Folic and Folinic Acids in Relation to the Development of Plasmodium lophurae

Abstract

Since the erythrocytes of ducks infected with Plasmodium lophurae contain much more folic and folinic acids than those of uninfected ducks, and since chromatography indicated that these growth factors were the same in infected and uninfected birds, it was of interest to follow changes in levels of folic and folinic acids with development of P. lophurae in red blood cells. Taking advantage of the synchronous cycle of P. lophiraae maintained in our laboratory, we found that when the parasites were in the uninucleate stage there was appreciable increase in folic acid content, and the ratio of folic to folinic acid was 3:1. However, within 24 hr when the parasites had developed to multinucleate and segmenter forms, there was a fiveto tenfold increase of folinic acid only, so that the ratio of folic to folinic acid was approximately 1:2. HIence, a marked increase in folinic acid was correlated with the development of the parasites from a uninucleate to a multinucleate stage, but there was no change in folic acid during that period. When P. lophurae was maintained in red cell suspension in vitro only a small increase in folinic acid occurred, despite the fact that the development from uninucleate to multinucleate forms was as great as that occurring in vivo. Earlier work has shown that the red cells of ducks infected with the bird malaria parasite Plasmodium lophurae contain much more folic and folinic acids than those of uninfected ducks of the same age (Trager, 1959; Siddiqui and Trager, 1964). Only a part of this increase could be accounted for by the folic and folinic acid contents of the parasites themselves. Furthermore, chromatography on paper has shown that the folic and folinic acid-active materials of normal erythrocytes and erythrocytes infected with P. lophurae are the same (Siddiqui and Trager, 1964). In view of these facts, and of the fact that the extracellular survival in vitro of P. lophurae was favored by the presence of folinic acid in the culture medium (Trager, 1958), it was suggested that the increased levels of folic and folinic acids might result from an altered metabolism of the host red cell, rather than from synthesis by the parasites. It was, therefore, of interest to follow the changes in the levels of folic and folinic acids with the development of P. lophurae in red blood cells. Received for publication 11 February 1966. * This paper was read at the Second International Conference on Protozoology, London, 1965. t Supported by Public Health Service Grant 5-T1AI-192. + Present address: Department of Preventive Medicine, Stanford University School of Medicine, Palo Alto, California 94304. MATERIALS AND METHODS Two-week-old ducklings were inoculated with P. lophurae in such a way as to give severe and highly synchronous infections. On the 4th day of infection (10 to 11 AMV), the parasitemias were from 60 to 100% and almost all the parasites were uninucleate (Fig. 1), but on the 5th day of infection (10 to 11 AM), most of the parasites had become multinucleate (Fig. 2). For each set of experiments a 2to 3-ml sample of blood was taken from each of three ducklings on the 4th day of infection. These samples were pooled and mixed with two volumes of uninfected duck blood to reduce the total parasite count to a level suitable for in vitro cultivation. Coagulation was prevented by mixing the blood with onetenth its volume of a solution containing 30 mg heparin and 0.85 g NaCl per 100 ml. A sample of this mixture was then prepared for microbiological assay. The rest was inoculated in 1.5-ml amounts to flasks holding 4.5 ml of a medium modified from that of Geiman et al. (1946) and previously used for maintenance of P. lophurae in erythrocyte suspensions in vitro (Trager, 1947; McGhee and Trager, 1950). The flasks were incubated at 40 C, rocked at 16 cycles per minute, and received a slow current of air with 5% C02. On the following day, the 5th day of the infection, the cells in the flasks were prepared for microbiological assay. At the same time samples were again taken from the same three ducklings bled on the previous day, and these were prepared individually for assay. Blood films stained with Giemsa were made from each sample. For all the assays the red cells were washed three times in physiological saline, hemolyzed, and incubated 90 min in a diluted buffer with ascorbic acid (Toennies, Bueding, and Phillips, 1956). The hemolysates were deproteinized by