Cultivation in vitro of Plasmodium gallinaceum oocysts

Abstract

Large numbers of viable infectious Plasmodium gallinaceum sporozoites, virtually free of mosquito tissue, were obtained by culturing somewhat younger stages of the sporogonous cycle. Twenty or more oocysts were placed in each hanging- or sitting-drop culture. Grace's insect tissue culture medium, slightly modified and supplemented with either vertebrate and/or invertebrate sera, was employed. In addition, approximately one half of the cultures contained mosquito cells, organs, or whole body extracts. Nine-day-old oocysts usually completed development within 24 hours and liberated sporozoites shortly thereafter. The extent of development in 8-day-old oocysts was dependent upon whether sporoblast formation with subsequent budding of the sporozoites had or had not occurred prior to explantation. If the former, the oocysts continued their development in 60% of the cultures; if the latter, less than 1% of the oocysts were capable of producing sporozoites. Attempts to culture 6- and 7-day-old oocysts were not successful. This failure of the younger oocysts to develop in vitro was attributed to insufficiencies in the culture medium, lack of tissue bulk, suboptimal conditioning of the medium, and the relatively static environment of the present system. The suggestion was made that such factors might be largely negated if the oocysts were cultured in the presence of an actively growing mosquito cell strain.