Gametogony and Sporogony in a Strain of Plasmodium berghei Preserved at Low Temperature

Abstract

A strain of Plasmodium berghei isolated from sporozoites of wild-caught Anopheles dureni and passed by blood transfers to young hamsters retained its gametocyte-producing ability after 310 days at -70 C. The infected blood was preserved by the addition of Alsever's solution and glycerol. Hamsters infected with the thawed infected blood showed large numbers of gametocytes and exflagellation readily occurred. Batches of Anopheles quadrimaculatus and A. freeborni which fed on these hamsters showed oocyst infection rates of 33.5% and 31.3%, respectively. Freezing of heparinized blood containing P. berghei gametocytes at -70 C without addition of Alsever's solution and glycerol abolishes and prevents exflagellation of microgametocytes. The addition of Alsever's solution with glycerol permits exflagellation of microgametocytes after thawing and preserves the viability and motility of the male gametes. Freshly isolated strains of Plasmodium berghei obtained from sporozoite-induced infections produce a characteristic abundance of gametocytes. In tree rats (Thamnomys surdaster) as well as in hamsters, young white rats, and white mice, gametocytes of these strains appear with the first waves of parasites detected in the peripheral blood. They continue present in varying numbers throughout most of the patent infection. Exflagellation of microgametocytes occurs readily in vitro and ookinete formation and oocyst growth may be observed in several anopheline species (Yoeli and Wall, 1951, 1952; Perez-Reyes, 1953; Rodhain, Wansom, and Vincke, 1955; Yoeli and Most, 1960). The ability of these strains to produce gametocytes declines as a result of prolonged and repeated blood transfers in experimental animals and eventually is lost. Such exhausted strains can sometimes be reinvigorated and Received for publication 23 April 1963. * This investigation was conducted under the sponsorship of the Commission on Parasitic Diseases, Armed Forces Epidemiological Board, and supported in part by the Office of the Surgeon General, Department of the Army, and by Research Grant AI 024-23 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service. their gametocyte production temporarily restored by a series of quick blood transfers in young tree rats, their natural biological hosts, or in baby hamsters-a biologically related and suitable experimental host (Jadin, Yoeli, and Pierreux, 1959). In general, however, loss of gametocyte-producing ability of blood passage strains is an irreversible process (Sergent and Poncet, 1956). Coggeshall (1939) first demonstrated the viability of malaria parasites preserved in the frozen state. His work was further extended by Manwell and Jeffery (1942), Jeffery and Rendtorff (1955), and Jeffery (1957), who maintained avian plasmodia and human infected blood and sporozoites for prolonged periods at low temperatures. Molinari (1960) and more recently Jeffery (1962) demonstrated survival of trophozoites of P. berghei in glycerolized whole blood at -78 C. The necessity of maintaining vigorous gametocyte-producing strains of P. berghei for prolonged periods in an attempt to establish cyclical transmission in the laboratory, and the great difficulty in obtaining freshly isolated strains from infected, wild-caught Anopheles dureni in the forests of Katanga, prompted us to preserve some of our newly isolated strains at low temperatures. The present paper describes the results of