Abstract
BackgroundLarge scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites. The initial step in this process is the production, in vitro, of ookinetes from gametocytaemic blood. Methods for isolation of these ookinetes from blood cells have been described; however, in addition to yield often being low, processing time and potential for contamination by erythrocytes remain high.MethodsThis study compares two procedures for retaining mature ookinetes from blood stage cultures, whilst removing red blood cells and other contaminants prior to further culture of the parasite. The well established method of isolation on Nycodenz cushions is compared with a novel method utilizing the innate magnetic properties of the haem pigment crystals found in the cytoplasm of ookinetes.ResultsYield and viability of ookinetes were similar with both isolation methods. However, in our hands magnetic isolation produced a cleaner ookinete preparation much more quickly. Moreover, decreasing the flow rate through the magnetic column could further enhance the yield.ConclusionWe recommend the enrichment of an ookinete preparation prior to further culture being performed using the magnetic properties of Plasmodium berghei ookinetes as an alternative to their density. The former technique is faster, removes more erythrocytes, but day-to-day costs are greater.
Highlights
Large scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites
Studies on ookinete stages of the malaria parasite were greatly aided by the development, over 30 years ago, of a method for the production of ookinetes in vitro following 24 h culture of gametocytaemic blood
Only 1% of ookinetes was recovered from either separation method
Summary
Large scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites. Studies on ookinete stages of the malaria parasite were greatly aided by the development, over 30 years ago, of a method for the production of ookinetes in vitro following 24 h culture of gametocytaemic blood. Inhibitors of DNA synthesis (such as mitomycin C, aphidicolin and Berenil [8,9]) have been successfully employed as tools to further enrich ookinete preparations by eliminating asexual stages from gametocytaemic blood. This objective has been achieved using pyrimethamine [7]. Magnetized beads coated in an antibody raised against a major ookinete surface protein, P28, have been used to isolate ookinetes from erythrocytes [10]
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