A quantitative long-term cryobiological study of malarial parasites*

Bulletin of the World Health Organization, 55 (2-3): 305-308 (1977) A quantitative long-term cryobiological study of malarial parasites* C. P. A STROME,' T. A. TUBERGEN,2 J. L. LEEF,3 & R. L. BEAUDOIN 4 A study spanning 10 years indicates that the erythrocytic stages of malarial parasites can be stored indefinitely in the vapour phase of liquid nitrogen without significant loss of infectivity. The results also suggest that loss due to the recovery procedures exceeds any loss that may have occurred during freezing and thawing. When feasible, large pools of material should be divided into smaller aliquots before storing the organisms at a low temperature, since freezing a second time produces significant losses of infectivity. Sporozoites and exoerythrocytic stages may be treated similarly, but the optimum conditions for freezing these forms have yet to be determined. The practice of freezing and storing the erythro- cytic stages of malarial parasites for various periods at low temperatures is not new (1-9). However, quantitative studies concerning the retention of infectivity of such organisms after freezing are few (10). The present study was initiated over 10 years ago to provide a quantitative estimate of the de- crease, if any, in the infectivity of Plasmodium fallax resulting from storage at a low temperature; aliquots have been assayed periodically from 1967 to 1977. The long-term preservation of sporozoites of Leuco- cytozoon simondi and short-term preservation of Plasmodium berghei are also reported. Little has been published concerning the freezing of sporo- zoites (1, 11, 12). Finally, we have included some preliminary results on the freezing of the exoery- throcytic stages of P. fallax. To our knowledge, there are no reports on the freezing of any malarial exoerythrocytic stages. * This work was supported by the Naval Medical Research and Development Command, Work Unit No. MF51.524.009.0077 and ONR Contract No. N00014-76- C-0724. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the US Navy Department or the naval service at large. 1 Head, Malaria Program, Biomedical Research Institute, Rockville, MD 20852, USA. 2 Biologist, Naval Medical Research Institute, Bethesda, MD 20014, USA. 3 Cryobiologist, Biomedical Research Institute, Rockville, MD 20852, USA. 'Head, Malaria Branch, Naval Medical Research Institute, Bethesda, MD 20014, USA. MATERIALS AND METHODS Blood stages of P. fallax were harvested from Beltsville white turkey poults, 28 days old at the time of inoculation; male and female birds were used in a random selection. Ten birds were given an intra- venous injection in the pectoral vein with 0.2 ml of heavily parasitized blood (40-50% of erythrocytes infected) and monitored daily for the onset of and subsequent increases in parasitaemia. Blood films were made daily, stained with Giemsa's stain, and examined. When parasitaemia reached 30% or higher, each bird was bled from the carotid artery with a 20-gauge needle and syringe containing 0.1 ml of sodium heparin. The blood was diluted with medium 199 and dimethyl sulfoxide (DMSO) to contain 2.5 x 107 of parasitized cells per ml at a DMSO concentration of 7.5% in medium 199; controls without DMSO were included. Prepara- tions to be frozen were equilibrated at 40C for 30 min prior to freezing in a dry ice-ethanol slurry at -79°C. Samples were shell-frozen by rapidly swirling the vials in the slurry. The vials were then stored in liquid nitrogen vapour in a Cryenco Biostat, model 22. The vials were thawed by rapid agitation in a water bath at 37°C. After thawing, serial dilutions with 0.9% saline were made to give 0.2-ml doses containing 5 x 106, 5 x 105, 5 x 104, 5 x 103, and 5 x 102 parasites, and these doses were inoculated into turkey poults. Parasitaemia was monitored to determine the infectivity of these doses after storage compared with that of unfrozen control preparations. 