this instance
simians, the transfer has failed also. For
does not necessarily signify rejection by the
example, Rodhain (1939) transferred parasitized
mosquitoes but rather it may mean that our
P. reichenowi blood to Cercopithecus schmidti
feedings were carried out at the wrong time in
without success and Bray (1963) failed to gain
the course of infections, or, that the
infection after two attempts using Cercocebus
physiological conditions of the vertebrate host
atys.
were other than ideal for the parasite. We
The situation in man generally follows the
believe that further trials, if they present
same line. Blacklock and Adler (1922) gave P.
themselves, will lead to success, for it is hard to
reichenowi to two Europeans using both the
imagine that under the right conditions this or
intravenous and subcutaneous routes without
any other primate malaria would fail to develop
obtaining infection. Later, Rodhain (1939,
in the virtually universal vector, A. b.
1939a) gave P. reichenowi blood to two patients
balabacensis or in one of the other experimental
and neither one became infected which led
vectors available to us.
Rodhain to say that P. falciparum and P.
 PLASMODIUM REICHENOWI 313

Immunity and Antigenic P. schwetzi in the chimpanzee. This point was

tested recently when we (Coatney, 1968 and
Relationships Contacos et al, 1970) transferred each of these
parasites to a splenectomized and to an intact
There is not much to be said on this subject chimpanzee by the inoculation of parasitized
because of the paucity of information. We know blood. Each of the animals developed good
that one, two, or all three of the chimpanzee infections but what was surprising was that 99
malarias may occur in a single animal at the percent of the parasites were P. schwetzi. Further
same time (Schwetz, 1933) or in tandem; but, study is needed before we can say more about
there is little information on the phenomenon of the phenomenon of dominance among the
dominance. In the human malarias, the malarias of chimpanzees.
dominance of P. falciparum over P. vivax is well
recognized and it would be expected that the
situation would prevail with P. reichenowi and

REFERENCES
ADLER, S., 1923. Malaria in chimpanzees in Sierra Leone. Ann. REICHENOW, E., 1917. Sobre el problema de la immunidad de
Trop. Med. Parasit. 17 : 13-19. los negros contra el paludismo. Bol. Inst. Nac. Hig.
BLACKLOCK, B., 1926. Plasmodium reichenowi Blacklock and Alfonso XIII 13 : 29-42.
Adler, 1924. Ann. Trop. Med. Parasit. 20: 145. REICHENOW, E., 1917a. Par6sitos de la sangre y del intestino de
BLACKLOCK, B., and ADLER, S., 1922. A parasite resembling los monos antropomorfos africanos. BoI. R . Soc.
Plasmodium falciparum in a chimpanzee. Ann. Trop. Espa'n. Hist. Nat. 17 : 312-332.
Med. & Parasit. 16 : 99-106. REICHENOW, E., 1920. Ueber das vorkommen der
BRAY, R. S., 1956. Studies on malaria in chimpanzees. I. The malariaparasiten des menschen bei den afrikanischen
erythrocytic forms of Plasmodium reichenowi. J. menschenaffen. aentralbl. f. Bakt. I. Abt. Orig. 85 :
Parasit. 42 : 588-592. 207-216.
BRAY, R. S., 1957. Studies on malaria in chimpanzees. III. RODHAIN, J., 1938. Les plasmodium des anthropoides de I'
Gametogony of Plasmodium reichenowi. Ann. Soc. Afrique centrale et leur relation avec les plasmodium
BeIge Med. Trop. 37 : 169-174. humains. Acta. Convent. Tertii de Trop. Atque Malar.
BRAY, R. S., 1963. Malaria infections in primates and their Morbis. 2 : 539-544.
importance to man. Ergeb. Microb. Immunit. Experim. RODHAIN, J., 1939. La receptivite du chimpanze Pan satyrus au
Therap. 36 : 168-213. Plasmodium vivax humain. C. R. Soc. BioI. 132 : 69-
BRUMPT, E., 1939. Les parasites du paludisme des chimpanzeés. 70.
C. R. Soc. BioI. 130 : 837-840. RODHAIN, J., 1939a. Les plasmodiums des anthropoides de I'
COATNEY, G. R., 1968. Simian malarias in man: facts, Afrique centrale et leurs relations avec les plasmodiums
implications, and predictions. Am. J. Trop. Med. & humains. Ann. Soc. BeIge Med. Trop. 19 : 563-572.
Hyg. 17 : 147-155. RODHAIN, J., 1941. Les plasmodiums des anthropoides de I'
CONTACOS, P. G., COATNEY, G. R., ORIHEL, T. C., Afrique centrale et leurs relations avec les plasmodiums
COLLINS, W. E., CHIN, W., and JETER, M. H., 1970. humains. Bull. Acad. Roy. Med. BeIge 6 : 21-60.
Transmission of Plasmodium schwetzi from the SCHWETZ, J., 1933. Sur une infection malarienne triple d'un
chimpanzee to man by mosquito bite. Am. J. Trop. chimpanze. aentralb. f. Bakt. I. Abt. Orig. 130 : 105-
Med. & Hyg. 19 : 190-196. 110.
GARNHAM, P. C. C., LAINSON, R., and GUNDERS, A. E., SCHWETZ, J., 1933a. Sur les parasites malariens (Plasmodium)
1956. Some observations on malaria parasites in a des singes superieurs (Anthropoides) africains. C. R.
chimpanzee, with particular reference to the persistence Soc. BioI. 112 : 710-711.
of Plasmodium reichenowi and Plasmodium vivax. SCHWETZ, J., 1934. Contribution a I'etude des parasites
Ann. Soc. BeIge Med. Trop. 36 : 811-821. malariens des singes superieurs africains. Riv. Malariol.
MESNIL, F., and ROUBAUD, E., 1920. Essais d'inoculation du 13 : 143-147.
paludisme au chimpanzé. Ann. Inst. Pasteur, Paris. 34 : SLUITER, C., SWELLENGREBEL, N., and IHLE, J., 1922. De
466-480. Dierlijke Parasieten van den mensch en van onze
PEEL, E., and RODHAIN, J., 1946. Contribution a l'étude des huisdieren. Scheltema and Holkema's Boekhandel,
Plasmodiums des anthropoides africains. La Amsterdam, p. 121.
schizogonie du Plasmodium reichenowi dans le sang VAN dEN BERGHE, L., CHARDOME, M., and PEEL, E., 1964.
peripherique. Ann. Soc. BeIge Med. Trop. 26 : 341- The filarial parasites of the eastern gorilla in the Congo.
348. J. Helminth. 38 : 349-368.
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 SECTION 6
Other Type Parasites
This page intentionally left blank.
 26
Plasmodium knowlesi Sinton and Mulligan, 1932
name Plasmodium knowlesi in honor of Dr. R.
IT is not unlikely that Knowles. In 1935, Mulligan wrote a more
Franchini (1927) was the first person to see detailed description of the parasite accompanied
Plasmodium knowlesi and to recognize that the by a well executed plate which gave increased
parasite he saw in the blood of Silenus stature to the parasite's distinctive nature.
cynomolgus (= Macaca fascicularis) was Malariologists have puzzled over a paper
different from P. inui and P. cynomolgi. Later by Ionesco-Mihaiesti et al (1934) in which they
(1931) it was seen by Dr. H. G. M. Campbell claimed to have found P. inui in the blood of a
who was working in kala-azar and had no baboon. The parasite was said not only to infect
particular interest, at the time, in the rhesus but, also, that it would infect man.
plasmodium he encountered in M. fascicularis. Baboons are not infected naturally with malaria
Dr. Napier, on the other hand, with whom Dr. and until recently, P. inui failed to grow in man.
Campbell was working, drew blood and The puzzle was cleared up in 1964 when
inoculated it into 3 other monkeys, one of which Professor Garnham visited Roumania and,
was a rhesus; it developed a fulminating through the kindness of Dr. G. Lupascu, who
infection. The original monkey was given to Dr. had kept the original slides, was able to examine
Das Gupta who maintained the strain for some the original material; the parasite in question
time by subpassage (see Knowles, 1935). Napier was actually P. knowlesi. The monkey-to-man
and Campbell (1932) investigated the tendency passage was thus cleared up because P. knowlesi
for the parasite to produce hemoglobinuria in will infect man as first shown by Knowles and
Cercopithecus pygerythrus (actually, M. Das Gupta (loc. cit.). The baboon had been
fascicularis) and M. rhesus (= M. mulatta). In given inoculations of emulsified spleen and
the same year (1932) Knowles and Das Gupta other organs from a M. fascicularis, the natural
described the blood forms of the parasite and host of P. knowlesi, which would account for its
showed that it could be transmitted to man. infection. The infection in the baboon was
From this vantage point, one wonders why recognized as mild which might be expected of
neither group elected to name the parasite. It an abnormal host except, as was shown in this
must be remembered, however, that not all laboratory, P. knowlesi will kill baboons when
investigators are taxonomic addicts and, too, infection is induced through the inoculation of
maybe they recognized that the literature on parasitized blood.
these parasites was already in a state of The true home of P. knowlesi is peninsular
disorganized chaos and elected to leave the Malaysia where monkeys, especially M.
naming to "the brave." Sinton and Mulligan fascicularis, are commonly infected. Their
(1932), after studying the Knowles and Das infections may include species other than P.
Gupta material and their own isolate from a M. knowlesi and their separation may require the
fascicularis, obtained in Singapore, noted the employment of several techniques and more
distinctive stippling in the red cells, the presence than a dash of patience. Its range extends east to
of an accessory dot, and the 24-hour schizogonic the Philippines (Lambrecht et al, 1961) and
cycle which convinced them that the parasite
represented a new species. They gave it the

