Thomas W. Holbrook

Immunity to exoerythrocytic forms of malaria. III. Stage-specific immunization of turkeys against exoerythrocytic forms of Plasmodium fallax.

A method for immunization of turkeys against exoerythrocytic infection with Plasmodium fallax was devised using formalin-killed merozoites (FKM) from cultures of infected turkey embryo fibroblasts. A single intramuscular injection of FKM combined with complete Freunds adjuvant (CFA) followed at weekly intervals by 2 intravenous (i.v.) injections of FKM in Earles balanced salt solution (EBSS) (a total of 5.0 X 106 FKM) resulted in 100% survival of turkeys after challenge with infective merozoites when blood stages were drug-suppressed. Two weekly i.v. injections totaling 3.5 X 106 FKM without CFA resulted in 80% survival after challenge. Only 20% of untreated control birds, and about 40% of birds receiving normal cell culture medium overlay, with or without CFA prior to challenge, survived infection. When FKM-immunized birds were challenged with infective merozoites from culture and received no drug suppression of blood stages, parasitemias were similar to those in control turkeys. Also, challenge of FKM-immunized turkeys with parasitized erythrocytes resulted in parasitemias similar to those of control birds. The development of exoerythrocytic stages subsequent to blood-stage infection was suppressed in most FKM-immunized turkeys, while control turkeys eventually died with heavy exoerythrocytic infections in brain capillaries. Past efforts to immunize animals against infection with malaria parasites have been directed at the use of erythrocytic stages or sporozoites as immunogens. Varying degrees of success have been achieved using killed erythrocytic stages of avian malaria parasites (Redmond, 1939; Gingrich, 1941; Jacobs, 1943; Richards, 1966). The use of adjuvants in combination with killed erythrocytic parasites increased the degree of immunity to subsequent infection (Freund et al., 1945; Thomson et al., 1947). The use of killed sporozoites for immunization was effective against mosquito-transmitted avian malaria (Mulligan et al., 1941; Russell and Mohan, 1942), although birds immune to sporozoite challenge were susceptible to infection induced with erythrocytic stages (Russell et al., 1942). Richards (1966) achieved more effective immunization of chickens with killed sporozoites than with killed blood stages of Plasmodium gallinaceum. The development of a culture system for the growth and maintenance of cells infected with exoerythrocytic stages of avian malaria paraReceived for publication 18 September 1973. * Supported by Research Grant AI-08988, and in part by AI-00092, NIAID, U. S. Public Health Service. t Deceased, 27 March 1973. sites (review by Huff, 1969) presents an opportunity for specific immunization with that stage. The results of studies utilizing exoerythrocytic merozoites from infected cell cultures as immunogens against infection of turkeys with P. fallax are herein presented. MATERIALS AND METHODS

Stimulation of resistance in mice to sporozoite-induced Plasmodium berghei malaria by injections of avian exoerythrocytic forms.

Mice received a series of injections of formalin-killed merozoites (FKM) of exoerythrocytic stages of Plasmodium fallax prior to challenge with sporozoites of P. berghei. In one study 4 of 16 FKM-immunized mice never exhibited parasitized erythrocytes after 2 challenges of 10(4) P. berghei sporozoites each, while all control animals died with high parasitemias. FKM-immunized mice were as susceptible as control mice to infections initiated with parasitized erythrocytes. In a second study, 14 of 16 mice immunized via the intravenous (i.v.) or combined intramuscular (i.m.) and i.v. routes were immune to an initial challenge with 10(4) sporozoites, but were susceptible to a second challenge. Three injections of FKM via the i.m. or intraperitoneal routes did not elicit a protective response against sporozoite challenge. Sera harvested from FKM-immunized and control mice prior to challenge produced no visible CSP reaction with P. berghei sporozoites, nor was infectivity of sporozoites altered after incubation in sera, showing that SNA was absent. In additional experiments results were less encouraging. An attempt to repeat the result of the second experiment failed. Each of 5 mice which received the same number of FKM by a similar schedule became infected after sporozoite challenge. In an additional study the immunization schedule was increased from 3 to 7 injections of FKM and 40% of FKM-immunized mice resisted challenge. However, mice which had received FKM prior to sporozoite challenge consistently displayed an increased prepatent period compared with control animals. A department from methods of the more successful studies was necessitated in these later studies in which FKM were harvested from cell cultures maintained for longer periods of time.

Leishmania in the chick embryo: IV. Effects of embryo age and hatching, and behavior of L. donovani in cultures of chick fibroblasts

Abstract On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10 6 L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching. Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.