3610 - 305- C. P. A. STROME ET AL. L. simondi sporozoites were obtained from black- flies collected in the field in Seney County, MI, USA, in July, 1970. Approximately 100 flies were collected and ground in a glass homogenizer in a solution of 5% glycerol in Hanks' balanced salt solution. The homogenate was diluted with the same solution so as to contain the equivalent of either 4 or 8 flies per ml, and was distributed in 1-ml volumes into 3-ml glass ampoules. The ampoules were heat-sealed and trans- ferred to a bath at 0°C for approximately 30 min. The samples were then frozen by means of a method designed for use in the field. Two 250-ml expanded- polystyrene cups equipped with a thermometer, one cup placed within the other, were tightly packed with ampoules and the empty space was filled with ethanol at 0°C. The cups were lowered into the vapour phase of a liquid nitrogen container at a rate allowing them to cool at approximately 1°C/min. When the samples reached - 50°C, they were abruptly immersed in liquid nitrogen and left there for storage. The ampoules were thawed by immer- sion and rapid agitation in a water bath at 370C. Fourteen-day-old White Peking ducks of either sex were infected with 1.0-ml doses of frozen-thawed sporozoites injected via the intraperitoneal route. The birds were monitored daily for parasitaemia for 3 weeks or until blood parasites were observed. P. berghei sporozoites were obtained by homo- genization of infected Anopheles stephensi in medium 199 and normal mouse serum, and centrifugation in a density gradient (13). Haemocytometer counts were performed and the sporozoite concentrations were adjusted to 30 000 sporozoites per 0.1 ml. The freezing medium consisted of medium 199 with DMSO and mouse serum in final concentrations of 7.5% and 50 %, respectively. Then 1-ml volumes were placed in 9-ml screw-capped vials and were shell-frozen and thawed as described above. NIH female white mice, at least 3 months of age, were used to demonstrate infectivity. The mice were injected via the tail vein with 0.1-ml doses each containing 30 000 sporozoites, and the onset of parasitaemia was monitored. Exoerythrocytic stages of P. fallax were obtained from infected embryonic turkey brain by means of the technique described by Pipkin & Jensen (14). Removal of the infected brain and trypsinization were carried out in accordance with the methods of Davis et al. (15). The digested turkey brains were pooled, divided into two equal parts, and centrifuged at 500 g for 10 min. One preparation was resus- pended in medium 199 and the other in medium 199 with 7.5% DMSO. The first portion was then diluted in tenfold decrements to N x 10-5 with medium 199. A 0.25-ml dose from each dilution was injected into the pectoral vein of Beltsville white turkey poults and the onset of parasitaemia was recorded. The second portion was programme-frozen at 1°C/min in a Linde Biological Freezer and stored in liquid nitro- gen vapour. Thawing was carried out by rapidly agitating the samples in a water bath at 37°C. The material was then diluted and injected into poults so as to determine infectivity. Exoerythrocytic stages of P. fallax also were cul- tured as described by Davis et al. (15), the material being frozen and its infectivity determined in turkeys as described above. RESULTS The results of long-term storage in liquid nitrogen vapour on the infectivity of P. fallax erythrocytic stages are presented in Table 1. With the inocula con- taining the two highest concentrations of parasites- 5 x 106 and 5 x 105-all the groups but one became infected and there were no differences between the Table 1. Turkey poults infected with P. fallax erythro- cytic stages following parasite storage in liquid nitrogen vapour for various periods (4 birds per test group) No. of parasites per injection Storage time 5x106 5x105 5x104 5x103 5x102 2 hours 4 4 4 1 0 7 days 4 4 2 2 0 1 month 4 4 1 0 0 3 months 4 4 0 1 0 6 months 4 4 1 0 0 12 months 4 0 0 0 0 18 months 4 4 4 0 0 24 months 4 4 4 0 0 30 months 4 4 1 0 0 36 months 4 4 2 0 0 73 months 4 4 4 3 1 124 months 4 4 4 1 0 Infectivity (%) 100 92 56 17 2 Unfrozen controls with DMSO 4 4 4 4 3 Unfrozen controls without DMSO 4 4 4 4 1 306 A QUANTITATIVE LONG-TERM CRYOBIOLOGICAL STUDY birds inoculated with unfrozen control samples and those inoculated with parasites frozen and stored in the gas phase of liquid nitrogen for 7 days to 10 years. The single exception was the group of birds into which parasites stored for one year had been injected. The sample tested in these birds was infec- tive only at the highest titre of 5 x 106 parasites per injection. Variability was greater at a dose of 5 x 104 parasites per injection. Infectivity at a dose of 5 x 104 parasites ranged from zero for samples stored for 3 months or 1 year to 100% for samples stored 18, 24, 73, or 124 months. The test dose of 5 x 104 parasites per injection produced fewer infections than unfrozen controls did. At the lowest dilu- tions-5 x 103 and 5 x 102 parasites per injec- tion-only 9 of 96 birds became infected with the frozen and stored material, 5 of these 9 infected birds having received injections with parasites that had been stored for