TH E315 ' 5‘. «9.01 huh" ‘4‘ ‘:‘“:W:tm- a; ,4 G u- I I ~ .4 K 4 at "t This is to certify that the thesis entitled STUDIES ON SERUM REQUIREMENTS FOR THE CULTIVATION OF PLASMODIUM FALCIPARUM: ANIMAL SERA AND MEDIUM ENRICHMENT presented by ALAN A. DIVO has been accepted towards fulfillment of the requirements for M.S. degreein M.P.HL \Wflkm Major pr fe Date 12/8/81 0-7 639 ('9 MSU LIBRARIES n RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. STUDIES ON SERUM REQUIREMENTS FOR THE CULTIVATION OF PLASMODIUM FALCIPARUM: ANIMAL SERA AND MEDIUM ENRICHMENT BY Alan A. Diva A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1981 ABSTRACT STUDIES ON SERUM REQUIREMENTS FOR THE CULTIVATION OF PLASMODIUM FALCIPARUM: ANIMAL SERA AND MEDIUM ENRICHMENT By Alan A. Diva Plasmod ium falc iparum human For continuous cultivation of serum requirements have been reduced from 102 to 52. Pooling a number of serum lots eliminates the variability between individual samples, subsequently reducing the amount of human serum required for optimum parasite growth. Freshly collected and pooled lots of various animal sera, as well as commercially available sera, were tested and compared to 52 pooled human serum. NeOpeptone and combinations of animal sera were examined as supplements to the culture medium, RPMI 1640. As an alternative to human serum, high quality 'bovine serum supplemented with neopeptone could support continuous parasite growth, but at reduced levels. Previous experiments indicated that dialysis of human serum removes low molecular weight components (6,000-8,000 MW) which are falciLarum. By making essential for continuous cultivation of g. comparisons using other media, richer than RPMI 1640, we were able to determine that hypoxanthine was the major dialzyable nutrient required Alan A. Diva for parasite development. Further’ experiments demonstrated that high quality bovine serum requires the addition of 3-12 x 10-)! hypoxan- of P. falciparum, eliminating thine to support continuous cultures neopeptone as a necessary supplement. This is dedicated to life. We search for the knowledge to give meaning to life. And, it is that life giving meaning to knowledge. Life is the essence of my existence, and yours. Life's drive for the preservation of life. With rational moralism and technically oriented realism. We search for the thread that is life's thread. To unite the universe and man into a state of mutual being. ii ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my major professor, Dr. James B. Jensen, for providing me with the Opportunity to study in his laboratory, and for his patience and guidance required for the com- pletion of my research and this thesis. I would also to thank Drs. Robert Moon, Robert Bull, and Jeffrey Williams for participating as members of my guidance committee. To my fellow laboratory workers and friends, Mike Boland, John VandeWaa, Tim Geary, Al Gabrielsen, and Tom Capps, a special thanks for their companionship and support. Without their presence the experience would have been much less fulfilling. Over and above all I thank my wife, Christine, and especially my son, Erich, fbr their continuing, unabating support and encouragement. For the sacrifices they have endured for my benefit; to them I owe the world. iii LIST OF TABLES . . . LIST OF FIGURES . . INTRODUCTION . . . . LITERATURE REVIEW . Nutritional Biochemistry of TABLE OF CONTENTS Introductory comments Carbohydrates Introduction TranSport and metabolism Erythrocytic Plasmodium Pentose-phosphate pathway 0 utilization and electron Nucleic acid precursors tranSport Pyrimidines O I C C C C C O O O O O O Purine tranSport and salvage Amino acids . Introduction Biosynthesis of amino acids . . . Digestion of host cell hemoglobin Exogenous amino acids . . Lipids . . . . . . . . . Vitamins and Cofactors Folates . . Pantothenates Vitamins A, BI’ 82, lg Vitro Cultivation of Intraerythrocytic Plasmodium Discontinuous cultivation Early attempts . Rocker-dilution/rocker-perfusion techniques Avain species Mammalian species B6, 0 . C, biotin, and niacin 0 Static cultivation techniques . . . . . . . iv Page vi vii mwvwa‘mmmb 15 15 15 17 18 19 20 Continuous cultivation of P. falciparum- Petri dish-candle ARTICLE I: STUDIES ON SERUM REQUIREMENTS Introduction . . . . Cultivation technique Medium . . . . . . . Erythrocytes . . . . jar Parasites for cultivation . serum 0 O O O O O O O technique . Serum reduction or replacement CULTIVATION OF PLASMODIUM FOR THE FALCIPARUM (I): ANIMAL SERA . Abstract . . . . . . . . . Introduction . . . . . . Materials and Methods . . . Results and Discussion . . Summary . . . . . . . . . . References . . . . . . . . ARTICLE II: STUDIES ON SERUM REQUIREMENTS FOR THE CULTIVATION 0F PLASMODIUM FALCIPARUM (II): MEDIUM ENRICHMENT Abstract . . . . . . . . Introduction . . . . . . . Materials and Methods . . . Results and Discussion . . Summary . . . . . . . . . . References . . . . . . . . BIBLIOGRAPHY . . . . . . . . . APPENDIX 22 22 22 23 23 24 25 25 28 29 30 31 33 41 42 43 44 45 46 48 59 61 62 77 Table: 1. Table: LIST OF TABLES Article I Comparison of 52 pooled human serum with 10% freshly collected, pooled but unsupplemented, animal sera . . . . . . . . . . . . . . . . . . . . . Comparison of the growth of P. falciparum in 52 freshly collected, pooled human serum (PBS) to growth in 10% freshly collected, pooled adult bovine serum (PBS) and "high quality" commercially prepared bovine sera. Three types of commercially processed human serum, with and without neOpeptone, are also included. All bovine sera were supplemented with neopeptone (Neo) . . . . . . . . . . . . . . . . Article II Growth of P. falciparum in RPMI 1640, Ham's F12, 199 with Egrle‘s Salts, and 199 with Hank's Salts. Each medium was supplemented with 101 pooled human serum. Exhaustive dialysis was 1:10,000, whereas control sera was minimally dialyzed 1:0.5 . . . . . . Comparison of P, falciparum growth, in RPMI 1640, between control PBS and exhaustively dialyzed PBS, with and without hypoxanthine (HX) added . . . . . . Comparison of P. falciparum growth, in Ham's F12 Supplemented wIth 5! PBS to growth in Ham's F12 supplemented with 102 PBS, 10% PCS, 10% PPS, or a combination of 3.31 of each . . . . . . . . . . . . Comparison of P, falciparum growth in RPMI 1640 and Ham's F12, each containing 5% PBS, 10% PBS, or 102 PBS + 1.2 m1 neOpeptone (15% w/v) . . . . . . . . Growth of P. falciparum in 102 PBS, using RPMI 8Upp1ement§d with minimally or exhaustively dialyzed neoPeptone, and the effect of supplementation of the medium with hypoxanthine (RX) . . . . . . . . . . . vi Page 37 39 49 51 52 53 57 Figure: Article I 1. LIST OF FIGURES Page Titration of pooled human serum using P. falciparum strain FCR3. Z Parasitemia represents the number of parasites per 10,000 erythrocytes. Values are the Mean :_S.D. for 4 observations . . . . . . . . . Figure: Article II 2. Titration of hypoxanthine using 102 freshly collected, pooled adult bovine serum (PBS) in RPMI 1640, P. falciparum strain FCR3 was used. Parasitemias represent the number of parasites per 10,000 erythrocytes. Values are the Mean :_S.D. for 4 observations . . . . . . . . . 55 vii INTRODUCTION Until recently, basic research on human malaria organisms has been limited by the lack of availability of the parasites. Initially most studies were carried out using either parasites taken directly from patients or, more often, animal Species of Plasmodium were used. Later certain simian hosts were found to be susceptible to the human malarias (1-3). Although employable, these methods were obviously inferior to maintaining parasites _1_r_1’ vitro away from an animal ghost. Attempts to culture both human and animal Plasmodium slowly gave way to methods for the continuous cultivation of P.fa1ciparum (4-—6). The Trager and Jensen method (4) has had by far the greatest impact on malaria research. With the advent of the Trager and Jensen method (4) continuous cultures of _P_. falciparum are routinely maintained in laboratories throughout the world. This technique has afforded innumerable Opportuni- ties to study parasite biology, biochemistry, and immunology; and these studies should facilitate the development of superior chemotherapeutic agents and possibly an effective vaccine based on antigens derived from it} 31319 cultures of the parasite. Currently the parasite cultures require the addition of 101 human serum to RPMI 1640 (7) for Optimum parasite growth. This requirement places a number of constraints on the investigator; some laboratories 2 are located in endemic areas where locally procured human serum may be inhibitory to the culture due to antimalarial antibody or antimalarial drugs present in locally procurred sera. Even in laboratories in developed nations fresh human serum maybe difficult to obtain. Further- more, any widely used vaccine should not be grown in human serum since the possibility of contamination with infectious agents is a real one. For these reasons, and others, a suitable replacement for human serum would be a beneficial development. The following points illustrate the objectives of this thesis: 1) It has been shown that human serum from different individuals varies dramatically in its ability to support optimum parasite growth; some samples require concentrations up to 152 while others are suitable at much lower concentrations (8). Thus, the minimum serum requirements could only be determined by using a pool of several randomly collected sera. Such would be a necessary first step in defining the nutritional contribution serum makes to the in 33333 cultivation of P. falciparum. 2) Commercially prepared human and animal sera were shown to be inadequate for supporting continuous parasite growth (8); because commercial processing is known to detract from the quality of animal sera for tissue culture purposes (9, 10), freshly collected and pooled animal sera were examined as replacements for human serum. 3) Reports (11, 12) indicating that human serum can be replaced using peptone- supplemented calf serum were not readily reproducible using commercial- ly available calf serum. It was of interest to determine if the quality of bovine serum affected the results when using peptone-supplemented medium; and to determine what factor(s) the peptone provide that are required for parasite growth. 