Biology 332 - Protistology Term 2 - 2002-2003
Plasmodium Falciparum; A Case Study
Throughout history, ancient as well as recent, it is widely speculated and in certain cases, such as the WWI troops engaged in the Macedonian Campaign (Garnham, 1966) that malaria epidemics were solely or partially responsible for the annihilation of established civilizations and communities.
Microscopic eukaryotic organisms P. vivax, P. ovale, P. malaria, and P. falciparum cause malaria in humans. Although all are unicellular in nature these organisms are far from simple and inflict varying degrees of the disease. However, only P. falciparum had proven to be fatal.
P. falciparum was first seen by Alfonse Laveran who examined the bodies of those who succumbed to malignant malaria in North Africa in the late 1800's. Laveran noticed large quantities of pigment in the blood, spleen, liver, and brain, which discolored the tissue. After two years of observation it was noted that spherical hyaline corpuscles and crescent-shaped bodies were present in the pigment. Though not certain if these were really parasites he had seen, on November 6, 1880 all doubt was lifted. By studying fresh blood samples of a soldier with chronic malaria, Laveran saw either filaments or flagella lining the exterior surface of the spherical and crescent shaped structures whose extremely rapid movements were almost certainly parasitic in nature. Laveran observed the crescent shape slowly metamorphasize into a spherical body which later exflagellated. He then called the organism that he saw after himself, Laverania falcipara, which later became known as Plasmodium falciparum (Garnham, 1966). Today, Plasmodium falciparum is place in the kingdom Protista, phylum Apicomplexa, class Sporozoea, subclass Coccidia, order Ercoccidiida, suborder Haemosporina, genus Plasmodium, genus Plasmodium and species falciparum (Karapelou, 1987).
Concentrated mainly in the tropics and subtropics, P. falciparum has spread up into the temperate zones and before eradication programs, up into the southern United States as well. Though now eliminated from developed countries, P.falciparum and therefore malaria tropica, the type of malaria caused by P. falciparum (Hausmann, 1996), run unchecked over most parts of Africa and elsewhere in the tropics and in turn this parasite is the greatest killer in these regions.
Surprisingly though, P. falciparum is not only bound by latitude, but also altitude as well. Due to various factors, the most important of these being temperature, there is little or no malaria above one thousand meters in the coastal belt of Africa, yet in the warmer interior, malaria runs rampant between 1200 to 1400 meters but, with limited exceptions, malaria transmission does not occur over 1500 meters (Garnham, 1966). At higher elevations and therefore cooler temperatures, sporogony in the mosquito, which will be subsequently discussed, can not be completed and therefore transmission does not occur thus placing a limit on the altitude at which malaria can be passed.
Plasmodium falciparum consists of a sexual and asexual form, which have hindered genetic analyses, but with conventional methods, such as micro-manipulation in which cells containing a single parasite can be identified, cloning and studying of the genome has been performed (Walliker, 1983) and information in regards to the genome of P. falciparum has been collected. The genome size ranges from 1x107 to ~ 4x108 base pairs (Pollack, 1982) and studies from restriction fragment polymorphisms show that blood stage parasites in general are haploid (Walliker, 1983; Arnot, 1988). P. falciparum do not undergo a stage of chromosome condensation but in general, malarial parasites have 14 chromosomes ranging in size from 0.7 to 3.5 Mbps numbered in ascending order of size with approximately 6000 to 7000 genes in their genome (World Health Organization webpage). Through restriction fragment length polymorphisms of genes for a histidine rich protein, it has been determined that Mendelian inheritance is exhibited in P. falciparum (Arnot, 1988).
The cell cycle in Plasmodium falciparum, as in any malarial parasite takes place in two stages or cycles as mentioned earlier, the sexual and asexual cycle. To accomplish this, two types of hosts are required, an invertebrate, in which the parasite reaches sexual maturity and a vertebrate where asexual multiplication takes place.. In turn, the invertebrate is deemed the definitive host and the vertebrate, the intermediate host. Figure one gives on overview of the life cycle of a malaria parasite. There are, as to be expected, differences among each species of malaria causing protozoan and P. falciparium is no exception.
