Avian embryos and tissue culture in the study of parasitic protozoa: I. Malarial parasites

Abstract

1. 1. Chicken and duck embryos have been used extensively and with considerable success in research with avian malarial parasites. Generally, infection has been via the vascularized membranes which connect intimately with the embryo itself, particularly through supra-chorioallantoic implantation, intravenous inoculation into the vessels of the chorioallantois, and injection into the yolk sac. Other infection routes used for viral research, such as intra-embryonic, intra-allantoic, intraamniotic, and inoculation into the extra-embryonic body cavity, have not as yet been successfully utilized. 2. 2. Advantages presented by the avian embryos over adult experimental animals, include those of (a) virtual freedom of the embryo from concomitant bacterial or parasitic infections, (b) relative ease of infection of the embryonic tissue because of absence of immune mechanisms, (c) freedom from danger of cross-infection from one embryo to another within the same experimental series, (d) easy access to the embryo through one of several infection routes, (e) relatively convenient experimental host of minimum size, requiring no feeding, cage space, or servicing other than incubation, and (f) a host so small that recovery of the infectious agent is markedly easier than in an adult animal. 3. 3. Use of avian embryos has contributed materially to the understanding of such phenomena as: age immunity; preferential susceptibility of the erythrocytic series to infection with the Plasmodium species; underlying basis of the incubation period; chemotherapy of malaria; pigment production in the erythrocytic series; species susceptibility to the malarial infections in the vertebrate hosts; pathology of the tissue phase of malaria, and comparison of sporozoite, blood, and tissue-induced infections. 4. 4. The observation and study of the living malarial parasite at the cellular and tissue level of organization has been made possible through the modem techniques of in vitro cell culture coupled with phase contrast microscopy and time-lapse microcinematography. Through applications of various methods of cell culture, exoerythrocytic stages of six species of avian Plasmodium have been cultivated, namely P. elongatum, P. gallinaceum, P. cathemerium, P. relictum, P. lophurae, and P. fallax. 5. 5. Most workers have utilized tissues from infected animals as a culture source, while a few have infected normal cells with sporozoites, infected blood, or exoerythrocytic forms. Fragments of tissue embedded in plasma clots were used most frequently and produced cultures too thick for microscopic examination. Hence, all these studies were made on fixed and stained material. Hanging drop preparations and monolayer cultures, used by more recent workers, have permitted microscopic studies of the parasites in vitro. By cultivating explants alternately in hanging drops and plasma clots, and by using roller tubes, parasites have been maintained in culture for as long as a year. 6. 6. The methodology of cell culture has been utilized in experimental malariology in the following ways: to study host-parasite relationships with special reference to the types of cells invaded by the parasites in vitro; to confirm the work of investigators who based results of their studies on the examination of fixed and stained material; to elucidate the development of exoerythrocytic schizogony, particularly in regard to the details of cryptozoite formation, nuclear division of the parasites, and maturation of the schizonts; to point the way to future research on mammalian species of Plasmodium, such as initiating infections in normal mammalian cells in tissue culture through the use of sporozoites from simian and human sources; and finally, to offer a method of direct observation of the effects which antimalarials have on cells and parasites in vitro.