4) Since dialyzed human serum has been 3 shown to be lacking in low molecular weight component(s) necessary for parasite growth in 3.1.53.9 (8), it was possible to examine some factors required for parasite deve10pment using dialyzed serum supple- mented with low molecular weight nutrients. The literature review has been divided into two broad catagories, I. Nutritional Biochemistry of Erythrocytic Plasmodium and II. _I_n_ vitro Cultivation of Intraery- throcytic P lasmodium. LITERATURE REVIEW Nutritional Biochemistry of Erythrocytic P lasmod ium Introduc tory comments The development of an adequate method for continuous cultivation of P. falciparum has been intimately associated with the knowledge of Plasmodium. biochemistry. Many biochemical studies on the malarial parasites have provided information directly applicable to in 2259 cultivation. The converse has been true for studies relating to the improvement of _1_r_r vitro culture techniques. Observations made on parasitized animals have also provided important facts. Together, the knowledge gained has provided the basic information required for the development of a culture system which not only meets the nutritional requirements of parasite and host RBC,.but also provides a physical environment which is conducive to survival of both. Further efforts to improve the Trager-Jensen method (5) by eliminating serum from the culture medium undoubtedly require a continuing appreciation for the biochemistry of the malarial parasite. Carbohydrates Introduction. The earliest studies of plasmodial biochemistry concentrated on carbohydrates; but many of these studies have been justifiably criticized for numerous reasons (13-15). Techniques were often inadequate for maintaining parasite viability £9. 1132; thus metabolic parameters were determined on dying parasites; and since early cultures contained host contaminants such as immature RBC's, platelets, and leukocytes, all having different carbohydrate metabolic requirements, it was impossible to dissect the parasites' metabolism from that of the other cells. Methods employed for removing unwanted blood cells from parasitized blood varied in their degree of sucess (16-19); even "free" parasites prepared by saponin lysis of the RBC remained contaminated with host cell membranes (14, 20, 21). For these reasons many of the earlier reports on carbohydrate metabolism were in error. TranSport and metabolism. Intraerythrocytic stages of Plasmo- gig require simple sugars as an energy source; they do not store glycogen as a carbohydrate reserve (16, 22, 23). This requirement was appreciated early; in 1912 Bass and Johns (24) demonstrated that glucose or maltose were required for'_i_n y_i_t_r£ deve10pment of P. falciparum and P. viVax. Infections of P. gallinaceum in chickens were much more severe when the animals received intravenous injections of glucose (25). In vitro studies indicated that substrate utilization was species Specific, but that all Plasmodium utilize glucose (13); furthermore infected RBC's consume dramatically more glucose than uninfected cells (14, 23, 26-33). To accomondate the needs of the parasite the infected RBC membrane undergoes permeability 6 changes (32, 34, 35); both simple diffusion and carrier-mediated processes appear to be affected. Although erythrocytic glucose metabolism varies between Species, similar pathways exist within the mammalian and avian groups of malarial parasites. In general, mammalian species incompletely oxidize glucose producing organic acids, predominantly lactate, as well as neutral volatiles, and small amounts of succinate, keto acids, and lipids (16, 18, 28-31, 33, 36-40). Avian malarias appear to oxidize glucose more competely to yield CO and organic acids (41-44). 2 Reports indicate that all Plasmodium possess the glycolytic enzymes necessary to carry out the reactions typical of the Embden-Meyerhoff pathway (23, 31, 45-53), and that only the avian Species appear to possess theerequired enzymes for the TCA cycle (44, 54-59). Electron microscopy has shown that the avian parasites have cristate mitochon- dria (60), whereas in mammalian species mitochondria are typically acristate (61). Product analysis indicates the plasmodia may possess alternative pathways for pyruvate oxidation (13, 23). Pentose-phosphate pathway. Product and enzyme analyses indicate that all malarial parasites examined lack a functional pentose- phosphate pathway (23, 31, 37, 38, 60-66); the manner in which the parasite compensates for this deficiency remains unknown. It has been proposed that the plasmodial parasites rely on the host RBC for pentose sugars and for providing reducing power to maintain reduced glutathione and NADPH levels. (13, 69); the growth of _I_’_. falciparum appears to be impaired in individuals with a G-6-PDH deficiency (67, 70-73). The identification of glutamate dehydrogenase in P. berghei (74) and E. lophurae (75) indicates that NADPH levels may be maintained by 7 the parasite. Pentose sugars may be derived from hostcell ATP meta- bolites taken up by the parasite (76). 9 utilization and electron transport. Early studies demon- 2 strated that all plasmodia take up 0 2; but the extent of O2 utiliza- tion was difficult to interpret because of the presence of host leuko- cytes and thrombocytes (26, 27, 30, 31, 42, 77-79). Attempts to culture 1:. lophurae (80) and E. knowlesi (81, 82) indicated that growth was favored at reduced 02 tensions and that high concentrations of 02 were detrimental to parasite survival. The Trager and Jensen method (5) has been used to determine that P. falciparum is an obligate microaerophile and optimum parasite growth occurred in an atmosphere of ‘32 02, and the balance N2; anaerobic conditions or when 02 was greater than 211 (83). The role of O2 in parasite biochemistry remains speculative; no growth occurred under uncertainties exist about the nature of electron tranSport in plasmo- dia. Cytochrome oxidase has been identified in both avian (83) and mammalian species (16, 34); but no other enzymes typical of electron transport. It has been postulated that cytochrome oxidase may be coupled to 33 m biosynthesis of pyrimidines. Other 02 re- quiring hydrolases and oxygenases may' be present in the malarial parasite (85). Meta110protein oxygenase inhibitors have been found to inhibit parasite growth (86). Nucleic acid precursors Pyrimidines. During erythrocytic development the malarial parasites synthesize large quantities of nucleic acids (87-89). Studies using both intraer throc tie and erythrocyte "free" parasites have V Y 8 demonstrated that plasmodia synthesize both DNA (and RNA nucleotides (90-94); when incubated in the presence of Na2l132PO4 the 32? label was recovered in both DNA and RNA. Early studies on _i_n_ 3122 cultivation of P. knowlesi indicated that a mixture of purines and pyrimidines was required for growth and development of the parasite (81). Technical difficulties did not allow the requirement for each to be determined individually, but the study did demonstrate that parasite nucleotides could not be synthesized entirely _d_e_ 3219’ Later, incorporation studies revealed that probably all malarial parasites synthesize pyrimidines 12 51219. With the exception of orotic acid, exogenously supplied pyrimidine bases are not utilized (95-99) and 14C-labeled bicarbonate was found to be incorporated into plasmodial DNA and RNA nucleotides (100). Enzymes associated with pyrimidines biosynthesis have been identified in both avian and mammalian plasmodia (13, 15,85,100, 101). Purine transport and salva£_. Both parasite and RBC utilize purine salvage pathways; by virtue of its juxtaposition the intraery- throcytic malaria parasite is dependent on uptake of purines by the host RBC. As indicated, early studies on 33 11332 cultivation of P. knowlesi alluded to the fact that' exogenously supplied purines are required for parasite growth (81). Enzymes associated with 513 mg purine synthesis have not been found in any plasmodial Species (13) and labeled glycine was not incorporated into the nucleic acids of E. knowlesi (phosphoribosylglycinamide synthetase reaction absent) (100). Incorporation studies have demonstrated that a number of purines may be salvaged by the host RBC and subsequently by the malaria parasite (89, 97-100, 102-109). 9 Biingener and Nielsen (106) were the first to demonstrate that malarial parasites utilize exogenous purines for nucleic acid synthe- sis; 3l-l-adenosine was readily incorporated into the nucleic acids of P. berghei infected cells. Other studies, using both intraerythrocy- tic and erythrocyte "free" parasites, have indicated that adenosine, inosine, and hypoxanthine are the major purine bases salvaged by the malarial parasite (102, 103, 105, 107, 108). Hypoxanthine is considered to be the preferred purine base. Data indicate that outside, or on the parasite surface, adenosine is deaminated to inosine, inosine deribo- sylated and hypoxanthine is taken up by the~ parasite (102-105, 109). Using continuous cultures of P. falcijarum (105), Webster et al. demonstrated that hypoxanthine is preferentially taken up when hypo- xanthine, adenine, and guanine are included in the culture medium; they also determined that hypoxanthine is the only purine present in signifi- cant quantities in the culture medium (RPMI 1640 + human serum) normal- ly used for continuous P. falciparum cultures. Considering that the majority of the purines in the host RBC are in the form of ATP, one might expect that the degradation of ATP may be an important source of purines for the parasite. Under the condition of nutrient deprivation host cell ATP 'levels do decline, but under normal conditions host cell ATP levels remain relatively constant for P. falciparum infected cells (105). Webster et al. demonstrated that exogenously supplied adenine was quickly salvaged by the entire erythrocyte population; their data indicate the RBC-ATP levels increase in response to the malaria infection. The importance of host cell ATP has been recognized for some. time, and has given rise to the ATP- Malaria hypothesis (110, 111). The hypothesis arose primarily from the 10 observation that malarial infections were much less severe when host RBC-ATP levels were low, and more severe when they were high (110-112), these observations were made for both human and animal malarias. Using erythrocyte "free" P. lophurae and intraerythrocytic P. falcigarum, Trager (113, 114) demonstrated that the ATPase inhibitor, bongkrekic acid, inhibited parasite growth, indicating that RBC-ATP may be involved in ATP dependent transport. Enzymes required for the purine salvage pathway have been identi- fied in P. berghei, P. chaubaudi, and P. lophurae (13, 102, 104, 115). Combining data from incorporation and enzymology experi- ments, purine salvage in the malarial parasites closely resembles that of the host RBC, with a few exceptions (13, 102, 104, 105). The plasmodial parasites must possess a pathway analogous to the adenlyosuc- cinate pathway, by which inosine monOphOSphate can be synthesized from hypoxanthine via adenylosuccinate synthetase and adenylosuccinate lyase reactions; host RBC's do not possess such a pathway (105). Cuanylates are also salvaged more actively by the parasite than by the host RBC. Details of purine salvage remain to be determined for many of the malarial parasites, but it is apparent the all Plasmodium examined so far possess similar purine salvage pathways. Amino acids Introduction. The erythrocytic stages of the malaria parasite acquire amino acids from three sources. They arise from either