317
318 PRIMATE MALARIAS

north to Taiwan (Yokagawa et al, 1941). If The species taiwanensis is surely a Hepatocystis
careful surveys were made, it probably would be to which, according to Hsieh (loc. cit.), Garnham
found in Java, southern Thailand, and possibly agrees. Another species variety Plasmodium
in similar climatic areas in Cambodia and South cynomolgi cyclopis Inoki et al, 1942 with
Vietnam. knowlesi affinities has also been described from
From time to time, variants and/or strains, Taiwan; it is discussed in Chapter 6. Because so
or subspecies, of P. knowlesi have been isolated little is known about the malarias on Taiwan,
and described. Sinton and Mulligan (1933) including a complete blank on the vectors, it is
isolated 5 different strains, but found no hoped that investigators will find time to pursue
significant points of difference between them the problem there.
and their original strain. In 1953, Edeson and This leaves us with P. knowlesi edesoni
Davey isolated a strain from a M. fascicularis Garnham, 1963. The parasite exhibits a
trapped in Negri Sembilan, Malaya; which, quotidian cycle with near absence of schizogony
following studies there, in India, and in En- in the peripheral blood, reminiscent of P.
gland, turned up no features that would coatneyi and P. falciparum up to the appearance
distinguish it. The strain isolated directly from of gametocytes which are spherical as against
Anopheles hackeri (Wharton and Eyles, 1961) is crescentic in P. falciparum. It is infectious to
now known as the 'hackeri' strain and it, too, rhesus monkeys, many of which recover unless
behaves like the earlier isolates. splenectomized.
Among the variants, the first to be The rings appear in the circulation about
described was by Brug (1934) who described midnight and many of them carry an accessory
variety sintoni from a M. fascicularis (actual chromatin dot; multiple infections may appear.
source is unknown but credited by some authors As growth proceeds, the parasites become drawn
to Java) which he considered different from the out, stretching across the host cell with the
typical P. knowlesi. The distinguishing nucleus on one side of the band. In late evening,
characteristics were absence of cellular the more compact parasites begin to leave the
distortion, rod-shaped pigment, and red-staining peripheral circulation only to disappear
rims around the schizonts which sometimes completely about 3 hours before sporulation
extended as septa between the merozoites. No pours young forms into the circulation again.
other like material has come to hand and so, for The mature schizonts, with condensed pigment,
the present, judgment is withheld as to whether carry in the neighborhood of 12 merozoites.
sintoni is a valid form. The mature macrogametocytes occupy the
Yokagawa (1941) gave the variety name entire red cell with a nucleus larger than
arimai to the parasite seen by Arima (1933) in ordinary; dark pigment is scattered in the
blood from a M. cyclopis, the only species of cytoplasm. The adult microgametocytes take up
monkey found on Taiwan. In the same paper, most of the host cell and support a large red-
Yokagawa offered the name Plasmodium staining nucleus which may be surrounded by a
taiwanensis for a new species which he said had thick rim of pigment.
an asexual cycle of 11 to 24 days. As one It is unfortunate that this strain is no longer
reviews the literature, difficult at best, but available to allow for comparison of the
cleared up somewhat by Hsieh (1960), it would sporogonic and other cycles with classical P.
appear that var. arimai was described again by knowlesi and with P. coatneyi. Because the
Yokagawa et al (1941, 1942) and Yokagawa original infection in M. fascicularis came from
(1942, 1942a). In 1951 according to Hsieh (loc. an area near Kuantan, Pahang, Malaysia, it is
cit.), Yokagawa mentioned that P. knowlesi var. hoped that it can be re-isolated. Until overall
arimai was close to P. knowlesi but that it would comparisons can be made, the subspecies is
not infect man. Because the data on the length of considered valid.
its asexual cycle is in doubt, its low
pathogenicity to monkeys, and its failure to
grow in man, the parasite is most likely P. inui.
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 PLASMODIUM KNOWLESI 321

