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Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Nonspecific and specific chemosuppression of the immune response to Plasmodium berghei protective antigens were investigated. Specific immunosuppression was defined operationally as the selective suppression of the protective response to the parasite in mice injected with a combination of gamma-irradiated infected mouse erythrocytes (gammaPb) and cyclophosphamide (CY) with continued responsiveness to sheep erythrocytes (SRBC). After initial treatment (gammaPb + CY), mice were injected with gammaPb in potentially immunogenic doses. These and appropriate control animals were later challenged with nonirradiated infected mouse erythrocytes. The influence of the initial treatment regimens on the protective response was evaluated by parasitemia, and mortality was observed after challenge. Specificity of suppression was measured by evaluating the ability of mice to produce antibody to SRBC. Both specific and nonspecific suppression of the protective response to malaria were noted. Initial treatment with drug alone resulted in increased parasitemia and mortality and suppression of the SRBC antibody synthesis in drug-pretreated immunized mice as compared with immunized mice not pretreated with the drug. On the other hand, suppression of the response to the parasite, but not to SRBC, in animals pretreated with gammaPb + CY was clearly greater than that induced by drug alone. Thus, animals treated with malarial antigen and cyclophosphamide develop a measurable specific immunosuppression. These studies indicate that immunity to malaria is influenced by both cyclophosphamide alone (general immunosuppression) and cyclophosphamide in combination with antigen (specific immunosuppression) in a manner analogous to other immune responses.

Parasitologic, hematologic, and immunologic parameters were monitored in intact (nonsplenectomized), adult chimpanzees infected with a "chimp-adapted" strain of Plasmodium falciparum. Following primary and secondary injections of 10(9) P. falciparum-infected erythrocytes, each chimpanzee developed a low grade parasitemia (up to 1,000/mm3) and maintained the infection without evidence of eliminating the parasites. Hematologic and serum biochemical values, as well as the majority of immunologic parameters tested, remained unaltered in infected chimpanzees. However, 2 weeks after infection T cells from infected chimpanzees demonstrated an enhanced response in vitro to stimulation with the mitogen PHA, and monocyte phagocytic activity for antibody-coated erythrocytes (Fc-mediated phagocytosis) increased significantly. During malarial infection, apes developed a strong T cell proliferative response to P. falciparum antigens and monocytes showed enhanced phagocytic activity for P. falciparum-infected erythrocytes in the absence of immune serum. These results suggest that cellular immune mechanisms, especially macrophage activation, may help control, but not eliminate, P. falciparum malaria in chimpanzees.

Five owl monkeys, Aotus trivirgatus, were immunized against falciparum malaria by the intravenous injection of gamma-irradiated parasitized blood from donor monkeys with high parasitaemias. Each animal received approximately 1 x 10(8) parasitized erythrocytes per immunizing dose. Three monkeys received 4 doses and 2 monkeys received a single dose. After challenge, 3 control monkeys that received saline and the 2 monkeys that received a single dose of parasitized blood died with fulminant parasitaemias. The 3 monkeys that received 4 immunizing doses had either no parasitaemia at all (one monkey) or low-grade (<2.5%) parasitaemias (two monkeys); one of the latter monkeys died after the malaria had run a prolonged course. No striking haematological abnormalities were noted in animals as a result of the immunization procedure. These results confirm that immunity to Plasmodium falciparum can be induced in owl monkeys by inoculation with parasitized erythrocytes that have not undergone culture and without the use of adjuvant.

The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.

Cryopreserved chimpanzee erythrocytes infected with Plasmodium falciparum were examined by electron microscopy directly and after a period of culture. Light microscopic observations on the viability of cryopreserved parasites in culture were also made. Parasitaemia data from a chimpanzee infected with parasites cryostored for more than 2(1/2) years are presented. Varying degrees of damage were apparent as the parasites were examined in vitro, for viability, and by electron microscopy. Schizonts appear not to survive the process, whereas the ability of "ring" trophozoite forms to develop into large trophozoites in vitro varies from 20% to 100% in different preparations. Cryopreserved material examined by electron microscope showed degenerative changes in most of the parasites, but after 72 h of cultivation most of the parasites appeared normal. These findings suggest that some organisms may sustain reversible damage during cryostorage.