de — novo synthesis, digestion of host cell hemoglobin, or uptake of exogenous amino acids provided by the host. Protein synthesis in 11 plasmodia to be typically eukaryotic, resembling that of other protozoa (13, 116). Biosynthesis of amino acids. Malarial parasites have been found to synthesize glutamic and aspartic acids, and alanine by C02 fixation (116, 117, 118). Only small amounts of the synthesized amino acids are incorporated into parasite proteins. It has been postulated that glutamic acid may be oxidized by a parasite specific (glutamate dehydrogenase (116); erythrocyte "free", P. lthurae was found to oxidize glutamic acid to 002 (119). Glutamic dehydrogenase has also been identified in rodent malarias (74, 75, 87) and is thought to be involved in NADP reduction (116). Piggstion of host cell hemoglobin. Host cell hemoglobin has been shown to provide the erythrocytic malaria parasite with amino acids required for protein synthesis (13, 116, 120). Radiolabeled RBC's were transfused into P. loghurae or P. knowlesi infected hosts; parasites which subsequently developed in the labeled cells were found toihave incorporated labeled host cell amino acids (19, 119, 121). Hemoglobin determinations have indicated that a large pr0portion of the host cell hemoglobin is destroyed over the period of parasite develop- ment (116). Electron micrographs have illustrated that hemoglobin is first phagocytized by a specialized organelle, the cytosome; a food vacuole is subsequently formed (122, 123). Enzyme analysis has indicated that hemoglobin probably first autooxidizes and is then acted on by proteases (116, 124). Released hematin accumulates and when the concentration increases, self-aggregates sto form hemozoin (malarial pigment). The exact composition of hemozoin has remained uncertain; isolation procedures appear to affect the degree to which proteins are 12 associated with hematin (116). The release of ammonia and CO2 from labeled amino acids indicates that amino acids are also oxidized for energy metabolism or NADP reduction (116). Exogenous amino acids. The importance of exogenously supplied methionine was recognized during early attempts to cultivate P. knowlesi (81); later both methionine and isoleucine were found to be essential for growth (125, 126). Similar results have been reported for P. falciparum (120). Glutamine has been found to be beneficial for E l_i_t_r_o development of P. knowlesi (127). Incorporation studies have indicated that most exogenously supplied amino acids are utilized to some degree by the malarial parasite (116). These investigations have demonstrated that isoleucine and methionine are consistently taken up by marmnalian species. Except for these two amino acids, there appears to be little correlation between the rate of amino acid uptake by the parasite and the amino acid content of the host cell hemoglobin. For the avian parasite, P. laphurae, amino acid incorporation was similiar to that of the mammalian species, with the exception of a higher rate for proline incorporation (13). Data from transport studies have shown that malarial parasites cause an alteration-of host cell permeability to amino acids (128). Lipids The entry of the merozoite into the host RBC and its subsequent deveIOpment is accompanied by large increases in the surface area of the malarial parasite. The expansion of both the parasite plasma membrane and the host derived parasitOphorous-vacuole membrane '9 lb [5“ 13 represent large increases in the overall lipid content of the parasite; a significant proportion of the parasite's total solids are made up of lipids (129). The lipid composition of the malarial parasite differs significantly from the host RBC. Generally the malarial parasites are richer in phOSpholipids and a greater percentage of the fatty acids are unsaturated as compared to the RBC (129). Evidence suggests that the plasmodial parasites synthesize lipids, but are incapable of ES 1919 fatty acid or sterol synthesis (129). The mammalian parasite, P. knowlesi has been investigated most throughly, thus the discussion is limited to findings with this species. Labeled glucose and glycerol were incorporated into phOSpho- lipids, but the label appeared predominantly in the glycerol backbone. Both intraerythrocytic and "free" parasites readily incorporated labeled palmitic, oleic, and stearic acids; most of the label appeared in phosphatidylethanolamine, phOSphatidylcholine, and phospha- tidylinositol. Choline and ethanolamine were incorporated into ph03pha- tidylcholine and phosphatidylethanolamine, reSpectively (130). $2 $12. cultivation demonstrated that exogenously supplied stearic acid was beneficial for parasite growth (131). Using labeled acetate, mevalonate, and cholesterol, Trigg (132).demonstrated that only choles- terol is incorporated. In addition, the growth and development of P. knowlesi _12 m was favored when cholesterol was included in the culture medium. Experiments using avian and rodent malarias general- ly support the findings in P. knowlesi (129). It would not be unreasonable to assume that lipid metabolism in P. falciparum will be found to resemble that of P. knowlesi. 2'” l. . .ll nJ' 14 Vitamins and cofac tors Folates. Numerous experiments and observations have demonstrated that the plasmodial parasites synthesize folate cofactors _de 3953, requiring only p-aminobenzoic acid (PABA) for folate synthesis. The importance of PABA was first recognized using sulfonamides to treat human malaria infections (134). PABA was found to reverse the effects of sulfonilamide on P. gallinaceum _in vitro (134). The growth of P. knowlesi _i_n y_it_r2 required the addition of PABA to the culture medium (81); inhibition of growth by sulfadiazine was reversed by PABA. Hawking (135) found that P. berghei and P. cynomolgi infections were suppressed in milk fed animals; dietary PABA reversed these effects. Similiar results were obtained using P. falciparum infected Aotus monkeys (l3). Enzymes associated with de novo folate biosynthesis have been identified in a number of Plasmodium species (133). Exogenously supplied folates do not appear to be utilized by the malarial parasites (133). Pantothenates. The importance of exogenously supplied panto- thenates was recognized during early attempts to culture P. lophurae; the parasites remained viable for much longer periods of time when calcium pantothenate was added to the culture medium (136). A dietary deficiency of antipantothenates were found to inhibit P. gallinaceum in infected chickens (137); E- “1038““! and B- coatneyi were found to be inhibited _i_n_ vitro by antipantothenates (108, 138). Trager (139), using erythrocyte "free" P. lophurae, found that coenzyme A (CoA), but not pantothenate, had a beneficial effect on extracellular parasite development; other precursors of 60A ,. irf RH! ac: Vi lrJ‘ 15 were also ineffective (140). Enzymes associated with CoA synthesis could not be identified in P. loghurae, although they were readily detectable in the duck RBC (141, 142). It was therefore concluded that the malarial parasites require preformed CoA from the host RBC. Vitamins A, 31’ B2,_ 36L C, biotin, and niacin. A limited number of reports exist on the requirements of vitamins for Plasmodium growth. Contradictory results have been reported for vitamin A; a vitamin A deficiency was found to depress parasite growth in P. lophurae (143) and P. berghei (144) infected animals, but another report has indicated that P. berghei infections are more severe in vitamin A deficient rats (13). Dietary deficiencies of B (thiamin) l and B6 (pyridoxine) (144) in rats, and niacin (nicotinic acid) (144) and B2 (riboflavin) (149) in chicks inhibited multiplication of P. berghei and P. laphurae, respectively. Nicotinamide was found to .favor the survival of erythrocyte "free" P. lophurae (144). When tested 3 _vi_vg a vitamin C deficiency depressed the growth of P. knowlesi (13), but when omitted from culture medium no apparent effect was observed (126). A biotin deficiency has been found to reduce the rated of growth of P. cathemerium in chicks (144), and biotin has been found to be beneficial for 'P. knowlesi growth i_n vitro (126). E Vitro Cultivation of Intraerythrocytic Plasmodium Discontinuous cultivation Early attempts. In 1912, Bass and Johns (24) reported a method by which to culture the asexual, erythrocytic stages of two species of 5 '3‘ 16 human malaria parasites, P. vivax and P. falciparum. Blood taken from patients infected with either parasite was defibrinated, supplemented with glucose, and allowed to incubate in stationary culture tubes at 37-410C. They stated that parasite develOpment not only included maturation of rings to schizonts, but also the invasion of new RBC's by merozoites. Optimum parasite deve10pment required the cultures to be supplemented with glucose or maltose. The work of Bass and Johns (24) received a great deal of attention from other malariologists. It was reproduced in part by Thompson and McLellan (146), Lavinder (147), Thompson and Thompson (148), and Black (149). Parasite growth through the first cycle was generally good, but very little reinvasion was observed, particularly for P. M39 As described by Black (149) reinvasion was favored for P. falciparum because of its preference for mature erythrocytes whereas P. m prefers reticulocytes which generally make up only a small fraction of the total number of RBC's. Although largely abandoned this simple technique has been found useful for chloroquine-sensitivity assays (150). For a period of time efforts had largely been abandoned to culture the human malaria parasites. Attention was directed toward develOping methods for culturing other mammalian and avian Plasmodium species for which animal models were readily available. Hewitt (151) found that stationary cultures of P. cathemerium infected canary blood complet- ed one cycle of development when incubated in medium composed of saline, glucose, and 5% rabbit serum, which was overlaid onto coagulat- ed egg. No reinvasion of uninfected cells was observed. Trager (80, 136) performed a series of eXperiments on the conditions affecting the Itfc 17 2:3 3:11:32 survival of the avian malaria parasite, P. lophurae. Using gently agitated cultures incubated at 39.5-420C it was demons- trated that red cell extract, chicken embryo extract, glucose, calcium pantothenate, glutathionine, homologous serum or plasma, a balanced salt solution high in potassium, a low parasite density, addition of fresh RBC's, and a low oxygen tension all favored survival. Under the best conditions small increases in the parasitemia occurred during the first few days in culture, but declined thereafter. Infective parasites were demonstrated after 16 days in culture. Rocker-dilution/rocker-perfusion techniques. A series of reports (36, 81, 87) indicated that P. knowlesi would develOp for brief periods _12 2132. Two methods were described for cultivation, the rocker-dilution and the rocker-perfusion techniques (81). For both systems the culture vessels were gently rocked at 38.50C and a gas mixture of 952 air-5% CO2 was passed over, but not through, the culture medium. The rocker-dilution technique required that the RBC's settle for the medium to be gently drawn off and changed daily; whereas for the rocker-prefusion technique the RBC's and the nutrient medium were separated by a permeable cellophane membrane, allowing for continued exchange of nutrients and metabolites. The medium contained normal whole monkey blood, a balanced salt solution resembling the inorganic composition of monkey plasma, purines, pyrimidines, amino acids supplied in the form of an acid hydrolysate of casein, and vitamins . Calcium pantothenate , p-aminobenzoic acid , thiamin , pyridoxine, riboflavin, ascorbic acid, and biotin were included in the "Harvard" medium (81). Most experiments were conducted over a 24-hr Period, but in one experiment the parasites grew _i_n vitro over a :l'ud' . 