Cycle in the Blood forms (Figs. 24, 25). The mature

macrogametocyte is generally spherical and fills
PLATE LI the host cell which may be enlarged to a
The young ring forms in the rhesus monkey
diameter of 8.5 µ. The cytoplasm stains a
and in man may appear in large numbers in the
distinctive blue and the nucleus, placed
circulating blood. They resemble P. falciparum
eccentrically, takes a deep pink stain enclosing a
rings but their nucleus is spherical and
heavier stained irregular area. The black pigment
prominent, many times lying inside the ring.
granules are prominent and scattered irregularly
Appliqué forms appear (Fig. 3) along with
in the cytoplasm (Fig. 24). The microgametocyte
regular rings harboring one or more accessory
is sometimes smaller than the distaff parasite but
chromatin dots (Figs. 4, 7-9). Sinton and
this is not always true. The cytoplasm stains a
Mulligan (1933) regarded the latter as diagnostic
medium pink with the nucleus a darker shade.
of P. knowlesi but we know now that these
The nucleus makes up about one-half the body
structures occur in other simian forms, too.
of the parasite and which is without pigment
When full grown, the non-amoeboid rings may
granules. The latter are jet black and scattered in
occupy half or more of the host erythrocyte. At
the cytoplasm (Fig. 25).
this stage of growth, band forms appear,
The asexual cycle in the blood occupies 24
reminiscent of P. malariae (Fig. 11). With the
hours, the only example of a quotidian cycle
loss of its vacuole, the parasite shrinks, becomes
among the primate malarias.
compact, and pigment appears in the form of
dark grains; the nucleus increases in size, and
takes a deep red stain. The cytoplasm stains a Sporogonic Cycle
deep blue. The host erythrocyte shows stippling PLATE LII
which some authors have called 'Sinton and The development of Plasmodium knowlesi
Mulligan's’ stippling, since it is not of the to the point of sporozoite-positive salivary
Schüffner type (Figs. 13-18). With the advent of glands has been reported in Anopheles annularis
schizogony, the nucleus divides and the process (Sinton and Mulligan, 1933; Singh et al, 1949),
continues until as many as 16 merozoites, in A. aztecus (Garnham et al, 1957), in A.
average 10, are produced. The process of stephensi (Singh et al, 1949; Hawking and
schizogony results in some contraction of the Mellanby, 1953; Hawking et al, 1957; and
parasite (Fig. 19) but with further development, Garnham et al, 1957), in A. atroparvus (Weyer,
it eventually fills the host cell (Fig. 20). At first, 1937; Hawking et al, 1957), and in A. b.
the pigment is scattered but now collects into balabacensis and A. freeborni (Collins et al,
one or more yellowish-black masses, and 1967). In A. stephensi and A. atroparvus, the
eventually into a single mass in the mature oocysts developed on the guts but sporozoites
schizont (Fig. 23). were rarely found in the salivary glands. In our
The early sexual forms may be recognized studies, we have followed the sporogonic
as small solid bodies which appear to grow more development in A. b. balabacensis, A. freeborni,
slowly than the asexual forms consuming A. maculatus, A. quadrimaculatus, and A.
probably 48 hours to complete their atroparvus (Table 40).
development. This parasite like some other In A. b. balabacensis, at day 4, the mean
species, notably P. eylesi and P. jefferyi,
displays a striking color difference in the sexual

PLATE LI.—Plasmodium knowlesi.

Fig. 1. Normal red cell. Figs. 16-23. Developing schizonts, nearly mature, and
Figs. 2-9. Young trophozoites. mature schizonts.
Figs. 10-12. Growing trophozoites. Fig. 24. Mature macrogametocyte.
Figs. 13-15. Mature trophozoites. Fig. 25. Mature microgametocyte.
322 PRIMATE MALARIAS

PLATE LII.—Developing oocysts and sporozoites of Plasmodium knowlesi in Anopheles b. balabacensis mosquitoes. X 580.

Fig. 1. 4-day oocyst showing scattered pigment. X 1300. Fig. 6. 10-day oocyst showing peduncle.
Fig. 2. 5-day oocyst. X 1300. Fig. 7. 11-day oocyst showing early differentiation.
Fig. 3. 7-day oocyst. Fig. 8. 11-day fully differentiated oocyst.
Fig. 4. 8-day oocyst. Fig. 9. Sporozoites near salivary gland tissue.
Fig. 5. 9-day oocyst.
 PLASMODIUM KNOWLESI 323

oocyst diameter was 8 µ, with a range of 5 to 12 mosquitoes (Fig. 70) indicates a close similarity
µ. The oocysts continued to grow and by day 10, between the two. It is surprising that the tertian
the mean size was 62 µ, with a range of 18 to parasite (P. cynomolgi) and the quotidian
103 µ sporozoites were present at this time in parasite (P. knowlesi) should have similar
the salivary glands. growth patterns when the growth phases in the
In A. freeborni and A. maculatus, the blood and in the fixed tissue are so dissimilar.
oocysts developed, but the mean diameters were
smaller than in A. b. balabacensis. In addition, Cycle in the Tissue
the sporozoites, although present in the salivary
glands of both species at day 12, were very PLATE LIII
scarce. The oocysts in A. quadrimaculatus were The tissue forms of P. knowlesi, like the
actually larger on comparable days than were other primate forms, develop in the parenchyma
those in the A. b. balabacensis. Sporozoites were cells of the liver and display structures which
present in the salivary glands on day 11. appear to be highly distinctive. Certain stages in
Sporozoites were not found in A. atroparvus the exoerythrocytic cycle were demonstrated by
although dissections were carried out through Garnham et al, 1957.
day 11. The extrinsic incubation periods in the The earliest forms were seen at 92 hours
mosquitoes ranged from 12 to 15 days (mean of after infection. At that stage, they occupied most
13.0 days). The sporozoites were shown to be of the enlarged host cell with the parasite oval in
infective in that transmission was obtained, by shape and with a smooth outline. The most
bites of A. b. balabacensis mosquitoes, in rhesus arresting feature of the interior was the decided
monkeys on 30 occasions. The prepatent periods separation of chromatin and cytoplasm with the
ranged from 6 to 9 days (mean 7.1 days). On 9 latter condensed into flocculi. Vacuoles were
other occasions, dissected guts and glands of A. present. The nuclei were large, numerous, and
b. balabacensis (2 times), A. freeborni (6 times), appeared as an aggregate of chromatin dots. In
and A. maculatus (once) were inoculated into the main, there was no continuity between
rhesus monkeys. The prepatent periods under nucleus and cytoplasm. The smallest EE bodies
these conditions ranged from 5 to 12 days with a measured 11 x 21 µ and the largest 29 x 29 µ.
mean of 7.2 days. Only one 117-hour (4¾ days) form was seen. It
A comparison of the growth curves of P. was an oval parasite which measured 33 x 50 µ.
knowlesi and P. cynomolgi in A. b. balabacensis In appearance, this form was much like the

