Observations on early and late post-sporozoite tissue stages in primate malaria. IV. Pre-erythrocytic schizonts and/or hypnozoites of Chesson and North Korean strains of Plasmodium vivax in the chimpanzee.

In a continuing reexamination of plasmodial tissue stages within the context of the hypnozoite theory of malarial relapse, 2 strains of Plasmodium vivax with distinct and disparate relapse characteristics in humans were studied in chimpanzees. Following intravenous inoculation of massive numbers of salivary gland sporozoites, both the frequently relapsing Chesson strain and a North Korean strain characterized by predominantly delayed relapses exhibited relapse patterns and antimalarial sensitivity in the splenectomized chimpanzee essentially indistinguishable from those seen in humans. Examination of hepatic biopsies obtained at 7 and 10 days after infection revealed both pre-erythrocytic (pre-e) schizonts and hypnozoites in tissue obtained from the animal infected with the Chesson strain, but only rare hypnozoites (no pre-e schizonts) at 7 days in the animal infected with the North Korean strain. These findings, combined with the comparability of relapse behavior--which indicates the suitability of the chimpanzee as a model for the natural (human) host-parasite relationship--are essentially as predicted by the hypnozoite theory, despite the small numbers of tissue forms seen. Pre-erythrocytic schizogony of the Chesson strain in the liver was essentially indistinguishable from that of other strains studied, also underlining the suitability of this model system for tissue stage studies of P. vivax.

Observations on early and late post-sporozoite tissue stages in primate malaria. III. Further attempts to find early forms and to correlate hypnozoites with growing exo-erythrocytic schizonts and parasitaemic relapses in Plasmodium cynomolgi bastianellii infections.

Rhesus monkeys were heavily infected with sporozoites of Plasmodium cynomolgi bastianellii in an attempt to demonstrate the site of invasion of sporozoites into tissue cells and their growth there. Further attempts were made to correlate the appearance and loss of hypnozoites with parasitaemic relapses. Hypnozoites were demonstrated and once again shown to decrease in numbers over 229 days during which time the infection showed parasitaemic relapses. Liver biopsies taken at two-day intervals for 12 days showed that hypnozoites decreased in numbers over-all and growing schizonts were demonstrated in the liver. At this time a parasite the size of a hypnozoite was seen with two nuclei and another was seen with an elongate, possibly dividing nucleus in one monkey. an attempt to find the location of the early intracellular exoerythrocytic forms in the liver at various times less than 40 hours after infection using smears and immunological staining with newly prepared anti-sera failed. Large numbers of sporozoites of P. knowlesi were also injected into a rhesus monkey the liver of which on the fifth day after infection showed no hypnozoites among 157 sections of growing schizonts and no parasites at all on the 42nd day after infection. In P. cynomolgi bastianellii infections parasites, mostly hypnozoites, were found in the liver up to 229 days after infection.

A new malaria parasite Plasmodium (Sauramoeba) heischi in skinks (Mabuya striata) from Nairobi, with a brief discussion of the distribution of malaria parasites in the family Scincidae.

A new species of malaria parasite, Plasmodium (Sauramoeba) heischi, is described from African skinks (Mabuya striata). Eleven individuals of 90 specimens collected in Nairobi were found to be infected. The new parasite is a large species, characterized by spindle-shaped gametocytes, the female often with a subterminal nucleus. The schizonts produce up to 65 nuclei and cause great hypertrophy and distortion of the host cell. Although similar to P. (Sauramoeba) giganteum in dimensions and merozoite numbers, P. heischi is easily distinguished by gametocyte and schizont shapes.

Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection.

Hypnozoites of two strains of the human relapsing malaria parasite, Plasmodium vivax, have been detected among maturing 7- and 10-day pre-erythrocytic schizonts in liver biopsies of chimpanzees infected by intravenous inoculation of sporozoites obtained from dissected salivary glands of heavily infected anopheline mosquitoes. As in the simian relapsing species, P. cynomolgi, the hypnozoites of P. vivax at 7 and 10 days are uninucleate forms of approximately 4-5 micrometers diameter, lying within the cytoplasm of individual hepatocytes. Their presence in this relapsing human species is added support for the hypnozoite theory of malarial relapse.

Observations on early and late post-sporozoite tissue stages in primate malaria. II. The hypnozoite of Plasmodium cynomolgi bastianellii from 3 to 105 days after infection, and detection of 36- to 40-hour pre-erythrocytic forms.

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

Observations on early and late post-sporozoite tissue stages in primate malaria. I. Discovery of a new latent form of Plasmodium cynomolgi (the hypnozoite), and failure to detect hepatic forms within the first 24 hours after infection.

Previous work in this laboratory has demonstrated the ability of the immunofluorescence technique to detect pre-erythrocytic stages of the primate malaria parasite, Plasmodium cynomolgi bastianellii, in hepatic tissue obtained as early as 48 hours after sporozoite inoculation. In an attempt to visualize still earlier post-sporozoite stages, hepatic tissue obtained from a rhesus monkey infected with 12,000,000 sporozoites was examined at 2, 12, 24, and 48 hours after inoculation, employing antisera reactive with both invertebrate and vertebrate stages of the parasite. Tissue was also obtained at 7, 50, 102, and 105 days after sporozoite inoculation, and was examined for adequacy of the hepatic infection and for the presence of late exoerythrocytic schizonts. Although a new, previously unrecognized, uninucleate latent stage of 5 micrometer diameter (the "hypnozoite") was detected among large maturing schizonts in the 7-day and later biopsies, no intrahepatic parasites were found in tissue taken at 24 hours or earlier, despite the presence of up to 61 7-day schizonts and eight hypnozoites per 5 X 8 mm section. Pre-erythrocytic forms again were detected at 48 hours, although in far smaller numbers than expected on the basis of the density of parasites at 7 days after infection. The significance of these observations is discussed in the context of previous negative findings.