1" J Alhl ‘Mi‘ [Hui . ' | bath 5.». .‘\. b- {‘6 18 6-day period. Methionine was later found to be an essential amino acid (152). Avian species. The rocker-dilution and rocker-perfusion tech- niques have been employed with varying degrees of success for the culti- vation of a number of avian and mammalian plasmodia. Using the rocker- dilution technique Trager (153) demonstrated that erythrocytic P. lophurae could be maintained for at least 8 days in culture, yielding an overall 170-fold increase in parasitemia. Using Harvard medium supplemented with pyridoxamine and higher concentrations of purines and pyrimidines it was not necessary to include red cell extract (137), but eliminating serum or major groups of nutrients had deleterious effects on parasite development. The use of commercially available Fischer's medium was tested and found to be inferior to the modified Harvard medium (154).. Using the rocker-dilution technique P. gallinaceum developed normally for one cycle (24 hrs) when using only normal chick serum for medium (155). Anderson (156) reported having continuously cultured P. ,gillinaceum by this method using Harvard medium supplemented with high concentrations of normal chicken plasma and red cell lysate. After 10 successive subcultures a 19,000-fold increase was reported. Although promising, Anderson's results were not reproducible (157). The rocker- perfusion technique and a modified Harvard medium were used by Manwell and his associates (158-160) to investigate a number of avian malarial parasites. They found that each species varied in its ability to be Cultured fl vitro and that P. hexamerium was best suited for 21 vitro cultivation (160). Under the best conditions with sub- culturing viable parasites were demonstrated afteri9 days in culture .358 mi 91““ I, , . . we '1:’ n) bt Ce 19 but there was a steady decline in the parasitemia. Mamalian species. Most of the subsequent studies using these techniques were conducted on mammalian malaria parasites. The simian parasites, particularly P. knowlesi, were used most frequently. P. falciparum was initially used only occasionally, when an infect- ed patient was available. The discovery that certain simian hosts could be experimentally infected with P. falciparum greatly facilitated research on the most important human malaria parasite (1-3). The culti- vation studies on the mammalian malarias were generally short-term experiments; usually only one cycle of development was monitored. Using the rocker-dilution technique, Trager (138, 157) demonstrat- ed that P. falciparum in human or chimpanzee RBC's or P coatneyi in rhesus monkey cells would develop for one complete cycle (48 hrs) when using modified Harvard medium supplemented with either 102 human or monkey serum. Polet (161) using the same technique was able to culture P. knowlesi for one cycle (24 hrs) when using Eagle's medium supplemented with 102 monkey or heat inactivated fetal calf serum; calf and horse serum were found to be detrimental to the cultures. Interestingly, it was observed that agitation of the cultures was deleterious to the integrity of the host RBC's, but beneficial to parasite develOpment; whereas standing cultures favored RBC integrity, but had negative effects on parasite growth. Using both the rocker-dilution and rocker-perfusion techniques, Geiman and Siddiqui (126, 131, 162-165) modified the basic Harvard medium in a number of ways, both improving and simplifying the method for short-term cultivation of a number of simian malarias, paricularly E. knowlesi, and the human parasite P. falciparum. Of their v " up!“ man ol‘ 20 findings the most significant contributions were the realization that in addition to NaHCO3, other more effective, organic buffers could be used in the culture medium to counter the effects of acidic metabolites produced by the parasites (163, 165); and that parasite development was consistently favored when the ratio of medium to RBC's was increased (163). Other of their findings, which was of yet have not been found to be beneficial for continuous cultivation of P. falciparum include the elimination of serum using Cohn's Fraction IV-4 (162), replacement of a complex mixture of amino acids derived from a casein hydrolysate with 14 defined amino acids (126), and the possible significance of stearic acid to parasite growth (131). Reports comparing commercially available tissue culture media demonstrated that modified Harvard medium was not the best possible medium for parasite cultivation. Trigg (166) found M199 and NCTC 135 media were as good as modified Harvard medium for P. knowlesi cultivation. Trager (6) found that both RPMI 1640 and Dulbecco's high glucose media were superior to modified Harvard medium for P. coatneyi cultivation, and that HEPES, an organic buffer, favored parsite development. In all cases media was supplemented with homologous serum. Static cultivation techniques. The first report of a static culture system was of course that of Bass and Johns (24). But it was soon tacitly assumed that all malarial parasites would grow better in an agitated system, being more comparable to the conditions _i_n li_tr_o_. Our experience has revealed that this assumption was not true for all plasmodia. In 1971, Trager (167) develOped a flow vial for cultivating P. coatneyi and P. falciparum. In his report he "as it"s, min 21 used modified Harvard medium, a gas mixture of 774 COZ/SZ 02/882 N2, and 102 heparinized rhesus monkey plasma for P. coatneyi and 102 heparinized human plasma for P. falciparum. The flow vials were constructed with an overflow 2-3 mm above the bottom of the vial; a suspension of infected RBC's was added to the vials, allowed to settle, and medium slowly added. The net result was a slow flow of medium over the settled layer of infected RBC's. With this method both species of parasites went through 2 cycles (96 hrs) of development without subculturing, but there was no increase in total number of parasites. Shortly after Trager reported his findings, Phillips et al. (168) demonstrated that P. falciparum from human patients could be maintained for up to 6 days, if subcultured and the pH adjusted at appropriate intervals. Some reinvasion had obviously occurred but the rate ofldilution from subculturing was by far greater than the rate of parasite multiplication. Their technique employed stationary sealed culture vessels with a relatively large surface area and an atmosphere of 952 air/5% CO . Parasites were incubated in M199 medium supplement- 2 ed with 51 fetal calf serum, glucose, and NaHCO3. Deve10pment through the first cycle appeared to be normal; there was a 5-fold increase in the parasitemia. For subsequent subcultures the rate of develOpment was slower and the rate of multiplication decreased; by the end of the 6th day most parasites appeared to be abnormal. Lilli R v- (e 3v.» VII: 51 fi-Ing‘ al. 22 Continuous cultivation of P. falc_i_parum - Petri dish-candle jar technique Introduction. Trager and Jenson (4), using the flow vial, an atmosphere of 72 COz/SZ 02/882 N2, and RPMI 1640 supplemented with HEPES (25 mM), NaHCO3 (0.2%), and 15% human serum were able to continuously culture P. falciparum. The technique was previously discussed and will not addressed in any further detail because it has essentially been superceded by a simpler, more efficient technique. Jensen found that P. falciparum could be continuously cultured by a much more convenient method, the petri dish-candle jar technique (5). Cultivation technique. The simplified method deve10ped by Jensen (5) uses 35m petri dishes for culture vessels. To each dish 1.5ml of a lO-lZZ RBC suSpension containing 0.12 infected cells is added. The dishes are then placed into a glass dessicator with a candle. The candle is lit and the cover replaced with the stopcock Open. When the candle flame goes out the stOpcock is closed and the dessicator placed into a 37°C incubator. Once daily the culture medium is changed by gently tipping the petri dish and drawing off the eXpired medium with a pasteur pipette; 1.5ml of fresh medium is then added back to the dish, gently agitated to redistribute the RBC's and then returned to the dessicator and incubated. The parasitemia of the cultures are monitored by counting Giemsa-stained blood films. After 96hrs of growth (2 cycles) the infected blood requires subculturing; generally the prasitemia increases 40-60 times its original 0.11. Subsequently, it has been found that a lower hematocrit will 8Upport a higher parasitemia (169). Variations of the basic technique ,- njfi‘,‘ ".lu‘. li‘O-l 5)) t“ I- r x _ a1: Ci bl 23 have since been reported. Other types of vessels have been used to replace the petri dish (170-173), and larger vessels have been used to accommodate greater volumes of cultured material. The dessicator and candle have been replaced by passing a gas mixture over the medium (170-173); the atmosphere in the candle jar was found to be 2-37. C0 2 and 14-172 02 and a similiar atmosphere will support parasite growth when passed over the culture medium (5). Optimum growth was determined to be with an atmOSphere of 32 O2 and l-ZZ CO2 (balance N2) (83). A semi-automated system has also been developed (173). Medium. Powdered RPMI 1640 (Gibco) with glutamine is the first medium found that would support continuous P. falciparum growth. It is prepared by dissolving 10.4g RPMI 1640 in 900 ml of glass redistill- ed water; to this 5.94g HEPES (Sigma) is added; the volume is brought to 960ml and sterilized by filtration. The medium can be stored for 1 month at 4°C. When ready for use complete medium is prepared by adding 4ml of 51 NaHCO to 96 ml of RPMI 1640 + HEPES (RP); to this 3 11ml (101) human serum is added (RP + H8). Other reports indicate that RPMI 1640 can be replaced with medium 199 with Earle's salts (171, 174) and HEPES with Tes buffer (174), but no direct comparisons have been reported. Attempts to improve parasite growth by supplementing RP with additional