TABLE 40.—Oocyst diameters of Plasmodium knowlesi in Anopheles b. balabacensis, A. freeborni, A. maculatus,

A. quadrimaculatus, and A. atroparvus.

Days after
A. b. balabacensis A. freeborni A. maculatus A. quadrimaculatus A. atroparvus
Infection
No. Range* Mean No. Range Mean No. Range Mean No. Range Mean No. Range Mean

4 72 5-12 8
5 246 8-24 15 145 8-22 14 36 8-18 14 52 12-18 14
6 266 11-35 20 306 11-38 19 56 12-28 19 6 26-41 31 166 12-32 19
7 244 12-53 34 155 9-60 33 177 9-50 23 28 24-47 40 72 20-53 33
8 226 14-77 45 215 14-63 39 129 18-63 37 19 35-74 55 113 19-64 42
9 309 13-92 57† 190 13-87 51† 155 18-74 43† 134 22-78 51† 75 24-74 42
10 195 18-103 62†** 242 18-101 58† 279 14-81 47† 122 25-100 72† 144 20-87 54
11 199 24-100 67†** 83 26-106 64† 136 27-89 53† 88 27-99 71†** 10 52-89 78†
12 50 27-79 58†** 5 44-67 54†** 104 20-83 53†**

Totals 1907 5-103 1341 8-106 1072 8-89 397 22-100 632 12-89

* Measurements expressed in microns.

† Oocyst differentiation.
** Sporozoites present in the salivary glands.
324 PRIMATE MALARIAS

FIGURE 70.—Range in oocyst diameters and the mean oocyst diameter curve of Plasmodium knowlesi and P. cynomolgi in
Anopheles b. balabacensis mosquitoes. (D = oocyst differentiation; SP = sporozoites present in the salivary glands).

earlier ones except that vacuoles were absent. cytoplasmic masses, and bars. The size of the
The biopsy material at 124 hours (5¼ EE bodies ranged from 38.2 x 25.5 µ to 52 x 52
days) caught the parasites just prior to the final µ.
division to form merozoites because 14 hours These authors also found a 141-hour (5¾
later, ring forms were in the circulating blood; days) ruptured form surrounded by phagocytes.
they appeared to be about 8 hours old. The infection had become patent some hours
The EE bodies were easily recognized before. The EE body area was approximately 75
because of their large size. They were oval x 110 µ; only a few merozoites were seen in the
bodies with an even border and prominent clefts center of the area.
or spaces in the cytoplasm. Sometimes these In our own studies, we have infected
parasites were pear- or hourglass-shaped. monkeys with the A. hackeri strain of P.
Cytoplasmic flocculi were present, and the knowlesi following the technique of Held et al
striking feature was the early differentiation of (1966) in which infected salivary glands from A.
the cytoplasm which is not seeded with nuclear b. balabacensis mosquitoes are injected directly
material until later. The nuclei of these 5-day into the liver. Beginning at 48 hours after
forms appeared to be of 3 types: clusters of dots, injection, biopsies were taken at 8 hour intervals
very small dots of chromatin scattered in the through 120 hours. Studies of the sections
 PLASMODIUM KNOWLESI 325

PLATE LIII.—Exoerythrocytic bodies of Plasmodium knowlesi in liver tissue of Macaca mulatta monkeys. X 580.
Fig. 1. 3-day body. Fig. 4. 4-day body.
Fig. 2. 3-day body. Fig. 5. 4-day body.
Fig. 3. 4-day body showing numerous flocculi.

revealed numerous EE bodies at each of the time resulting, almost always, in the death of the
periods. At 120 hours young ring forms were animal. Studies on sporozoite-induced infections
present in the circulating blood and, at the same (Fig. 71) show that parasites are first apparent in
time, fully mature EE bodies were demonstrable the peripheral blood by day 6. The median
in the liver sections. The greatest rate of growth parasitemia curve exhibits a dramatic rise
appeared to take place between 72 and 96 hours beginning on day 10, which reaches a median
(Plate LIII). Numerous flocculi were present in infection level of approximately 3.5 parasites per
the sections, but vacuoles were not 100 RBC on day 11. At this time, the first
demonstrable. animals died. The level of parasitemia continued
It is quite apparent that the EE cycle of to rise until day 13, after which it leveled off to
Plasmodium knowlesi, at least in this strain, is approximately 12 parasites per 100 RBC. The
less than 120 hours. mean time of death was 13.6 days with a range
of 11 to 16 days.
Course of Infection One of our M. mulatta monkeys (T-722)
was inoculated with parasitized blood which had
been frozen for approximately one year. The
In the rhesus monkey (M. mulatta), infection was slow to develop, not reaching its
Plasmodium knowlesi is a fulminating infection
326 PRIMATE MALARIAS

FIGURE 71.—Parasitemia and times of death of 19 Macaca mulatta monkeys infected with sporozoites of Plasmodium knowlesi.

