G. A. Butcher

Structure and invasive behaviour of Plasmodium knowlesi merozoites in vitro.

The structure and invasive behaviour of extracellular erythrocytic merozoites prepared by a cell sieving method have been studied with the electron microscope. Free merozoites contain organelles similar to those described in late schizonts of Plasmodium knowlesi. Their surface is lined by a coat of short filaments. On mixing with fresh red cells, merozoites at first adhere, then cause the red cell surface to invaginate rapidly, often with the formation of narrow membranous channels in the red cell interior. As the merozoite enters the invagination it forms an attachment by its cell coat to the rim of the pit, and finally leaves this coat behind as it is enclosed in a red cell vacuole. Dense, rounded intracellular bodies then move to the merozoite periphery, and apparently rupture to cause further localized invagination of the red cell vacuole. The merozoite finally loses its rhoptries, the pellicle is reduced to a single membrane and the parasite becomes a trophozoite. Invasion is complete by 1 min after adhesion, and the trophozoite is formed by 10 min.

Lamellar membranes associated with rhoptries in erythrocytic merozoites of Plasmodium knowlesi: a clue to the mechanism of invasion.

In merozoites of Plasmodium knowlesi, rhoptries have a dense substructure of fine (2.5 nm diameter) granules and short rods. These are not altered by lipid extraction, and stain with ethanolic phosphotungstate indicating a proteinaceous composition. Various types of fixation also show multilamellar whorls with a periodicity of 5-7 nm in the tips of rhoptries or extruded at the merozoite apex. In merozoites fixed during invasions of red cells, membrane continuity typically occurs between the rim of the rhoptry canal and the red cell membrane, but where this contact has apparently been lost, extensive membranous whorls and blebs are often found at the apex of the parasite. Similar structures occur at the apices of merozoites within late-stage schizonts. It is suggested that the same mechanism which generates these lamellae forms the parasitophorous vacuole by inserting membranous elements formed by the parasite into the red cell membrane, so causing its invagination. A similar mechanism may be responsible for the release of merozoites from the late-stage schizont.

Freeze fracture studies on the interaction between the malaria parasite and the host erythrocyte in Plasmodium knowlesi infections.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.

In vitro isolation of Plasmodium knowlesi merozoites using polycarbonate sieves.

A culture chamber fitted with a polycarbonate sieve has been used to isolate Plasmodium knowlesi merozoites as they are released from schizonts. A 3 mum pore-size sieve allows passage of normal erythrocytes and red cells containing rings and trophozoites and can be used to concentrate schizonts from a mixed cell population. A 2 mum pore-size sieve retains normal and parasitized cells and provides uncontaminated merozoites in high yield (5 x 10(10) merozoites per ml schizonts). Merozoite viability diminishes rapidly during 30 min after isolation. These preparations should prove valuable for studies of the biochemical, physiological and antigenic properties of this transient phase of the malaria parasite.

MEROZOITE VACCINATION OF DOUROUCOULI MONKEYS AGAINST FALCIPARUM MALARIA

Erythrocytic merozoites of Plasmodium falciparum (Gambia) were isolated from cultures of schizont-infected human red cells on CF 11 cellulose columns. Douroucouli monkeys vaccinated with such preparations stored in liquid nitrogen and then emulsified in Freunds complete adjuvant (F.C.A.), were resistant to successive challenges with West African (Lagos) and East African (Uganda Palto-Alto) strains of P. falciparum. The induced immunity is specific since vaccination with P. knowlesi merozoites in F.C.A. does not modify the course of P. falciparum infections in douroucouli monkeys.

Structure and development of the surface coat of erythrocytic merozoites of Plasmodium knowlesi

SummaryThe surface of extracellular merozoites of P. knowlesi is covered with a coat 15–20 nm thick, made up of clusters of filaments standing erect on the plasma membrane. Filaments have stems 2 nm thick, the peripheral ends of which are complex, branching or ending in long trailing threads. Coat filaments occur on the surface of the parasite in regular rows at an early schizont stage, and persist until well after merozoite release. They are sensitive to trypsin and papain, and bind ethanolic phosphotungstate, indicating a proteinaceous nature. They are also removed by exposure to phosphate-buffered saline. Filaments bear negative charges, binding cationised ferritin throughout the depth of the coat and staining with ruthenium red. They cover the whole merozoite surface and mediate intercellular adhesion at distances of 15–150 nm, membrane to membrane. It is suggested that these filaments correspond to a major merozoite surface protein, and are important in the initial capture of red cells.

The in vitro culture of the blood stages of Plasmodium berghei

Abstract Plasmodium berghei was cultured in medium 199 with 10% serum at various temperatures. At 31° and 37°C the parasite numbers decreased. Experiments at 15°C achieved consistent multiplication rates greater than one, with a maximum of three-fold.

Plasmodium knowlesi infections in a small number of non-immune natural hosts (Macaca fascicularis) and in rhesus monkeys (M. mulatta)

The natural host of Plasmodium knowlesi is the kra monkey, Macaca fascicularis, but this parasite, initially mistaken for P. malariae, is now infecting humans in some areas of Southeast Asia. Here we present data from experiments performed in the 1970s in which sera from a few naive M. fascicularis, taken in the course of a first infection, exhibited rapidly rising inhibition of in vitro replication of P. knowlesi. The results were compared with sera from P. knowlesi-infected rhesus monkeys that usually die if left untreated.

Immunization Against Erythrocytic Forms of Malaria Parasites

It is almost 20 years since a worldwide program of malaria eradication was formally adopted by the 8th World Health Assembly and its implementation coordinated by the World Health Organization (WHO). The strategy of eradication involved widespread indoor spraying with residual insecticides and elimination of remaining foci using antimalarial drugs and further spraying. The success of this international endeavor can be judged from a recent WHO report showing that malaria has been eradicated or controlled in almost 80% of originally malarious zones.* However, the remaining areas where eradication could not be implemented include 66 countries covering vast tracts of the developing world, notably in Africa, inhabited by about 500 million people. Reliable data are not available from these areas, but it seems likely that malaria at present produces a morbidity rate of 100 million and mortality of about 1 million per annum, mainly among young children. Moreover, the resurgence of malaria in Pakistan, Bangladesh, Sri Lanka, and several Indian provinces that all had advanced eradication programs is a source of much alarm. It is clear that tropical areas still harbor a vast reservoir of malarial infection and that its eradication or even containment is no longer feasible with available health facilities.

Culture of malaria parasites

A human malaria vaccine that will protect against the blood stages of Plasmodium falciparum is dependent upon the continuous cultre of the parasite. Scientists have demonstrated that it is now possible to achieve this on a small scale. Present expertise needs to be expanded to produce larger quantities of material for antigenic studies and potential vaccine production.