Invasion characteristics of a Plasmodium knowlesi line newly isolated from a human

Abstract

Plasmodium knowlesi is extensively used as an important malaria model and is now recognized as an important cause of human malaria in Malaysia. The strains of P. knowlesi currently used for research were isolated many decades ago, raising concerns that they might no longer be representative of contemporary parasite populations. We derived a new P. knowlesi line (University Malaya line, UM01), from a patient admitted in Kuala Lumpur, Malaysia, and compared it with a human-adapted laboratory line (A1-H.1) derived from the P. knowlesi H strain. The UM01 and A1-H.1 lines readily invade human and macaque (Macaca fascicularis) normocytes with a preference for reticulocytes. Whereas invasion of human red blood cells was dependent on the presence of the Duffy antigen/receptor for chemokines (DARC) for both parasite lines, this was not the case for macaque red blood cells. Nonetheless, differences in invasion efficiency, gametocyte production and the length of the asexual cycle were noted between the two lines. It would be judicious to isolate and characterise numerous P. knowlesi lines for use in future experimental investigations of this zoonotic species.

Figure 1. Isolation, expansion of the P. knowlesi, UM01 line.

The UM01 line was isolated from a knowlesi malaria patient and expanded by passaging it through M. fascicularis (macaque A, B, C and D). The ring stages of the UM01 line obtained from the expansion were cryopreserved until further use. Parasites obtained either from in vivo or ex vivo maturation were used for invasion and inhibition experiments.

Figure 2. Invasion studies with P. knowlesi.

P. knowlesi (UM01 and A1-H.1 strains) invasion in macaque and human normocytes and reticulocytes; bars = median values (black for the UM01 line and red for the A1-H.1 line). The effect of red blood cell species (human vs macaque) and age (normocyte vs reticulocyte) was compared using a 1Way ANOVA and Tukey’s Multiple Comparison Tests.

Figure 3. Giemsa stained blood smears of UM01.

Gametocytes (red arrow) were observed at different days of culture.

Figure 4. Characterising the DARC dependence of P. knowlesi merozoites for the invasion of human and macaque normocytes.

Inhibition of P. knowlesi (UM01 and A1-H.1 lines) invasion into human (Hu) and macaque (Mc) normocytes by MAb Fy6 and anti-Fy^b (Duffy negative human blood was used as a positive control). Bars show the percentage grand median inhibition levels normalised to the Ab-free control of each independent experiment for the UM01 line. The effect of MAb Fy6 and anti-Fy^b in invasion inhibition in both human and macaque blood was compared using a 1Way ANOVA and Tukey’s Multiple Comparison Tests.