Plasmodium knowlesi: secondary processing of the malaria merozoite surface protein-1.

Secondary processing of the Plasmodium falciparum malaria merozoite surface protein-1 (MSP-1) is defined as a single proteolytic cleavage within the carboxy-terminal membrane-bound component of the MSP-1 protein complex on the free merozoite surface. The N-terminal cleavage product (MSP-1(33)) is shed from the parasite surface along with a number of other polypeptides, whereas the C-terminal processing product remains bound to the merozoite surface and is the only part of MSP-1 detectable in the newly invaded host cell. We report that secondary processing of MSP-1 takes place in a similar manner on invasive merozoites of the simian malaria parasite Plasmodium knowlesi. Processing can take place to a limited extent in pure isolated merozoites; however, within 10 min of the addition of purified invasive merozoites to rhesus erythrocytes, processing and shedding of MSP-1 has gone to completion only in those parasites which have undergone invasion; residual free merozoites remain uniformly reactive with antibodies against MSP-1(33). Successful invasion is therefore associated with complete shedding of MSP-1(33) from the merozoite surface. The nucleotide sequence of the 3' domain of the P. knowlesi MSP-1 gene is also presented.

Retrospective study of diseases in a captive Lemming Colony.

Fifty-four ill or nonproductive lemmings (Dicrostonyx spp.) were evaluated for signs, lesions and causes of disease for 5 yr in a domestic colony. Parasitic granulomas caused by Encephalitozoon cuniculi were the most common finding and were seen in 22 lemmings. The disease was characterized by circling and torticollis with granulomas in many tissues, especially the central nervous system. Suppurative otitis occurred in 12 lemmings and was associated with Klebsiella pneumonia infection; circling was the common sign. Hepatic microabscesses were present in seven lemmings but a cause was not identified. Five lemmings had neoplasms and 14 had either suppurative processes, aspermia, or ovarian cysts.

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.

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

The 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.

Long term cultivation of a simian malaria parasite (Plasmodium knowlesi) in a semi-automated apparatus.

An apparatus is described and illustrated for the continuous semi-automated cultivation of Plasmodium knowlesi. The change of medium was automated and involved six operations. Parasites were maintained for 12 weeks, at which time the experiment ended. During this period, parasite density, morphology and serological specificity were monitored. Parasites retained synchromy and normal morphology only for the first cycle in culture. The maximum degree of morphological abnormality (90%) was reached at three weeks. Lysis of infected erythrocytes seemed to occur before full maturation of the parasites. Infected cells cultured for 31 days produced a normal, fulminating infection in a rhesus monkey.

Antigenic analysis of sequential erythrocytic stages of Plasmodium knowlesi.

The antigenic composition of sequential erythrocytic stages of Plasmodium knowlesi has been compared by crossed immuno-electrophoresis using a pool of immune rhesus monkey antiserum. Eleven major parasite antigens have been identified; 9 are stage-independent, and 2 stage-dependent. Differences in the relative amount of the stage-independent antigens have been demonstrated and quantified. The distribution of antigens between parasites and schizont and infected red cell membranes has been examined. Only 6 of the 11 parasite antigens were exhibited by a schizont membrane preparation, all these antigens were also expressed by the intracellular parasite. Antigens exclusive to the schizont membrane were not demonstrated.

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.

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.