Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement.

Gametocytes are the intraerythrocytic stages of malaria parasites that infect mosquitoes. When gametocytes of the chicken malaria parasite Plasmodium gallinaceum are ingested by a mosquito they become extracellular in the mosquito midgut, form gametes, and fertilize within 10 to 15 min after the insect has taken a blood meal. Gametocytes of P. gallinaceum were infectious when fed to Aedes aegypti mosquitoes in blood meals containing native serum from chickens or from the non-host species, man or sheep. Gametocytes stimulated to undergo gametogenesis and to fertilize in vitro were also infectious when fed to mosquitoes in native chicken serum. However, native serum from most non-host species, including sheep and man, suppressed the infectivity of newly fertilized zygotes to mosquitoes and lysed the zygotes in vitro. These effects were shown to be due to the activity of the alternative pathway of complement (APC) in the serum of the non-host species. After mild trypsin treatment, the zygotes of P. gallinaceum no longer infected mosquitoes in the presence of native chicken serum, although in heat-inactivated chicken serum their infectivity was normal. We conclude that trypsin-sensitive components on the zygotes surface protect them from destruction by the APC of their native host. The ability of gametocytes of P. gallinaceum to infect mosquitoes in the presence of native human serum is probably due to proteases that inactivate the APC of human serum before the gametes and zygotes emerge as extracellular parasites in the blood meal.

Plasmodium gallinaceum: density dependent limits on infectivity to Aedes aegypti.

In acute, blood-induced infections of chickens, the malarial parasite Plasmodium gallinaceum is most infective to the mosquito Aedes aegypti 1 day before gametocyte numbers peak. In an effort to account for this disynchrony , daily changes in parasite infectivity, parasitemia, hematocrit, and hemoglobin were measured during the course of infections. Three events were correlated with the loss of infectivity: (1) In the 24 hr between park infectivity and peak gametocytemia , schizont-induced hemolysis reduced the red blood cell volume 22%. (2) P. gallinaceum zygotes, fertilized in vitro and mixed with heavily infected red blood cells from which all viable, mature gametocytes had been removed, produced 67% fewer oocytes than when combined with uninfected red blood cells. (3) Zygotes fertilized in vitro on the day of peak parasitemia produced 47% fewer oocysts than zygotes prepared 24 hr earlier. It appears that high parasite density reduces infectiousness by destroying, through hemolysis and intraerythrocytic metabolism, a substance necessary to the sporogonic stages, and that there is also an intrinsic loss of infectivity, possibly due to decreased efficiency of fertilization.

Plasmodium knowlesi: persistence of transmission blocking immunity in monkeys immunized with gamete antigens.

Eight rhesus monkeys immunized with a partially purified preparation of Plasmodium knowlesi gametes were monitored for over 6 years to determine the extent of transmission blocking immunity. Monkeys were challenged regularly, and anti-gamete antibodies were assayed by in vivo and in vitro mosquito feedings. Transmission blocking immunity persisted at high levels in most of the monkeys. In those animals in which protection waned between challenges, a challenge infection provided a sufficient booster effect to prevent infection of mosquitoes. Immunity to other stages of malaria (i.e., sporozoites and asexual erythrocyte forms) failed to induce immunity against gametes.

Plasmodium gallinaceum: erythrocyte factor essential for zygote infection of Aedes aegypti.

Zygotes of Plasmodium gallinaceum, fertilized in vitro and fed to Aedes aegypti mosquitoes through a membrane, formed oocysts only when a substance in the cytoplasm of uninfected erythrocytes was present. The relation between erythrocyte volume and infectivity was linear (1:1.2) up to a 50% hematocrit. The intraerythrocytic substance was both nondialyzable and poorly soluble in plasma. By carboxymethyl cellulose chromatography, cytoplasmic constituents eluted at pH 8.6 supported the same infection as control blood did; but higher and lower pH eluates supported none. Dialyzable factors present in the plasma, but absent from M199, enhanced infection but were not essential. Zygotes developed normally to ookinetes in the gut of plasma-fed mosquitoes, or when cultured in plasma or M199. Ookinetes from culture formed normal oocysts when fed to mosquitoes in blood or when injected with M199 into the hemocoels of unfed females. Mosquitoes fed infected blood containing lima bean or soybean trypsin inhibitor were unable to digest the erythrocytes and, although normal ookinetes developed, no oocysts formed. It appears from this and histological evidence that an erythrocyte substance, released by mosquito digestion, is needed for ookinete invasion of the gut epithelium.