nutrients have failed (5). Erythrocytes. Human blood preserved with either acid citrate dextrose (CPD) or citrate phosphate dextrose (CPD) at 4°C may be used for culture purposes (5). The ABC type used will depend on which type of serum the medium has been prepared with, but generally type A+ or 0+ cells are used. Erythrocytes may be used immediately after collection, but parasite growth is superior in RBC's which have been stored for the min 161 24 some period of time (1+ weeks) (5). Because RBC's may be used for up to 5 weeks after collection, cells outdated for transfusions can still be used for cultivation. To prepare RBC's for culture an apprOpriate volume of blood is Spun down, the plasma and buffy coat removed, and the cells resuSpended in 2-3 times the volume of packed RBC's with RP. The cells are washed twice and resuspended in RP 4» HS to a 52 hematocrit. Parasites for cultivation. The parasite, P. falciparum, can be obtained from either an infected culture, a patient, or an $33 monkey (4). Parasites may also be preserved indefinitely in liquid N2; Rowe's cry0preservant is used (175). Parasitized blood taken from a patient or w monkey must be heparinized to prevent clotting of the serum (6). Parasitized blood is prepared in the same manner as uninfected~ blood, except RP + H8 is used instead of RP for washing the cells (5); cryOpreserved cells must first be washed with 3.52 NaCl to prevent lysis of the parasitized cells (176). To set up cultures an appropriate volume of a 502 suspension of infected RBC's is added to a 502 suspension of uninfected RBC's to yield a final 0.12 parasitemia; RP + HS is then used to dilute the 502 suspension to the desired hematocrit (5). Experience has shown that not all strains of P. falciparum are suitable for cultivation (177). The parasite strains that do develop 13 m appear to have normal ultrastructural characteristics (123), although changes do occur during cultivation. Gametogenesis can be observed in freshly isolated strains, but the frequency diminishes over time (178). It has been reported that gametogenesis can be induced by nutrient deprivation (179). Cultured parasites have been found to - 'vng [I .,. ny- 25 become increasingly less "knobby" over time; normally P. falciparum alters the RBC membrane to form "knobs" which adhere specifically to the endothelial cells of the capillaries (180), accounting for sequestration of the parasite during an infection. in £33.29. both "knobby" and "knobless" parasites are formed; the "knobby" parasites have the selective advantage of being sequestered and not being cleared from the blood by the Spleen. Therefore when a parasite strain is first isolated it is predominantly "knobby". But _i_n yi_t_1:g the selective advantage is reversed and the "knobless" parasite p0pulation increases faster than the "knobby" pOpulation (181). It has also been reported that P. falciparum strain FCR3. Originally isolated and characte- rized to be chloroquine-sensitive, became drug resistant after 4 years of continuous cultivation without any selective pressure to become so (182). Sefl. In the original reports (4) 152 (v/v) human serum (HS) was used, but Jensen (5) found that 102 HS was as good as 152. Serum should be obtained from freshly drawn blood without anticoagulant added; the blood is allowed to stand for at least a day to ensure adequate shrinkage of the blood clot. The serum and, unavoidably, some cells are run into centrifuge tubes and spun; the serum is drawn off and should be stored at -20°C. When new cultures are set up from a patient or an Aotus monkey type AB+ serum should be used; this type of serum is compatible with any type of human cell and with Aotus mviratis erythrocytes (6). If aseptic technique is not maintained, complete medium (RP + H8) may be filter sterilized; RP + H8 may be stored for up to a week at 4°C. S_erum reduction or replacement. Jensen (183) found that human 'E 53 'i 11. TIC 42655 azure the l .9 .29 J: , nah ’ ’95 A\~ grew Slip 26 serum varies significantly between individuals; some samples will support Optimum parasite growth at less than 102, but not all; to ensure Optimum parasite growth at least 102 HS must be included in the medium. Jensen also found that plasma which contains CPD as a preserva- tive may be used for cultivation if the clotting factors are removed by the addition Of CaClZ, but the parasite growth is only 702 Of that in 102 normal HS (183). Interestingly, while trying to determine if the excess CaCl2 [could be removed by dialysis, he found that essential low molecular weight growth factors or nutrients are removed by the pro- cedure. Using normal serum it was determined that dialysis resulted in the retentate being defective; when the retentate was recombined with the dialysate parasite growth was significantly improved (183). The dialysis experiments indicate that HS provides low molecular weight factors that are not provided by RP but are required for parasite growth. Efforts tO reduce or eliminate HS by the addition of a variety Of SUpplements have proved to be fruitless. The addition of excess glucose, isoleucine, methionine, and HEPES, or including RBC extract, adenosine, or vitamin E did not have a serum sparing effect (5). The use Of other reported serum substitutes, including fatty acid-free bovine or human serum albumin, or a mixture of Bacto-peptone, Yeastolate, Lactalbumin hydrolysate, bovine insulin, and polyvinyl- pyrrolidone did not support parasite growth or have a serum sparing effect (183). Commercially available bovine (adult and newborn), ecluine, ovine, porcine, or even human serum were found to be inadequate for the replacement Of HS human serum, as was freshly collected fetal calf serum (183). 27 TO date a few reports do exist indicating the successful replacement of HS. Ifediba and Vanderberg (11) have reported that HS can be replaced with NeOpeptone or Proteose-Peptone NO. 3 supplemented calf serum in RP. Their report indicates that the parasite requires an adaptation period (1 month) over which time the concentration of human serum is decreased with a concomitant increase in the concentration Of calf serum. This report has been reconfirmed by Siddiqui (12). Zhengren (172) has reported using medium 199 supplemented with ATP, adenosine, COA, creatinine, inositol, glutathione, glyglycine, glucose, vitamin C, and newborn calf serum to replace human serum. Sax and Rieckman (184) have demonstrated that RP + rabbit serum will support continuous P. falciparum growth with only a minimal adapation period. In all Of these reports the growth of P. falciparum is purported tO be as good as growth in RP + HS. It has also been demonstrated that umbilical cord serum is an adequate replacement for adult HS (185). Studies on Serum Requirements for the Cultivation of Plasmodium falciparum (I): Animal Sera A. A. Divol and J. B. Jensenl * This investigation received the financial support of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 1 . Present Address: Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Michigan Agricultural Experiment Station Journal Article No. 10022. 28 .A o‘- ,. :. II-v- 1 . I . a V, n b s 0". m' than. pl .5 -‘. b‘.1¢.u {are rec 29 ABSTRACT For continuous cultivation Of Plasmodium falciparum human serum requirements have been reduced from 102 to 52. Pooling a number of freshly collected lots Of human serum eliminates the variability observed between individual serum samples, subsequently reducing the amount of human serum required for Optimum parasite growth. Freshly collected and pooled lots of bovine, porcine, goat, equine, and ovine sera, as well as commercially available fetal and young calf sera were tested and compared to 52 pooled human serum. NeOpeptone and various combinations of animal sera were examined as supplements to the basic culture medium, RPMI 1640. As an alternative for human serum only bovine serum supplemented with neopeptone could support continuous parasite growth, but at significantly reduced levels. Parasites can be transferred directly from human serum into neOpeptone-supplemented, freshly collected, pooled bovine serum, eliminating any adaption period required for continuous parasite growth. l .11’""’ "mu": “lug: In) Liv ."')'.‘ vpd‘v‘a on Al vac: COll E? 30 INTRODUCTION With the advent Of the Trager and Jensen method (1), continuous cultures of Plasmodium are routinely maintained in various labora- tories throughout the world. This technique has afforded innumerable Opportunities to study parasite biology, biochemistry, and immunology; and these studies should facilitate the development Of superior chemotherapeutic agents and possibly an effective vaccine based on antigens derived from _ig $353.9. cultures Of the parasite. Currently, the parasite cultures require the addition Of 102 human serum in RPMI 1640 for Optimum parasite growth. This requirement places a number of constraints on the investigator; some laboratories are located in endemic areas where human serum may be inhibitory to the cultures due to antimalarial antibody or antimalarial drugs present in glocally procurred sera. Even in some laboratories in developed nations fresh human serum is difficult to obtain. Futhermore, any widely used vaccine should not be grown in human serum when the possibility of contamination with infectious agents is a real one. For these reasons, and others, a suitable replacement for human serum would be a benefi- cial develOpment. Previous experiments have indicated that commercially available animal sera are inadequate for continuous cultures of P. falciparum (2); additional reports support the claim that commercial processing Of animal sera detracts from their quality in tissue culture applications (3). At the same time, it was demonstrated that commercially prepared human serum was inferior to that freshly Obtained from local blood banks. In addition, freshly collected human serum varies significantly an”; .u- M. D‘nn' pict- | .'