Sporozoites - in the definitive host (figure 1, step 1)
The definitive host for P. falciparium is the mosquito. At the end of the sexual cycle sporozoites, (figure 2) the final stage of division of the oocyst, invade the mosquito's salivary glands causing the glands to become friable, discolored, and swollen (Garnham, 1966). As seen in the figure, the sporozoite is a sickle shaped hyaline body, which is able to bend and stretch. A diagram of the structures seen in the anterior end in photo 1 of figure 2, such as the concentric rings is shown in figure 2.
The sporozoites cannot remain indefinitely in the salivary glands and after roughly 40 to 55 days they become non-infective (Garnham, 1966). Not unlike other sporozoites, P. falciparum is very sensitive to changes in osmotic pressure and heat.
Exoerythrocytic Schizogony (figure 1, steps 2-5)
Exoerythrocytic schizogony is the separation of the nucleus but not of the cytoplasm until final segmentation by parasites developing in tissues outside the red blood cells in the vertebrate host. This process is limited to a single generation, the progeny directly proceeding the sporozoites. To follow the development of the schizont, chimpanzee liver samples of infected apes were obtained through a laparotomy. After the fourth day of infection, the schizont causes an enlargement of the parenchyma cell, displacing its nucleus (figure 4.1). The parasite takes a spherical shape 30um in diameter, surrounded by a membrane that encompasses the cytoplasm and nuclei (figure 4.2). Eventually the cytoplasm condenses around each nucleus.
On the sixth day of infection, the schizont grows very rapidly and irregularly in every direction (figure 4.3&4.4) and now measures 50-60 um in length. The nuclei have multiplied asexually, being in the intermediate host, and are interspersed thoroughly within the cytoplasm. Towards the end of schizogony, the parasites break apart into small pieces of cytoplasm, roughly 2um in diameter containing two nuclei. At approximately the end of the fifth day of infection, a final division occurs and the final end products of schizogony called merozoites are made. Roughly 40,000 merozoites (Karapelou, 1987) measuring 0.7um across containing a nucleus and trace of cytoplasm is released (figure 4.5) into the sinusoid, bringing the prepatent phase to an end.
To see this in better detail, refer to figure 5. In this diagram, it can be seen that a large vacuole, around the end of the fifth day, develops in the interior of the multinucleated cytoplasm pushing it outward forming a thick ring. Due to the irregular shape of the vacuole, secondary rings may develop and then eventually these rings are cut into multinucleated fragments. A similar process repeats again and these fragments are further broken down into smaller bits containing fewer nuclei as seen in diagram 5. The process by which fragment contains eight nuclei, then four, then two, then only one, has been termed aposchizogony which simply put, is repeated divisions of a multinucleated mass to a uninucleated mass.
Exoerythrocytic schizogony proceeds at the fastest rate in P. falciparum, so quickly in fact that it made the final details of this process difficult to accurately observe but nevertheless it was done in the 1960's (Garnham, 1966).
Asexual Cycle in the Blood (figure 1, steps 1-9, 10-20)
After the merozoites have released from the exoerythrocytic schizont, they are free to enter the blood stream and attach to any red blood cells they come across. Since P. falciparum is able to attach to erythrocytes at any stage of development, this accounts for its ability to multiply rapidly (Garnham, 1966). P. falciparum can look quite variable in blood smears, from a signet ring, to a large fleshy parasite or to a bizarre tenuiform trophozoite (Garnham, 1966). Its appearance all depends upon which strain of P. falciparum is being observed and under what temperature conditions or time in its cycle.
After a single parasite enters the erythrocyte, a ring forms which is 1.2um in diameter and contains a nucleus, cytoplasm, and a vacuole which stores nutrients derived from the blood (figure 6.1). The nucleus then splits into two unequal parts, which may remain by each other or migrate to opposite ends of the ring (figure 6.2). Multiple P. falciparum may enter a single erythrocyte (figure 6.4) though there is not much difference if single or multiple parasites infect the blood cell.