FIGURE 72.—Infectivity of Plasmodium knowlesi gametocytes, in a Macaca mulatta monkey, to Anopheles b. balabacensis
mosquitoes.

peak parasitemia until day 15 (Fig. 72) and infected on 35 of 47 feeding days. There
thereafter slowly declined for the remainder of appeared to be 4 distinct waves of mosquito
the 60-day observation period. No treatment was infections which were correlated, partially at
employed. When the nature of the infection least, with gametocytemia.
became apparent (day 10), daily feeding of A. b. Plasmodium knowlesi was first shown to
balabacensis mosquitoes was initiated and infect man by Knowles and Das Gupta (1932)
continued, with few interruptions, for the next followed by the report of Ionesco-Mihaiesti et al
50 days. During this period, mosquitoes were (1934). Van Rooyen and Pile (1935) employed
 PLASMODIUM KNOWLESI 327

P. knowlesi therapeutically for the treatment of Milam and Kusch (1938) offered P. knowlesi
general paresis and reported that non-immunes infections to a series of 35 patients, of whom 20
accepted the infection readily but those with had not experienced malaria before, while the
previous experience with P. vivax were resistant. remainder had had mild attacks, or had failed to
An editorial following the von Rooyen-Pile accept infection with P. vivax. Included in the
paper called attention to the loss of virulence series were 6 Negroes. Each of the 29 Caucasian
following continued passage in man. The next patients developed infections while among the 6
week, Nicol (1935) in commenting on P. Negroes, 4 experienced only mild infections and
knowlesi infection in man mentioned the loss of 2, none. However, the latter 2 did have low-
virulence following man to man passage, also. grade infections because subinoculation of their
Chopra and Das Gupta (1936) used P. knowlesi blood to normal monkeys revealed parasites for
transferred directly from a Silenus rhesus (= M. up to 3 weeks following their inoculation.
fascicularis) monkey, for the treatment of Clinically, the course of the disease followed
neurosyphilis in 2 patients. They were satisfied closely that of P. vivax except the duration was
with the results and pointed out the advantages shorter. Initial fevers were about 102.2° F but
of the procedure over one employing P. vivax. In later ones had peaks of 104 to 105.8° F which
1937, Ciuca et al published two papers (1937, appeared daily for about 10 days and then 'tailed'
1937a) which dealt with a total of 321 patients off to normal. Paroxysms varied from 2 to 15
exposed to infection with P. knowlesi. In the with an average of 10; definite chills were
first group, probably non-immunes, 79.8 percent experienced by only about half of the patients.
developed fever and parasites in their blood. In The highest parasite counts seldom exceeded
the second group, most of whom were thought to 100 parasites per 10,000 RBC. However, one
have had experience with malaria previously, patient showed 1,200 parasites per 10,000 RBC.
only 46 percent became infected. Following Relapses (recrudescences) occurred which were
these reports, Ciuca and his colleagues both clinical and parasitological; they terminated
continued to employ P. knowlesi for the within 3 days.
treatment of general paresis until in 1955 they Through all the work enumerated above,
reported that after 170 transfers, the infection the infection was passed solely by the
became so virulent it had to be terminated with inoculation of parasitized blood although
drugs. Shortly, thereafter, they abandoned the attempts were made to pass the infection via
use of the strain. If they were satisfied with the mosquito bite on occasion (Coggeshall, 1941).
efficacy of the treatment, which is obvious since Later (1957) Dr. Lainson, according to Garnham
they continued to use it for so many years, one (1966) received 90 bites from a lot of A.
wonders why they failed to obtain a new isolate, labranchiae mosquitoes, showing 84 percent
and use it. In contrast to the increased virulence infected with P. knowlesi. Although he was
aspect in man encountered by Ciuca et al, Jolly observed for months, no infection developed.
et al (1937) reported that although P. knowlesi The question of transferring P. knowlesi to man
produced fulminating infections in their via mosquito bite, either experimentally or in
experimental lower animal hosts, it produced nature, remained in limbo until a fortunate
only mild infections in C. papio after being circumstance occurred in 1965.
passed through man. They characterized the Following the accidental sporozoite-
disease in man as mild with a tendency toward induced infection of man in this country with P.
spontaneous recovery. cynomolgi in May of 1960 (see Chapter 6),
Milam and Coggeshall (1938) carried out investigations were begun in Malaysia where the
duration of infection studies in Caucasians and infecting parasite had originated. That study had
Negroes in this country and produced several objectives; the one which concerns us
corroborative evidence as to the mildness and here was the possible zoonotic potential of the
short duration of the initial infection. In general, simian malarias. We were confident this
the infections in Negro patients were milder than phenomenon could be demonstrated in the field,
those occurring in Caucasians. In the same year, and the senior author had gone so far as to cast
328 PRIMATE MALARIAS

P. cynomolgi in the starring role. This was not to known that our group was looking for a strain of
be, as shown in the following account of an P. malariae, blood was drawn and (refrigerated)
episode which under reasonable circumstances where it remained until sent to our installation at
could not happen--but did! the U.S. Penitentiary in Atlanta, Georgia, on
In the spring of 1965, a 37 year old Monday. There it was put into a volunteer who
American male was detailed by the Army to subsequently developed malaria. One can
peninsular Malaysia for a short while and, as imagine our surprise when the parasite turned
part of his assignment, he spent 5 days alone in out to be P. knowlesi. The ring was joined--
the bush on Bukit Kertau, working by night and simian malaria is a zoonosis (see Chin et al,
sleeping by day. He returned directly to Kuala 1965). Needless to say, the original patient was
Lumpur, the Capitol, and after about a week he cured of his infection and later visited our
left for home. Enroute, he stopped off in laboratory on several occasions to fill us in on
Bangkok, Thailand, and on the third morning he the many details. One other facet might be
felt ill (anorexia, fatigue, and some nausea). He mentioned as frosting on the cake. Before the
decided home was the best place for him and so patient left for Malaysia he obtained some
he departed. He arrived at the Travis Air Force chloroquine tablets and later, even though he
Base in California on Friday night where he was suspected he had malaria, he refrained from
seen by a base physician. He complained of sore taking them because of an admonition that
throat, chills, fever, and profuse sweating. He "drugs should be taken only on advice of a
was treated for an upper respiratory infection physician." If he had taken one tablet this tale
and departed immediately for his home in Silver would have died with the parasite.
Spring, Maryland. He was still sick the next Subsequent to the original blood-induced
morning (Saturday) whereupon he consulted the infection at the U.S. Penitentiary (Atlanta, Ga.),
family physician. When seen by the doctor, he the disease has been passed, by the same route,
was having a chill. When questioned, he offered 11 times (Chin et al, loc. cit. and later) and on 8
the information that he might have malaria since occasions by the bites of infected mosquitoes
he had been in Malaysia recently. When his (Chin et al, 1968). The daily parasite counts in
blood smear was examined, the doctor saw only the volunteers infected by the inoculation of
rings and jumped to the conclusion that the parasitized blood and those infected through the
patient had falciparum malaria. He told the bites of infected mosquitoes showed no
senior author later, that he did not want to treat appreciable difference, so the data were
the patient because he was unfamiliar with the combined, and are shown in Figure 73 along
disease, not having seen a case since his intern with the median parasitemia curve. The latter
days, but remembered that falciparum was shows that the peak parasite count was reached
deadly. on day 8 following which the parasitemia fell
The doctor decided to refer the patient to rapidly to a low level by day 13. Although
the Army's Walter Reed Hospital in parasite counts as high as 1200 per mm3 were
Washington, D. C., because the physicians there encountered as late as the 28th day of
were familiar with the treatment of the disease parasitemia, most of the patients exhibited no
and the man was their dependent. Saturday was parasitemia after day 16.
not an admitting day, and the doctor was told to The salient features of the blood-induced
hold the patient until Monday; this he was afraid infections were: the quodidian asexual cycle in
to do. He next turned to the NIH Clinical Center the blood, temperatures as high as 104.8° F, and
in Bethesda, Maryland, where, luckily, the parasite counts as high as 20,850 per mm3. The
physician on duty was interested in malaria and clinical manifestations were moderate to severe
was well aware of our interest, too. His with attacks terminating spontaneously after two
comment was "send him over." When a blood weeks. In the series of sporozoite-induced cases,
smear was examined at NIH, some 6 hours later, the course of infection was not much different
numerous band forms were in evidence. The from that of the blood-induced cases.
diagnosis was P. malariae. Because it was
 PLASMODIUM KNOWLESI 329