-O ”as? Q 'I. ~31” 113 new 31 between individuals in its ability to support continuous parasite growth (2). Other investigators (4, 5, 6) have reported the replacement Of human serum with a variety Of supplements. Their data indicate that enriched fetal or young calf serum yields parasite growth comparable to that Obtained in human serum. In our hands the bovine alternates give variable results, but growth is always inferior to that obtained using human serum. In this report we discuss the‘minimum amount of freshly collected and pooled human serum required for optimum parasite growth, and compare this to various animal sera prepared in the same manner. Bovine serum from a number Of different sources was supplemented with neopeptone according to the method of Ifediba and Vanderberg (4), and examined as a human serum replacement. MATERIALS AND METHODS All sera were tested in cultures Of P. falciparum maintained using the petri dish-candle jar method (7). Experimental groups of 4 petri dishes each were subcultured from a common pool Of infected blood which had been grown in RPMI 1640 (Gibcoa) supplemented with human £998 A Rh+ serum; initial parasitemias of 0.12 were used. All experi- ments were conducted using P. falcipgrum strain FCR3 (8) and parasite development monitored over a 96 h period. Parasitemias were determined by counting parasites per 10,000 erythrocytes. aGibco Laboratories, Grand Island, New York -1 ' .nln "Cu ”p.14 .0 w u I l" a .3 real.» ““II F “L.I1( - ”II a \u ‘l uh "1!. "Ci 1 "Q , Baum: ,trzi: Re 9 53' D: .3856 ‘u I", 32 Freshly collected sera Human serum was Obtained from freshly clotted whole blood purchas- ed from the local American Red Cross Blood Bank. A pooled human serum lot (PHS) was prepared by combining equal portions of 18 serum samples. Since initial experiments suggested that 52 PHS was as good as most individual samples at 102, this level became the standard for compari- son with animal sera. Fresh bovine and porcine blood were cOllected from a local abattoir. A pooled lot of bovine serum (PBS) included samples from 15 adult holstein cows and 1 black angus steer. The porcine pool (PPS) contained serum from 8 different animals, all sows. The equine (PES), goat (PCS), and ovine (POS) sera were kindly provided by Dr. J.F. Williams (MSU Vet. Clinic) from farm animals in his care. These sera were tested in pools of 2 to 6 samples each. The animal sera were tested as supplements to the RPMI medium at 102 concentrations. Combinations of these sera were tested using equal portions of each sera, to a total Of 102. All animal sera were sterilized by filtration. Commercially prepared sera Some‘CO-ercial sources of fetal or young calf sera provided a "high quality", specially processed product which is reputed to be a superior material for culture purposes. We tested sera of this type from Biocellb (Newborn Calf Serum), and AMI“c (ZetaSera-D) and com- pared these to our freshly collected pooled adult bovine serum (PBS). A _ --—----——--~-----.~--- Biocell, Carson, California c AHF Imuno-Reagents, Sequin, Texas cane 1 .‘vlv ‘ . 1.....An LCMUQ. 116 u: (w t___v '—C D O u 33 sample from Gibco (Calf Serum) was also tested as a representative of "standard commercial" bovine serum. Three types of processed human serum from AMF were also tested, these include Human Serum Defibrinated (HSD), Clarified Human Serum Defibrinated (CHSD), and T3/T4 Free Human Serum (TFHS). These human sera were Obtained from plasma separated from outdated blood. NeOpeptone (Difcoe) was prepared at a 152 (w/v) concentration and used at a level Of 1.2 ml per 100 ml Of complete medium according to Ifediba and Vanderberg (4). All animal sera were heat inactivated at 56°C for 30 min and stored at -20°C until being tested. To prevent RBC agglutination, the swine serum required absorption against human erythrocytes be fore use . RESULTS AND DISCUSSION The data illustrated in Fig. 1 indicated that 52 pooled human serum (PHS) supports Optimum parasite growth. Our laboratory now routinely uses 52 PHS to supplement RPMI 1640 with all strains Of P. ECiParum. We have no reason to believe that 52 PHS would not SUpport newly isolated parasite cultures, but this has yet to be 1 l O . n demonstrated. The Observed reduction In the pooled serum concentratIo ' ' at required for Optimum parasite growth was expected, considering th Jensen (2) found many individual serum samples would support growth at e. . .. Dich, DetrOIt, Michlgan 34 Fig. 1. Titration of pooled human serum using P. falciparum strain FCR3. 2 Parasitemia represents the number of parasites per 10,000 erythrocytes. Values are the Mean : S.D. for 4 Observations. VlNSllSVHVd °/o I——o——~| l-0'-{ l—o—I ro-i \I—C-i‘ |-O il-OK k 1 1 ”'1‘,” <2 <2 0. IO N - l 2.0 9.0 6.0 HYPOXANTHINE x 155M 3.0 . 1 0:1" - 1M ‘655 "Q "q" 1'.“ "rl 10 36 much less than the 102 level, with only the poorest samples requiring 102. Work in our laboratory indicates that 15 to 20 individual samples should be pooled in order to ensure an adequate pool of serum. We presently divide each serum lot into 50 m1 aliquots added to one liter bottles which are kept frozen until 20 samples have been combined into one liter. This pool Of serum is then melted and aseptically distribut— ed into 5 ml portions and refrozen until used. Importantly, it has been found that high parasitemias can be maintained in the same manner using 52 PHS as is required for cultures using 102 human serum. When the culture medium is continuously renewed or changed daily, 52 PHS performs as well as 102 serum in all applications so far tested. Furthermore, 2.52 PHS will support continuous parasite growth consis- tently at the rate indicated in Fig. 1, but lower concentrations give variable results after repeated subcultures. The data illustrated in Table 1 indicate that 52 PHS is superior to any of the freshly collected pooled animal sera tested. Initially the PPS, PCS, and combinations of PBS, PPS, and PCS were encouraging, but after. the second subculture these sera failed to support signifi- cant parasite growth. Of the animal sera tested, only PPS supported sig— nificant growth beyond 96 h, and of the' various combinations, only the PBS/PCS and PBS/PPS/PGS supported significant parasite growth. It was expected that all combinations which included PPS should have promoted parasite growth for at least two subcultures. Thus it was surprising that combinations Of these sera would not support continuous cultures. A review of the reports on human serum replacement indicate that the work reported by Ifediba and Vanderberg (4) most closely parallels our objectives, which are to replace the human serum required for I IH‘IU :.V at -.W:- In F-l‘ .vxlzl I I I a»... — nu.cul a I-.v o ,vcv - - 1.: \l» - unfit-.55 ”AV fl Film ‘3 RES... 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M .l "" iv “an“ ' . Imu' . t " 1!... _14‘ t..t;{ace 1.».xw A, a" Inn-«u: h.v--" 381190 " l- r «4.11 k-‘ 38 continuous cultures Of P. falciparum without adding complicating factors to the system, or reducing parasite growth. They have reported that RPMI 1640 supplemented with calf serum plus neopeptone or, proteose peptone #3 will support parasite gorwth as well as RPMI plus 102 human serum. The drawback of their system as reported is that the parasites apparently require an extensive adaptation period to the bovine serum-in order for continuous growth to be maintained. In our experience, even after 3 months of laborious adaptation, cultures in neopeptone-bovine serumnever grew as well as those in 52 PHS supple- mented medium. It was of interest to determine if the source Of bovine serum used affected both the rate of growth of the parasite and the length of time required for the parasite to become successfully adapted. Table 2 illustrates data from experiments using bovine serum from a number of different sources. Three types of specially processed human sera were also tested. Some of our eXperiments with neOpeptone were run using goat, porcine, and bovine sera and it was found to have a positive effect only when used in bovine serum. The data presented in Table 2 indicate that PBS (freshly collected and pooled adult bovine serum) and the "high quality" commercially processed bovine sera will support parasite growth at about 60-652 the rate obtained with 52 PHS when supplemented with neOpeptone. Standard bovine serum sources generally supplied 8 131'0‘1'4‘:t 13°01‘91“ in quality Which gives inconsistent results in parasite cultures. We have found that freshly collected adult bovine serum requires no adaptation period for maintenance of continuous parasite cultures. Furthermore, after an adaptation period Of 3 months, the rate of growth is not significantly different from that observed when the parasites were initially intro- .n. :0 ,‘v 1"- 1,...uo n ‘ Lr'. ‘5‘ b“" .13] + l I. (z- Irv e w 39 Table 2. Cuparison of the growth of P. falciparum in 52 freshly collected, pooled human serum (PHS) to growth in 102 freshly collected, pooled adult bovine serum (PBS) and "high quality" comercially prepared bovine sera. Three types of commercially processed human serum, with and without neopeptone, are also included. All bovine sera were supplemented with neopeptone (Neo). Sera 2 Growth in Comparison 1 Comments to 52 PHSb PHS 100 i 2.1 Represents a 51-fold increase in parasitemia over a 96 h period PHS + Neo 99 i 6.1 PBS + Neo 65.1 1 4.4 Growth continues at initial rate for subsequent subcultures Adapted PBS + Neo 68.2 i 4.5 Ibid Biocell + Neo 63.7 i 2.1 Ibid AMF + Neo 64.5 1 3.3 Ibid Gibco + Neo N. A. G. ' d Processed Human Serum CHSD N. A. G. 01151) + Neo N. A. G. TFHS 16.5 -_0-_ 1.4 Causes slight hemolysis over 96 ' h period TFHS + Neo 31.4 _+_ 2.7 Ibid HSD + Neo 31.2 + 2.8 — ‘2 a . . PHS (Pooled Human Serum), PBS (Pooled Bov1n Serum), Biocell (Newborn Calf Serum), AMP (ZetaSera-D), Gibco (Calf Serum), CHSD (Clarified Human Serum Defibrinated, TFHS (T3,T4 Free Human Serum), HSD (Human Serum Defibrinated) Values refer to the Mean 1 S.D. for 4 Observations c No appreciable growth, less than 52 of the control (PHS) Serum derived from outdated CPD-preserved blood. . ‘ .n, 2:? ”153 . act-5i ‘v .i-n-H ,‘N' 3;,451»! , u .gv-vn '\ .~o=t 5v nip. ‘ ' a :5..E “‘0‘” 1.“ Lu. ut : y —.1 . '7' r1 (‘2 I... 40 duced into the supplemented bovine serum (652 when compared to 52 PHS for non-adapted parasites as compared to 682 for adapted ones). An adaptation period appears to be required only for bovine serum of inferior quality, such as the Gibco Calf Serum tested. Elimination Of an adaptation period has the advantage of not selecting for any particular subpopulation of P. falciparum and also allows the parasite to be freely transferred from bovine serum into human serum with no adverse effects. Our laboratory has also found that high parasitemias can be maintained using 102 PBS plus neOpeptone. The practicality of using bovine serum. for continuous parasite cultures must still be rigorously evaluated. We do not know if growth of P. falciparum in bovine serum. alters its antigenic Characteristics, or whether or not it has an effect on drug sensitivity when tested _i__n The three types of AMP brand Of processed human sera were inadequate for culture purposes (Table 2). It is interesting that neopeptone has a positive effect on parasite growth when used with these sera, whereas it does not when 52 PHS is supplemented. Commercial processing must remove or degrade essential components required for parasite growth. Currently, our laboratory is investigating the basis of the improvement in parasite growth in bovine serum seen with neopeptone. Obviously, the neOpeptone must enrich the culture medium with a required nutrient which is not pmesent in bovine serum. In eXperiments to be reported elsewhere, hypoxanthine appears to replace neopeptone for continuous parasite cultivation in bovine serum. ::""- l ”ski I! S‘fl‘ :SKVUr h . 