more than 6 years. Parasites that had been frozen and thawed more than once showed a marked loss in infectivity. Samples frozen and thawed twice retained infectivity only in the two highest dilutions (2 out of 4 and 1 out of 4 birds infected at concentrations of 5 x 106 and 5 x 105 parasites per injection, respectively). Those frozen and thawed three times were not infective at any concentration used in this study. Results with frozen L. simondi sporozoites (Table 2) indicate that there was infectivity in sam- ples stored as long as 7 years. There was a decrease in the infectivity of only one of four separately prepared and frozen batches when the samples assayed during the first year were compared with those stored for 7 years in liquid nitrogen. At the dose level of 30 000 P. berghei sporozoites per dose, there was no significant loss of infectivity after freezing and storing for 24 h: all 10 mice inoculated with unfrozen control samples were infected, as against 19 out of 20 inoculated with parasites that had been frozen and stored. There was Table 2. Ducklings infected with L. simondi sporozoites following parasite storage in liquid nitrogen for 7 years Flies per No. of birds infectedSample dolsperSampledose 1970 1977 1 4 10/10 0/1 2 8 1/1 1/1 3 8 2/3 1/1 4 8 1/1 1/1 Table 3. Turkey poults infected with activated P. fallax exoerythrocytic stages following storage of parasites in liquid nitrogen vapour for 2 h Dilution of Unfrozen Following original samples controls storage 1:10 2/2 2/2 1 :100 2/2 2/2 1 :1000 2/2 0/2 1:10 000 2/2 0/2 1:100 000 0/2 0/2 a considerable loss of infectivity of exoerythrocytic stages of P. fallax following freezing (Table 3). Unfrozen preparations remained 100% infective at a dilution of 10-4 of the original suspension, whereas frozen material was infective only at the 10-1 and 10-2 dilutions of the original sample. DISCUSSION The most useful information in Table I is that concerning the dose level of 5 x 104 parasites per injection. Above that level, all samples of P. fallax erythrocytic stages were uniformly infective except for the sample stored for 12 months and for samples frozen more than once. This indicates that any differences between samples were undoubtedly masked by the large number of organisms injected. At the level of 5 x 104 parasites per injection, a representative end-point in the titration seems to have been reached, although, at this dose level, the preserved material varied in infectivity from 0 to 100%. Samples stored for 2 h, and for 18, 24, 73, and 124 months were 100% infective, as were the unfrozen controls, yet samples stored for other times were less infective. Among the possible explanations for the variability observed are differ- ences in cooling and warming rates among samples, in the natural resistance of test animals, and in the injection technique although the injections were all performed by the same individual. Nevertheless, it is clear from the results that the variability observed was not related to the duration of storage. L. simondi sporozoites from blackflies were col- lected and frozen in the field, so that it was not technically feasible to establish an unfrozen control. However, no appreciable changes in infectivity were shown at the dose levels used when the infectivity of the material stored for 7 years was compared with that of the material stored only for the duration of the collection trip. 307 308 C. P. A. STROME ET AL. Preliminary short-term studies show that delete- rious effects on the infectivity of P. berghei sporo- zoites owing to freezing were not detectable at the dose level used. The large decrease in infectivity seen in the exoerythrocytic stages of P. fallax does not necessarily indicate that those stages are more sensi- tive to freezing than are those of the erythrocytic stage, since many factors, including the cooling and warming rates, type and concentration of preserva- tive, inherent variability in host susceptibility, and variations in technique, may be important. Our results indicate a need for detailed analysis of these factors before definite conclusions can be reached. The studies show the reliability of long-term stor- age at low temperature of malaria parasites in the erythrocytic stage. The results indicate that similar storage of sporozoites and exoerythrocytic stages may eventually be possible. ACKNOWLEDGEMENTS The authors thank Dr Joseph Armstrong and Ms Nancy Pacheco for their editorial assistance, and Mr Fred Mitchell for his excellent technical assistance. 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