As the rings grow, extensions of the cytoplasm are extended past the margin of the ring (figure 6.5) and after 24 hours the ring has increased its diameter to roughly 4 um while becoming thicker and fleshier. A large bead like nucleus will project inward from the edge of the ring and a few hours later the vacuole begins to disappear and the nucleus metamorphasizes to a curved or semilunar bar that is hollow in the center (Garnham, 1966). Shortly thereafter, specks of pigment begin to appear in the cytoplasm (figure 6.8) and now the parasites recede into the internal organs, out of the peripheral circulation to almost undetectable levels. Since there are numerous parasites, they are obviously not all at the same stage of development and therefore some are always detectable in the periphery circulation.
During this time, the erythrocyte has undergone significant changes, and structures known as Maurer's cleft (figure 6.7&6.8) comprising of three distinct features can be seen (Garnham,1966). These features comprise of one, a purplish tint that develops, darkening the erythrocyte. Two, a red edging forms around the erythrocyte and three, clefts appear inside the corpuscle. The erythrocyte may shrink slightly and distort or increase in size, depending upon the strain.
At a stage of growth associated with Maurer's clefts, the infected corpuscles become sticky and cling to each other, leukocytes, and especially the endothelial cells of capillaries where blood flow is naturally slower. Organs particularly affected by this are the spleen, bone marrow, brain, intestine, and heart (Garnham, 1966). This also accounts for the decreased number of parasites in the peripheral circulation. At this point, schizogony again occurs but this time in the internal organs. The vacuole disappears and the nucleus divides while the purple pigment concentrates into a single mass (figure 6.9). The parasite at this point grows and the nucleus divides again and again.
Merozoites, which are small oval bodies, are the end product of schizigamy and the exact number and size of them vary, but generally they measure 5um across with about 16 per mature schizont. The merozoites may remain in an unruptured corpuscle for an undefined length of time but occasionally the corpuscle may rupture, releasing merozoites into the peripheral blood, creating a new generation of tiny rings. This asexual portion, including schizogony, occurs with great rapidity in a 48-hour cycle (Garnham, 1966).
Gametocytes (figure 1, steps 21-28)
The gametocyte starts as a round body measuring 2um across (figure 6.11) with a denser cytoplasm on the surface than in the interior and the nucleus is a spongy mass. As the gametocyte grows, it becomes more oval in shape but eventually assumes bizarre forms, the most characteristic of which is a straight border on one side and a bow shaped border on the other (figure 6.12) (Garnham, 1966). Other shapes include spindles (figure 6.14), diamonds, oats, and cigars.
The immature female parasites are considered to be the ones with a more fusiform outline, or ones that stretch in a long thin band with tapering ends across the corpuscle. The males on the other hand, are plumper or sausage shaped. At this point of development in both sexes, the density of the nucleus and distribution of pigment are of slight value.
Next seen are twin crescents (figure 6.15) found in the peripheral blood or in the spleen with the tapering ends close together at one extremity. Masses called Garnham bodies, which are actually thick filaments of considerable size, form (figure 6.16). It is hypothesized that Garnham bodies were a vestigial supporting framework shed by the developing gametocyte or else they are the products of the disintegration of the erythrocyte. A parasitic origin of these structures is though to be more likely (Garnham, 1966).
The gametocytes enter the peripheral blood approximately 8 to 11 days after the initial infection. Their numbers can get as high as 100,000 crescents per cubic mm, and after three weeks are no longer detectable in the blood (Garnham, 1966). Crescents can persist for up to 3 months or even longer in extreme cases.
The mature male gametocyte or microgamete (figure 6.20) measures 9-11 um in length and has a diffuse nucleus spreading over half the total length of the parasite (Garnham, 1966). The cytoplasm varies from a light blue to a pale mauve and the remains of the host cell remains attached to the parasite. The female parasite, or macrogamete, is more slender (figure 6.19) pointed, and measures 12-14um in length. The nucleus is compact but is obscured by pigment granules that surround it. Initially the females outnumber the males 3:1 but towards the end of the infection their numbers become more or less equal (Garnham, 1966).
