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Sporozoites of Plasmodium gallinaceum Brumpt were preserved in the mosquito Aedes aegypti (Linn.) for 767 days in liquid nitrogen at -197 C without apparent loss of viability or infectivity. Prepatent periods which averaged 7 days and parasitemias in baby chicks which were used to challenge the infectivity of the sporozoites were essentially the same as those produced from infected unrefrigerated mosquito controls. Advantages of this method of preserving and storing the sporozoites are: (1) simplicity, (2) indefinite preservation time, and (3) freedom from blood pathogens other than malaria. The present study was undertaken to develop a simple technique for preserving strains of malaria parasites for an indefinite period of time in order to make feasible the establishment of "banks" of malaria strains that could be available to investigators anywhere at any time. The methods of low temperature preservation of malaria parasites now in use have been of great value but the time and skill required to prepare the materials limits their usefulness. The technique described in this report is simple and could be utilized in most places where malaria research is conducted. The parasites are preserved in the mosquito host in liquid nitrogen at -197 C. The senior author (1952) stated that the sporozoite is the mature stage or the end product of the sexual cycle, and to a degree, may be comparable to the resting or infectious stages of other parasites. It therefore should be more capable of withstanding an adverse environment than other malaria forms. The salivary glands in the mosquito should afford the most ideal physiological environment for storing sporozoites at low temperatures. Jeffery and Rendtorff (1955) listed several apparent advantages that sporozoites might have over other forms for preservation of malaria strains. These included resistance to adverse conditions, smaller size, freedom from host cells, concentration in smaller volume, and freedom from other vertebrate-host pathogens. The value of long-term preservation of malaria parasites at low temperature primarily Received for publication 23 January 1967. * This investigation was supported in part by Public Health Service Research Grant AI-05253, from the NIAID. lies in the facts that: (1) continual propagation from host to host of malaria strains is not required. Many valuable strains have been lost when research projects were terminated and the time-consuming process of propagation could no longer be justified; (2) the alteration of strains through prolonged serial blood passage is minimized. Several malaria strains have been altered and some have lost the ability to produce gametocytes due to the lack of passage through a mosquito host. If a large volume of parasites from the original host is frozen and stored, the original strain may be returned to at times when changes become apparent in the working strain. MATERIALS AND METHODS A laboratory strain of Aedes aegypti (Linn.) and the 8A strain of Plasmodium gallinaceum Brumpt were used throughout this investigation. Several hundred young adult female A. aegypti were infected with P. gallinaceum by allowing them to feed on an infected chick with a high gametocyte count. Unfed mosquitoes were removed from the cage and only heavily engorged mosquitoes were retained. The mosquitoes were kept as described by Terzian et al. (1949) in a climate room maintained at 26 C and 70% relative humidity for 8 or more days to permit maturation of sporozoites. After the sporozoites had developed, sample salivary glands were examined to determine the degree of infection. Only groups of mosquitoes with a high percentage of infection were selected for freezing. These mosquitoes were immobilized in a refrigerator at 4 C, taking care that ice crystallization did not occur. Ten whole mosquitoes were placed in each '1/-dram screw-cap vial and the vials of mosquitoes were immersed rapidly in liquid nitrogen for freezing and storage. Thawing usually was accomplished rapidly in a water bath at 40 C until it was noted accidentally that sporozoites from mosquitoes that thawed at room temperature were infective. The viability of the sporozoites was challenged at regular in |