1... . , .4 i‘ use» ‘i-A “were. star): A! 1:15 c 41 SUMMARY For continuous cultivation Of Plasmodium falciparum the human serum requirements have been reduced from 102 to 52. This was accomplished by pooling 18 freshly collected individual serum lots and determining the -minimum amount Of the pooled material that would support Optimum parasite growth. Pooling a number Of freshly collected lots Of human serum, between 15 and 20, eliminates the variability Observed between individual serum samples, and reduces the amount of human serum required for optimum parasite growth. For adaptation of newly isolated strains, higher serum concentrations may be useful. Freshly collected and pooled lots Of bovine, porcine, goat, equine, and ovine sera, as well as commerically available fetal and young calf sera were tested and compared to the 52 pooled human serum control. The results indicated that porcine and goat sera, and combinations of porcine, goat, and bovine sera would support parasite growth for a limited) number of cycles; these sera failed to support continuous parasite growth. NeOpeptone and combinations Of animal sera were examined as supplements to the basic culture medium,- RPMI 1640. As an alternate for human serum only bovine serum supplemented with neOpeptone could support continuous parasite growth, but at significantly reduced levels. When high quality bovine serum was used, parasites could be transferred directly from human serum into bovine serum, eliminating any adaptation period required for continuous parasite growth. The freshly collected, pooled adult bovine serum was as good, or superior to any of the commercially prepared fetal or young calf sera tested. 42 REFERENCES TRACER, w. a JENSEN, J. 8. Science, 193:673-675 (1976). JENSEN, J. B. Bulletin of the World Health Organization, _5_7_(Supp1.):27-31 (1979). BOONE, c. w. ET AL. 1313553, 1(3):174-189 (1972). IFEDIBA, T. & VANDERBERG, J. P. Journal of Parasitology, 99(2) :236-239 (1980). ZHENGREN, c. ET AL. Chinese Medical Journal, g;(1):31-35 (1980). SIDDIQUI, W. A. Continuous _i_p vitro Cultivation of Plasmo- dium in Human Erythrocytes: Description of a Simple Technique to Obtain High Yields Of Parasites. In: Practical Tissue Culture Applications. New York, Academic Press, 1979, pp. 267-277. JENSEN, J. B. & TRACER, W. Journal Of Parasitology 63:883-886 (1977). JENSEN, J. B. & TRACER, W. American Journal of Tropical Medicine and Hygiene, ~2__7_:743-748 (1978). Studies on Serum Requirements for the Cultivation of Plasmodium falcipgrum (II): Medium Enrichment A. A. Divo1 and J. B. Jensen1 * This investigation received the financial support Of the UNDP/World Bfink/WHO Special Programme for Research and Training in Tropical Diseases. 1 . Present Address: Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Michigan Agricultural Experiment Station Journal Article No. 10023. 43 11...»..FJ- .~ . 1 ' 1 r: E :2? o e em to“\ . . 9. a $111.01.. pap-s P- "hc3l wt “'11, ‘1 Vin-Ii ”u &_I\ O. ‘ grant SEMI 44 ABSTRACT Previous eXperiments have indicated that the dialysis Of human serum removes low molecular weight components (6,000-8,000 MW) which are essential for continuous cultivation Of P. falciparum; these experiments used RPMI 1640 when testing the dialyzed serum. TO determine which low molecular weight components were important for parasite development, we compared growth in normal serum to dialyzed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. Through these comparisons, we determined that hypoxanthine was the major dialyzable nutrient required for parasite develOpment. High quality bovine serum requires 3-12 x 10 -5 M hypoxanthine as a supplement to support continuous culture Of P. falciparum. Thus far we have been unable to attain parasite growth in supplemented bovine serum which is as good as growth in human serum. 1 W ' .Zathllj l. 01 l n. 5“,. “MSBL': 1:5 13.5 Lfij ‘2‘: In 91%.} 3113 45 INTRODUCTION Currently, the continuous cultivation Of Plasmodium falciparum requires the addition of 52 pooled human serum to ensure Optimum parasite growth (1). This requirement for human serum reflects the inability Of the basic culture medium, RPMI 1640, to meet the nutrition- al or regulatory needs of the parasite. By comparison to other cell culture techniques, the cultivation Of P. falciparum is still in its infancy. Until the development of the Trager-Jensen method (2) for the continuous cultivation Of the parasite, most research on basic parasite biology Of Plasmodium spp. was carried out using animal ‘ models for study. Only a limited number of reports have addressed the nutritional status Of P. falcijarum (3-8). Reports indicate that human serum may be eliminated from continu- ous cultures by using supplemented bovine serum (4-6), but our experience is that parasite growth is inferior to that which is Obtained using human serum. By more thoroughly defining the nutritional requirements Of P. falcifiaarum, it may be possible to eliminate the human serum now required for continuous cultivation, or allow animal sera to be used without detracting frOm the growth characteristics obtained with human serum. The advantages of using a system free of human serum have been previously addressed (1). Since dialyzed human serum has been shown to be lacking in small molecular weight component(s) which are necessary for parasite develOp- ment (3), this Observation has made it possible to determine some of the factors required for parasite develOpment by using dialyzed serum SUpplemented with low molecular weight nutrients. We have also examined .L-r ,..1.Cb I a’fi '1“ bu.) 6H ;-a:a '9- L) “e 46 other commercially available culture media that are nutritionally richer than RPMI 1640, and have supplemented these media with dialyzed human and selected animal sera as well as selected nutrients. MATERIALS AND METHODS Continuous cultures Of P. falciparum, strain FCR3 (9), were maintained using the petri-dish candle jar method (10). All eXperiments were begun using parasites grown in RPMI 16408 supplemented with 52 type A Rh+, pooled human serum (1). To begin each eXperiment a common pool Of 0.12 parasitized blood was divided into test groups Of 4 dishes each. Experiments were monitored over a 96 h period; the culture medium was renewed once daily. Parasitemias were determined by counting parasites per 10,000 erythrocytes. Human and animal sera The sera used included pooled human serum (PHS), and pooled lots of freshly collected bovine (PBS), porcine (PPS) and goat (PCS) sera. All sera were collected and pooled as reported by Divo and Jensen (1). Malia and supplements The preparation of RPMI 1640a (hereafter referred to as RPMI) has been previously described by Jensen (10). Ham's I-‘12a (hereafter referred to as F12) with L-glutamine was prepared by dissolving 10.6 g 0f powdered medium into 900 ml 3X glass distilled HOH; 5.94 g HEPESb a . Gibco Laboratories, bSigma Chemical Company I‘d N.- s 0 VA 1 0 .klu .u» 11 . n 11 :. U and »I a I . iZCEI 1 C o 1.3 9 21313.4: 1 le‘; 47 and 1.0 g glucose were added dissolved into solution; the total volume was brought to 1,000 ml and 1.176 g of NaHCO3 added. Medium 199 with Earle's Saltsa and L-glutamine (hereafter referred to as 199E) was prepared by dissolving 9.9 g of medium into 900 m1 HOH; 5.94 HEPES and 1.0 g glucose were added; the volume brought to 1,000 ml and 2.2 g NaHCO3 added. Medium 199 with Hank's Saltsa and L-glutamine (here- after referred to as 199H) was prepared by dissolving 11.0 g of medium into 900 ml Of HOH; 5.94 g HEPES and 1.0 g glucose were added; the volume brought to 1,000 m1 and 0.35 g NaHCO3 added. The pH of all media was adjusted to 7.2-7.4 using 10N NaOH. The following reagents were prepared in 3X glass distilled HOH at 100X the concentration to be used in formulating complete medium. When preparing supplemented medium the volume Of HOH initially added was treduced by the corresponding volume of the supplements to be added. The reagents and 100x concentration include: inosineb (0.805 g/l), adenosineb (0.802 g/l), adenineb (0.405 g/l), hypoxanthineg (0.41 g/l), L-prolinec (3.45 g/l), L-alanined (0.89 g/l), Na pyruvatecl (11 g/l), vitamin B12b (0.13 g/l), putrescine HClb (0.0158 g/l), linoleic acidb (0.0084 g/l) dissolved 0.1 g into 1 ml abs. EtOH, then diluted with HOH, alpha-lipoic acidc (0.021 g/l) dissolved 0.0106 g into 6 drops of 1N NaOH, then diluted with HOH, reso47aoue (0.0834 g/l), 0.1304511011‘3 (0.00025 g/1),' and 211304f (0.086 g/l). NeOpeptoneg (152 W11) was PTEPaI’Ed and used - ---~--—‘-—-—--—-~—_—--——-~ Sigma Chemical Company , cNutritional Biochemical Corporation, d . e . f . Calbiochem , J . T. Baker Chemical Company , Mal linckrodt , Incorporation, 8Difco Laboratories AA“: 5 l 1 a “(in .9 -~‘-:"§ 1i “v.1... l...' 4' .. fl . 513.)“ . r-‘l 48 at a concentration of 1.2 ml per 100 ml RPMI 1640 (4). Media and reagents were sterilized by filtration. Dialysis of human serum and neOpeptone The dialysis Of human serum was carried out using dialysis tubing (Spectrapor) with a 6,000-8,000 MW pore size. Twenty ml of pooled human serum (PHS) were exhaustively dialyzed for 48 h against 2 volumes of 1,000 ml each Of RPMI 1640 plus HEPES and NaHCO3, the dialyzing medium being renewed after 24 h. Nenty ml of neopeptone (152 w/v) were dialyzed in the same manner as the PHS. Control PHS and neopeptone were minimally dialyzed against 10 m1 of the same medium in a 50 ml graduat— ed cylinder. Dialysis was carried out at 4°C. RESULTS AND DISCUSSION As indicated in Table l, RPMI and F12 media were superior to the two 199 media tested when 102 pooled human serum (PHS) was used. In addition, exhaustively dialyzed human serum was defective when used to supplement RPMI, but less defective when used with F12 and the two 199 media. When human serum was dialyzed against F12 medium and then used to supplement RPMI, it was not defective. Since Jensen (3) has previous- ly shown that dialysis Of human serum results in the loss of low molecular weight factors (less than 6,000-8,000 MW) which are essential for parasite growth, these results indicate that F12 and the 199 media aIPPear to contain factors that promote parasite growth which are not Present in RPMI. Because the F12 appeared to be the superior medium under these conditions it was used to make further comparisons. The 49 Table 1. Growth of P. falciparum in RPMI 1640, Ham's F12, 199 with Earle's Salts, and 199 with HankTs Salts. Each medium was supplemented with 102 pooled human serum. Exhaustive dialysis was l:10,000, whereas control sera was minimally dialyzed 1:0.5. 2 Parasitemia 2 Reduction in Growth Control Exhaustivelya Between Control and Media PHSa Dialyzed PHS Dialyzed PHS RPMI 1640 3.6 i 0.2 1.2 i 0.1 ' 67 Ham's F12 4.4 1 0.1." 