Mosquito Stages (figure 1, steps 25-35)
The microgametes and macrogametes are transferred to the mosquitoes when the mosquito ingests male and female pre-sex cells and blood from an infected intermediate host such as a human (Karapelou, 1987). Exflagellation, which is the extrusion and liberation of microgametes by microgametocytes, starts very soon after the mosquito has fed. The male gamete rounds up while the nucleus is dividing and 4 to 6 microgametes are released. The warmer the surrounding temperature, the sooner exflagellation commences (Garnham, 1966). The microgametocyte, after undergoing some internal changes, forms four or five flagella that lash at the erythrocyte in their vicinity, but are unable to break free. The microgametocyte measures 16 to 25um in length (figure 7-1) and has a slender form. During this time, the macrogamete has rounded up into a sphere and shed the red cell envelope. The nucleus of the female parasite moves to the surface where a small protuberance is formed and into this, penetrates the microgamete forming a zygote.
Ookinete and Oocysts (figure 1, steps 30&31)
Within a half hour, the zygote elongates and 12 to 18 hours after the mosquito fed, a fully developed ookinete with fused nuclei is found in the mid gut (figure 7.2&7.3). The ookinete is 11-13um long and roughly 2-5um wide. As the ookinete grows, it is classified as an oocyst, measuring 15-20um at maturity after five days. From this point, division is rapid and imperfect cellulation, which is a zone of cytoplasm that becomes incompletely differentiated, occurs. As nuclear division progresses, the nuclei diminish in size and are arranged on the sides of the polygonal masses and sporoblasts or sporozoitoblasts, which are small projections of cytoplasm, develop. These sporoblasts eventually become nucleated and break free as sporozoites thereby completing the whole life cycle of Plasmodium falciparum (Garnham, 1966).
Out of P. vivax, P. ovale, P. malarie, and P. falciparum, P.falciparum is the only one that is fatal in humans. This is due to the fact that P. falciparum is able to migrate into the small blood vessels of vital internal organs and is not confined to peripheral blood unlike the other human malaria parasites. And because P. falciparum can enter the small blood vessels and capillaries where it interferes with the flow of blood, the situation is compounded by venous constriction due to the "malaria toxin".
Blockage begins when the sticky corpuscles, which were able to adhere to each other, erythrocytes and leukocytes, are also able to stick themselves to the endothelial lining of the blood vessels. These particular endothelial cells, through contact with the parasite, become unhealthy, swell up and further impede blood flow. Anoxaemia and malnutrition occur and in turn the endothelial wall weakens and a hemorrhage ensues. This cuts off the blood supply to tissues distal to the blockage/hemorrhage and necrosis takes place. Eventually after enough tissue hemorrhaging and necrosis has occurred, the corresponding organ and organ systems shut down, resulting in death (Garnham, 1966).
Subspecies in falciparum malaria
Falciparum malaria is due to a variety of subspecies or strains. The idea of subspecies has been accepted since the time of the early Italian malariologists. There are several strains today including but not limited to immaculata, quitidiamum, tenue, perniciosa, and aethipicum. Immaculata was identified in 1891 by Grassi because he incorrectly thought that pigment was absent from the organism. In 1892, quotidinamum was named by Celli and Sanfelice because they mistook two broods of parasites for a variety that segmented every 24 hours. Perniciosa was named by Ziemann in 1896 when he found this parasite in the Cameroons. He noted several similar key characteristics between perniciosa and the parasites from east Africa. Also, through comparing life cycles and other characteristics, Raffaele and Lega in 1937 named aethiopicum in present day Ethiopia. The last well-known subspecies, P. tenue, was described by Stephens in 1914 from a blood film out of West Africa based on its morphology and life cycle.
Plasmodium falciparum has proven itself to be extremely resilient and highly resistant to any form of immunization. By means of an elaborate and complex life cycle, this protist has managed to wade all attempts at irradiation and continues to thrive and threatens the lives of millions of people each and every year (Schaechter, 1993).