Plasmodium gallinaceum: avian screen for drugs with radical curative properties.

Existing primary screens for radical curative antimalarial drugs fail to adequately detect many compounds which affect the latent, exoerythrocytic hypnozoite, the stage of the parasite responsible for relapse. At the same time, these screens falsely identify a wide range of compounds with no radical curative activity. The avian malaria, Plasmodium gallinaceum, and Aedes aegypti mosquitos were used in a screen which measures the effects of candidate compounds on gametocytes and their development within the mosquito. Sporontocidal and gametocytocidal effects could be differentiated by this screen. In a blind study, those compounds shown to be exclusively gametocytocidal were those same drugs which had previously been shown to have radical curative effects against true relapsing malarias. The chicken malaria gametocyte screen was more sensitive than the rodent screens in detecting useful compounds, with a minimum of false positive identifications.

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.

Infection of Aedes aegypti with zygotes of Plasmodium gallinaceum fertilized in vitro.

Female gametes of Plasmodium gallinaceum fertilized in vitro, cleaned of all other blood constituents, resuspended in blood, and fed to Aedes aegypti through a membrane were infective. At the lowest zygote concentration, 10(4)/ml, nearly every ingested parasite produced an oocyst. As the concentration ingested increased, efficiency to infect diminished, until above 10(7) zygotes/ml the number of oocysts produced became constant. This method should be valuable for determining the nutrient requirements of the ookinete and early oocyst and for studying the effect of immune sera on these stages in vivo.

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.

Malaria in asplenic mice: effects of splenectomy, congenital asplenia, and splenic reconstitution on the course of infection.

In investigations on the role of the spleen in host defense against malaria, we studied the course of murine malaria in three groups of mice with altered splenic function: congenitally asplenic mice, adult-splenectomized mice, and adult-splenectomized mice which were reconstituted with spleen-cell suspensions. Intact mice infected with either Plasmodium yoelii or P. chabaudi adami experienced infections which resolved spontaneously, with low mortality. Congenitally asplenic and splenectomized-reconstituted mice were unable to clear their primary infections, and experienced high mortality; infections in the latter two groups of mice differed little from those in splenectomized, nonreconstituted controls. However, when asplenic and splenectomized mice were treated with cloroquine during their primary infections and then rechallenged with the homologous Plasmodium species, they experienced mild infections similar to those of intact controls. These observations support the concept that the host defense in primary malaria infections requires an architecturally intact spleen, and therefore is not solely dependent upon the presence of a subpopulation of immune spleen cells.

Influence of malaria infection on the elaboration of soluble mediators by adherent mononuclear cells.

MALARIA RESULTS IN TWO SEEMINGLY PARADOXICAL PERTURBATIONS OF THE IMMUNE RESPONSE: polyclonal B-cell activation and immunosuppression. To determine what immunoregulatory role mediators secreted by adherent cells might play in these alterations, we cultured adherent cells from uninfected mice and from mice at different times during infection with Plasmodium berghei or P. yoelii. Culture supernatants obtained from these cells were tested for their ability to enhance the in vitro proliferative responses of thymocytes to suboptimal concentrations of concanavalin A or to inhibit the mitogen-stimulated proliferation of normal spleen cells. Supernatants obtained from adherent cells of mice early in infection (days 1 to 3) contained significantly elevated levels of enhancing activity which on Bio-Gel P-100 chromatography resembled lymphocyte-activating factor. Later in infection (days 4 and 5), these supernatants contained inhibitory activity. Normal adherent cells, when cocultivated in vitro with parasitized erythrocytes, ingested parasite debris and were stimulated to produce the enhancing factor. At high parasite/adherent-cell ratios, cells elaborated an inhibitory factor. These findings suggest that during malaria, adherent cells are converted from a nonspecific helper role to a nonspecific suppressor role. This modulation in function may be due to the direct interaction between adherent cells and parasitized erythrocytes.