FIGURE 73.—Median parasitemia curve and individual parasite counts in 20 Plasmodium knowlesi infections in man (8
sporozoite-induced and 12 blood-induced).

In the main, the data supplied by the Host Specificity

investigators who observed the parasite in man
agree. At the same time, there are certain points
The natural host of P. knowlesi is Macaca
of difference which probably should be
irus (= fascicularis) from Malaysia (Sinton &
mentioned: 1) Van Rooyen and Pile (1935) and
Mulligan, 1932, 1933) and the Philippines
Nicol (1935) commented on the loss of virulence
(Lambrecht et al, 1961). It has also been found
when P. knowlesi was passaged to man, but the
in M. nemestrina (Eyles et al, 1962) and
more extensive work of Ciuca et al (1955)
Presbytis melalophos (Eyles et al, 1962a) in
showed quite the opposite. The work of Chin et
Malaysia.
al lends support to that thesis. 2) Milam and
Experimentally, the parasite readily infects
Kusch (1938) remarked about the difficulty of
M. mulatta as demonstrated by many authors.
infecting Negroes with P. knowlesi but Chin et
Experimental infections in other simians are
al, in their work, were able to infect Negroes
given below:
easily and saw no difference between infections
in Caucasians and non-whites. What should be SPECIES REFERENCES
stressed is that here, for the first time (Chin and Callithrix jacchus Cruz and de Mello, 1947
Cebus spp. Garnham, 1966
colleagues), P. knowlesi was transferred to man Cercocebus fuliginosus Rodhain, 1936
by sporozoites, at each attempt, (in one case, Cercopithecus cephus Rodhain, 1936
following the bite of a single mosquito) with Cercopithecus grisio viridis Rodhain, 1936
Cynocephalus papio Jolly, Lavergne and
prepatent periods of 9 to 12 days. It is of Tanguy, 1937
interest, too, that not only was the infection Hylobates hoolock Garnham, 1966
transferred from man to man via mosquito bites,
but also, back, to the rhesus monkey.
330 PRIMATE MALARIAS

SPECIES REFERENCES against an acute attack following superinfection

Hylobates lar Eyles, 1963
Macaca cynomolgus (= fascicularis) Jolly, Lavergne and with a different strain of the same parasite.
Tanguy, 1937 Multiple heterologous superinfections with
Macaca nemstrina Eyles, 1962
Macaca radiata unpublished data
certain strains of P. knowlesi appeared to
Macaca speciosa (= arctoides) Eyles, 1963 produce a marked degree of tolerance to other
Papio doguera unpublished data heterologous strains which had common
Papio jubilaeus Rodhain, 1936
Papio papio Garnham, 1966
immunologic factors, but in the absence of such
Presbytis cristatus Eyles, 1963 common factors, multiple heterologous
Saimiri sciureus Chin et al, 1965 superinfections produced no effective tolerance.
Semnopithecus entellus Garnham, 1966
Shortt et al (1938) found that P. knowlesi
A natural vector of P. knowlesi in Malaysia infections which had been cured by
is Anopheles hackeri as shown by Wharton and administration of drug, gave no residual
Eyles (1961). In addition, we have found A. immunity to infection with the homologous
vagus, A. sinensis, A. b. introlatus, A. maculatus, strain of the parasite. Voller and Rossan (1969)
A. kochi, A. b. balabacensis, and A. were able to show there was no relationship
quadrimaculatus mosquitoes, all but the latter between prior total parasite experience and
indigenous to peninsular Malaysia, susceptible immunity. A chronic infection, even at a low
to infection. Other species which have supported level, elicited a more effective immunity than
growth of the parasite, at least the presence of frequent cure and challenge. The actual duration
oocysts on the gut, are: of previous parasitemia seemed to be more
important than the density of parasitemia in
SPECIES REFERENCES determining the ability of an animal to control
Anopheles annularis Singh et al, 1949
infections or to resist challenge.
Anopheles atroparvus Weyer,1937; Hawking et Brown et al (1968) reported that a number
al, 1957 of antigenic stabilates are produced during the
Anopheles aztecus Garnham et al, 1957
Anopheles freeborni Collins et al, 1967
course of an infection with P. knowlesi. It was
Anopheles labranchiae Garnham, 1966 shown, however, by Voller and Rossan (1969)
Anopheles stephensi Mulligan, 1935; Singh et that although populations of parasites isolated
al, 1949; Hawking and
Mellanby, 1953; Garnham from different recrudescences, of chronic P.
et al, 1957; Hawking et al, knowlesi infections, were antigenically distinct,
1957
the immunity produced by repeated exposure to
Relative susceptibility studies, using eight one antigenic variant was effective against
species of Anopheles, (Table 41) indicated that challenge with heterologous variants.
A. b. balabacensis was the most susceptible and No cross-immunity between infections due
that A. albimanus was refractory to infection. to P. knowlesi and those due to P. cynomolgi
Other species reported to be refractory are A. was found by Mulligan and Sinton (1933).
fluviatilis (Singh et al, 1950), A. punctipennis Voller et al (1966) however, showed that
(Coggeshall, 1941), and A. subpictus (Singh et monkeys previously infected with P. knowlesi
al, 1949). were protected against subsequent challenge
with P. cynomolgi or P. coatneyi. Later work
(Voller and Rossan, 1969a) indicated that
Immunity and Antigenic monkeys with chronic infections of P. knowlesi,
Relationships although refractory to homologous challenge,
were susceptible to infection by P. cynomolgi
Mulligan and Sinton (1933, 1933a) found
and by P. coatneyi. Infections of P. inui
that a chronic or latent infection with one strain
developed somewhat more slowly in monkeys
of P. knowlesi conferred an effective immunity
with chronic P. knowlesi infections than in
against the clinical effects of superinfection with
control animals.
the same strain of parasite. However, such
In man, Ciuca et al (1937) demonstrated
infections did not confer effective immunity
 PLASMODIUM KNOWLESI 331