3.7 1 0.2 16 P“; 199 with 2.1:01 1.7101 19 Earle's Salts 199 with 1.5 i 0.1 1.3 i 0.1 17 P Hank's Salts :: . EJ a . Values represent the Mean 1 S.D. for 4 observations. b . . . The data indicate that F12 is superior to RPMI when usrng minimally dialyzed PHS, but from our experience we cannot conclude that F12 is superior to RPMI when using normal non-dialyzed PHS. 50 factors present in F12 but not in RPMI were used to supplement RPMI. These included ZnSOA, FeSO47HOH, alpha-lipoic acid, linoleic acid, putrescine HCl, hypoxanthine, L-proline, L-alanine, and Na pyruvate. Of all the supplements tested (individually and in various combinations) only hypoxanthine was found to contribute to increased parasite growth. The data in Table 2 indicate that the addition of hypoxanthine to RPMI restored parasite growth in exhaustively dialyzed PHS to the level seen before dialysis. The results infer that hypoxanthine was the primary component required for parasite growth that was removed by exhaustive dialysis, and that the serum concentratiOn Of purines are critical for parasite development. These conclusions are supported by Webster et al. (11) who demonstrated quantitatively that the concentra- tion Of purines, primarily hypoxanthine, in serum-supplemented medium ' decreases significantly during parasite growth. To determine if F12 Offered any advantages for parasite cultiva- tion when alternate animal sera are used, 52 PHS was compared to 102 PBS (pooled bovine serum), 102 PGS (pooled goat serum), 102 PPS (pooled porcine serum), and a combination of all three. The data in Table 3 indicate that F12 supplemented with 102 PBS was superior to the other sera tested, and that only the PBS would support continuous parasite growth. These results were interesting in consideration Of our previous findings using RPMI supplemented with 102 PBS and neOpeptone (4), which demonstrated that, Of a number Of animal sera tested, only bovine serum SUpplemented with neOpeptone would support continuous parasite growth. In this instance, F12 plus PBS required no neopeptone to support the falciparum cultures. As indicated by the data in Table 4, and with all Of our '1" 1:16 C 51 Table 2. Comparison Of P. falciparum growth, in RPMI 1640, between control PHS a d exhaustively dialyzed PHS, with and without hypoxanthine (HX) added" 2 Reduction in Growth Due Media 2 Parasitemiaa to Dialysis RPMI 1640 + Control PHS 3.8 i 0.2 - RPMI 1640 + Exhaustively 0.8 + 0.1 78 Dialyzed PHS RPMI 1640 + Exhaustively 3.5 1 0.2 8 Dialyzed PHS + BK aValues represent the Mean -_I-_ S.D. for 4 Observations. bThe concentration Of hypoxanthine ws 3 x 10"5 mol/l. :Puigfist“ “"‘.d¥ 99" (AP! I)! 117111 Sera ’ {D an 4! ms (9‘ 111‘ . LL, (1‘) 52 Table 3. Comparison Of P. falciparum growth, in Ham's F12 supplemented with 52 PHS to growth in Ham's F12 supplemented with 102 PBS, 102 PCS, 102 PPS, or a combination of 3.32 of each. 2 Growth in Comparison Sera to 52 PHS Comments 52 PHS 100 i 4.6 Represents a 40X increase in parasitemia over 96 h 102 PBS 60.9 :_4.6 Supported continuous parasite growth 102 PPS 27.6 :_2.8 Subsequent subculture failed 10: Pcs N.A.G.b 102 PBS/PPS/PGS 53.9 :_2.1 Subsequent subculture failed a . Values represent the Mean :_S.D. for 4 observations. b . No appreciable growth. c . , ExPeriment terminated after 100+ days. _‘Afl 103' .1. 1.. 53 Table 4. Comparison Of P. falciparum growth in RPMI 1640 and Ham's F12, each containing 52 PHS, 102 PBS, or 102 PBS + 1.2 ml neOpeptone (152 w/v). 2 Growth in Comparison Media to RPMI 164 + 52 PHSa Comments RPMI + 52 PHS 100 1 2.8 RPMI + 52 PHS was superior to F12 + 52 PHS F12 4» 5% PBS 87.6 I 5.8 RPMI +102 PBS 6.2 11.0 F12 + 102 PBS 55.1 :_3.7 F12 + 102 PBS was superior to RPMI + 102 PBS RPMI + 102 PBS + 68.4 i 3.4 NeOpeptone promotes neopeptone significant growth in RPMI but not in F12 F12 +101 PBS + 46.8 1 2.6 neOpeptone g a . Values represent the Mean + S.D. for 4 Observations. 54 experience comparing these two media, RPMI was generally superior to F12 when supplemented with 52 PHS. In addition, RPMI is less costly and easier to prepare; we have found no distinct advantages for using F12 on a regular basis. Although F12 was not superior when using PHS, when 102 PBS was used to supplement both media, F12 was by far superior to RPMI but still inferior tO RPMI + PHS. When neOpeptone was used with the PBS, it contributed significantly to parasite develOpment in RPMI, but appeared to detract from parasite growth when used in F12. The results suggest that a factor required for parasite growth may be in common with both neopeptone and Ham's F12. TO determine the factors present in F12 that accounted for its ability tO support continuous parasite growth in PBS, RPMI was supplemented with the components present in F12, but not in RPMI. The components tested were previously listed, and again the only supplement which promoted parasite growth in PBS was hypoxanthine. TO determine (in: Optimum concentration of hypoxanthine, a titration curve was constructed, Figure 1. The data indicate that the Optimum concentra- tion lies between 3 x 10..5 M and 12 x 10.5 M‘, the upper limit was not determined. Twice the concentration found in F12 (6 x 10-5 M) was used for making further comparisons. The results indicated that PBS was deficient in utilizable purines, and that these must be supplied for PBS Ix) support continuous parasite growth. TO determine if neOpeptone was acting as a purine source for parasite growth, it was dialyzed in the same manner as PHS. The data in Table 5 indicate that exhaustively dialyzed neopeptone was defective when used to supplement PBS, and that hypoxanthine would 55 Fig. 1. Titration Of hypoxanthine using 102 freshly collected, pooled adult bovine serum (PBS) in RPMI 1640, P. falciparum strain FCR3 was used. Parasitemias represent the number of parasites per 10,000 erythrocytes. Values are the Mean 1 S.D. for 4 Observations. H- F—9—H q l-o—i ‘ 1—-—4 i-O-l - F..——‘\ H.—(\ t—o—i l 1 L l 1 0. o o. o. o. '0 st to on .. VINBLISVUVd '7. I 5.0 l0.0 5.0 POOLED HUMAN SERUM ‘7. 57 Table 5. Growth of P. falciparum in 102 PBS, using RPMI supplement- ed with minimally 1», F.) tic su; 60 59 explained by the fact that the media each described contained purines or neOpeptone. Ifediba and Vanderberg's work with neOpeptone has been discussed. Zhengren et al. (5) reported using calf serum with medium 199, which contains significant concentrations of hypoxanthine, adenine, and guanine. Siddiqui reported that RBC extract and bovine serum in RPMI would support parasite growth and it is commonly known that the erythrocyte extract contains high concentrations Of purines. In summary we have determined that hypoxanthine is the major dialyzable component in human serum which is essential for the continu- ous cultivation of P. falciparum. And that freshly collected, pooled adult bovine serum (PBS) can be supplemented with hypoxanthine to attain superior growth when compared, to growth in PBS supplemented ' with neOpeptone. We also found that there were no advantages Of using F12 or either of the 199 media when culturing parasites in human serum. Thus far we have not been able to reduce the pooled human serum below 52 by addition Of hypoxanthine, indicating that at this serum concentra- tion some other growth factor becomes limiting. In our eXperience, PBS supplemented with hypoxanthine supports continuous parasite growth at 60-702 the growth rate of 52 PHS, and even after 4 months of continuous cultivation in PBS, parasite growth was still not equal to that usually seen with human serum. SUMMARY Previous experiments have indicated that the dialysis Of human serum removes low molecular weight components (6,000-8,000 MW) which are essential for continuous cultivation of P. falciparum; these L. artisan éeteniine arasite serum 0' :cnsiie superic tempo: 1113i paras 9c pa: 60 experiments used RPMI 1640 when testing the dialyzed serum. To determine which low' molecular weight components were important for parasite development, we compared growth in normal serum to dialyzed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. We found that RPMI 1640 was superior to other media tested when using normal serum, but that Ham's F12, and Medium 199 with Hank's or Earle's Salts, were superior to it when using dialyzed serum. By supplementing RPMI 1640 with the components present in the other media, but not RPMI 1640, we determined that hypoxanthine was the major dialyzable nutrient required for parasite growth. We also compared parasite growth in RPMI 1640 to growth in Ham's F12 when supplemented with freshly collected, pooled. adult bovine, porcine, and goat sera. Ham's F12 *was found to support continuous parasite growth when supplemented with bovine serum, but not when using Porcine or goat sera. Again, by supplementing RPMI 1640 with the components present in Ham's F12, but not RPMI 1640, we determined that bovine serum supplemented with hypoxanthine (3-12 x 10“5 M) would support continuous parasite growth. Other reports describe continuous cultivation using bovine serum supplemented with a variety of nutrient mixture; our results indicate that these supplements probably serve as a purine source required for parasite development. Hypoxanthine supple- mented bovine sera support continuous parasite cultures, but a reduced rate when compared to growth attained with human serum. Addition of hypoxanthine to human serum does not improve parasite growth nor does it have a serum-sparing effect. I _ nfiw I'htll' 10. ll. 12. 61 REFERENCES DIVO, A. A. S JENSEN, J. B. Bulletin of the World Health Organization, (Manuscript in press). TRACER, w. a JENSEN, J.B. Science, 122:673-675 (1976). JENSEN, J. B. Bulletin of the World Health Organization, 5_7_ (Suppl. 1):27-31 (1979). IFEDIA, T. & VANDERBERG, J. P. Journal of Parasitology, PP (2):236-239 (1980). ZHENGREN, C. ET AL. Chinese Medical Journal, 93 (1):31-35 (1980). SIDDIQUI, W. A. Continuous Pp vitro cultivation of Plasmodium falciparum in human erythrocytes: Description of a simple technique to obtain high yields of parasites. Pp Practical Tissue Culture Applications, New York, Academic Press, 1979, pp. 267-277. DRUILHE, P. ET AL. TrOpenmedical Parasitoggy, 3_1_:409-4l3 (1980). BUTCHER, C. A. Bulletin Of' the ‘World Health Ogganization, ‘21 (Suppl. 1): 17-26 (1979). JENSEN, J. B. S TRACER, W. American Journal of TrOpical Medicine and Hygiene, _2__7_:743-748 (1978). JENSEN, J. B. & TRACER, W. _J_O_prnal of ParasitOPpgy, 63:883-886 (1977). WEBSTER, H. K. ET AL. Clinical Research, P§:326-336 (1980). SEIDER, M. J. 6 RIM, H. D. American Journal Of Physiology, P32 (5):C262-C267 (abstr.) (1979). 62 BIBLIOGRAPHY is"— it“— ‘ 10. ll. 12. 63 BIBLIOGRAPHY Collins, W. E., P. C. Contacos, E. C. Cuinn, M. H. 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Lambert, and M. Gentilini. 1980. Plasmodium falciparum ig_vitro culture: Improvements using um- bilical cord serum and medium.modifications. Tropenmed. Para— sitol. 31:409. 77 APPENDIX The above articles "Studies on Serum Requirements for the Cultivation of Plasmodium falciparum 1. Animal Sera 2. Medium Enrichment" have been accepted for publication by the Bulletin of the World Health Organization. Permission has been granted by the publisher to include these articles within this Master's thesis. .-.-| Q