79.8 percent of those individuals with little or no only partial resistance to inoculation with P.
previous history of malaria were susceptible to knowlesi.
infection with P. knowlesi. Of 29 patients Antisera to P. knowlesi gave a fluorescent
subsequently reinoculated with the parasite, antibody cross-reaction at a relatively high level
none developed a durable infection although a to P. fieldi and P. cynomolgi antigens (mean
few parasites were found for a limited time. In reciprocal titer ratios of 100:87 and 100:41), but
those individuals with a probable previous reacted at a much lower level to other primate
history of malaria, the infectivity rate with P. malaria antigens (Collins et al, 1966). In the
knowlesi was only 46 percent. In these patients, reverse procedure, P. knowlesi antigen cross-
a previous infection with P. knowlesi gave reacted higher to P. cynomolgi than it did to P.
complete immunity to reinfection. Patients fieldi antisera (mean reciprocal titer ratios of
whose first experience was to P. vivax displayed 100:54 versus 100:12).
TABLE 41.—Comparative infectivity of Plasmodium knowlesi to eight species of Anopheles.

Number of Percent
Mosq. species Number mosquitoes infection GII**
comparison* tests ratios
Standard Other Standard Other
Bal 100
Bal : F-1 33 224 946 44.8 23.7 34.7
Bal : Koc 1 26 34 17.1 11.5 19.0
Bal : St-1 19 243 255 35.0 12.9 14.9
Bal : Atro 27 335 379 47.2 15.6 12.3
Bal : Mac 68 1093 2034 38.2 21.7 5.2
Bal : Q-1 24 293 883 52.6 8.0 3.9
Bal : Alb 4 121 88 38.0 0.0 0.0

* Bal = Anopheles b. balabacensis, F-1 = A. freeborni, Koc = A. kochi, St-1 = A. stephensi, Atro = A. atroparvus, Mac = A. maculatus, Q-1 =
A. quadrimaculatus, Alb = A. albimanus.
** GII = Gut Infection Index = average number of oocysts per 100 guts; the GII ratio is the relationship of the GII of the A. b. balabacensis to
another species where the GII of A. b. balabacensis = 100

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MILAM, D. F. and KUSCH, E., 1938. Observations on text). J. Med. Assoc. Formosa. 40 : 2185-2186.
Plasmodium knowlesi malaria in general paresis. YOKOGAWA, S., 1942. Plasmodium knowlesi var. arimai and
Southern Med. Jour. 31 : 947-949. Plasmodium taiwanensis, naturally infected, in
MULLIGAN, H. W., 1935. Descriptions of two species of monkey Macacus cyclopis (Swinhoe, 1862) (Japanese text).
Plasmodium isolated from Silenus irus. Arch. f. Protist. Nitsushinigaku. 31 : 34-38.
84 : 285-314.
 PLASMODIUM KNOWLESI 333

REFERENCES—Continued
YOKOGAWA, S., 1942a. On the classification of the plasmodia Swinhoe, 1862) of Formosa. Taiwan Igakkai Zasshi 40:
found in the indigenous monkey (black-leg monkey) of 2173-2181. (NS).
Formosa found by us previously reported (Japanese YOKOGAWA, S., KOBAYASHI, H., RO, M., and YUMOTO, Y.,
text). Nipponigaku and Kenkohoken. 204-205. 1942. On the two malaria parasites newly found in
YOKOGAWA, S., 1951. On the exo-erythrocytic development of Macacus cyclopis (Swinhoe, 1862) (Japanese text).
malaria parasites. New Parasit. 2 : 45. (NS). Nipponigaku and Kenkohoken. 5-8. (NS).
YOKOGAWA, S., KOBAYASHI, H., RO, M., and YUMOTO, Y.,
1941. On two species of malaria parasites found for the (NS) = Not seen
first time in the indigenous monkey (Macacus cyclopis,
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 27
Plasmodium girardi Buck, Coudurier, and
Quesnel, 1952
the parasites is accompanied by a beautifully
WORKERS in Madagascar have colored plate painted by Dr. Foley.
long suspected that the lemurs were infected The lemurs of Madagascar are protected
with malaria but the parasite was not seen until animals and consequently there are few
1951 when it was discovered in the blood of a opportunities for studying their parasites.
Lemur fulvus rufus. The parasites were scanty, However, a few animals were examined by
so the animal was splenectomized. A heavy members of the University of California
infection developed 12 days later. The infection expedition to Madagascar in 1948, and another
was studied daily; after about a week, a second malaria parasite (P. lemuris) was discovered; it
species appeared. Each organism was is discussed in Chapter 28.
recognized as a new species. The first one was According to Garnham (1966) the original
given the name Plasmodium girardi in honor of material on P. girardi is no longer available. In
Dr. G. Girard, the former director of the Pasteur 1962, at his request, Drs. Raymond and Brygoo
Institute of Tananarive. The other parasite was splenectomized two animals; one of them
given the name Plasmodium foleyi in honor of exhibited a low-grade infection with P. girardi
Dr. H. Foley of the Pasteur Institute of Algeria. and that material was used for a more extended
The latter parasite is now considered to be a study of the parasite. Only the blood stages of
hepatocystis and therefore it will not be the parasite have been seen.
discussed in its entirety here. The description of

335
 PLASMODIUM GIARDI 337

Cycle in the Blood The macrogametocyte is spherical and

stains a deep blue with granular cytoplasm and
PLATE LIV heavy dark pigment in an aggregation of distinct
The youngest forms are small solid bodies,
grains. The nucleus is deep red and found near
approximately 1.8 µ in diameter, with a large
the periphery of the cell. The host cell is not
nucleus and a small rim of cytoplasm which
enlarged. The cytoplasm of the microgametocyte
stains a deep blue. The youngest stages exhibit a
takes a lilac stain. The pigment is granular to
granule of dark pigment which the original
bacilliform and is located in 4 or 5 aggregates of
authors stated did not appear until the parasite
unequal size. The nucleus takes a deep red stain
occupied about a fourth of the host cell. The
and is located peripherally. The parasite fills the
pigment body lies on the edge of the cytoplasm.
host cell without host cell enlargement.
Projections of cytoplasm may occur but for the
most part, the parasite is compact and dense
even though a vacuole occurs later.
It is interesting that cirrhosis of the liver of
As the trophozoite grows, it may occupy
the host animal was known long before malaria
one-half the cell whereupon it loses its vacuole
parasites were found in them. Whether this
and the parasite as a whole shrinks; the amount
cirrhotic change is due to, or is connected with,
of pigment increases and takes up a position on
their malarias is not known. The spleen appears
or near the periphery of the parasite. The nucleus
normal when the infection is latent which makes
becomes enlarged, sometimes diffuse, but
it appear that these animals handle the infection
always with lighter and darker areas. Very small
easily. If the spleen is removed, the infection
vacuoles may be seen in the cytoplasm.
comes to the fore and then again subsides.
With continued growth, the nucleus divides
Plasmodium girardi may have a distinct
in to two rather large portions, each with a dense
place in the evolution of the malarias of
core area. Pigment continues to form and now
primates, but the extent is in limbo until more is
comprises 4 or 5 dark grains arranged on the
known about its life cycle. The blood phase of
periphery of the parasite. As division continues
the infection leads one to suspect that it probably
beyond the 4-nucleate stage, the host cell
belongs with the quartan type parasites and, if
becomes pallid, distorted, and develops
so, it is their most primitive example. One thing
fimbriated projections. The mature schizont fills
appears certain, and that is, that the distribution
the host cell without enlargement, and displays
of the pigment in these parasites is not like what
10 to 12 merozoites; the pigment is still placed
is found in the malarias of simians. We shall
eccentrically. Host cell stippling appears to be
have to wait for an explanation as to why this
absent.
difference occurs.

REFERENCES
BUCK, G., COUDURIER, J., and QUESNEL, J. J., 1952. Sur GARNHAM, P. C. C., 1966. Malaria parasites and other
deux nouveaux plasmodium observes chez un lémurien haemosporidia. Blackwell Scientific Publications,
de Madagascar splénectomise. Arch. Institut. Pasteur d' Oxford, pp.1114.
Algerie 30 : 240-243.

PLATE LIV.—Plasmodium giardi and P. lemuris.

P. giardi : Figures after Bück et al, 1952. P. lemuris (1000X) : Figures after Huff and Hoostraal, 1963.
Fig. 1. Normal erythrocyte. Figs. 1-5. Uninucleate trophozoites.
Fig. 2-4. Uninucleate trophozoites. Fig. 6. Binucleate schizont.
Figs. 5-7. Schizonts. Fig. 7. Multinucleate schizont.
Figs. 8, 9. Macrogametocytes. Figs. 8, 9. Macrogametocytes.
Fig. 10. Microgametocyte. Fig. 10. Microgametocyte.
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 28
Plasmodium lemuris Huff and Hoogstraal, 1963
they appear to flow around other cells in the
HOOGSTRAAL and Lawless, in the smear.
course of studies of the fauna and its parasites of The macrogametocytes have lavender to
Madagascar, made a blood film from a black purple cytoplasm. The pigment is made up of
lemur, Lemur collaris, housed in the Tananarive small dark brown granules within vacuoles. The
Zoo. Some years later the slide was examined at microgametocytes have red-staining nuclei and
the Naval Medical Research Institute in slate-gray cytoplasm. The pigment is like that in
Bethesda, Maryland, where thirty malaria the macrogametocytes.
parasites were found. The morphology of the
parasite was different from any of the described
species and, therefore, the authors created a new
species, Plasmodium lemuris. Only the blood
stages are known for this species.
An interesting sidelight to the discovery of
Cycle in the Blood this parasite is that, when Dr. Huff first saw the
PLATE LIV macrogametocytes, he thought he was seeing a
leucocytozoon in a mammal; strange, indeed, if
The young trophozoites are small and it were true. He discovered later, however, that
occupy three-tenths to four-tenths of the the bizarre form was in reality a distorted
erythrocytes. The nucleus stains rose-red. Larger gametocyte inside the reddish stained sac of the
trophozoites are more irregular tending toward host cell.
amoeboidity. The pigment is in granules; there is It should be relatively simple to distinguish
no stippling of the host cell. The schizonts are in between the malarias of lemurs. Plasmodium
enlarged and distorted erythrocytes and display foleyi is probably a hepatocystis (see Chapter
irregularly shaped nuclei. The pigment is brown 27) since only gametocytes made their
and clumped into a diffused mass. Mature appearance in the peripheral blood. Plasmodium
schizonts have not been seen. girardi is smaller than P. lemuris but shares
The gametocytes are of large size and other characteristics with it. Both cause
irregular in shape. The nuclei are band-like or distortion of the host erythrocyte and appear in
lobed irregularly. The host erythrocyte is greatly bizarre shapes. Plasmodium lemuris has the
enlarged and in many instances is almost larger gametocytes, of the two but is without the
completely filled by the parasite. In some of pigment located at the edge of the parasite as is
these forms, the rim of host cell cytoplasm, true with gametocytes of P. girardi.
around the parasite, stains pink. It is assumed So little is known about the malarias of
that the cells infected with this parasite are more lemurs, especially since the description of P.
pliable than normal cells because of the way lemuris is based on only thirty specimens, that

339
340 PRIMATE MALARIAS

one hesitates to comment on them. It may be REFERENCES

that there is only one species. Should that be
true, P. lemuris would become a synonym of P. HUFF, C. G., and HOOGSTRAAL, H., 1963. Plasmodium lemuris
girardi. n. sp. from Lemur collaris. J. Inf. Dis. 112 : 233-236.
SECTION 7
Indexes
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