Louis C. Koontz

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.

Engorgement response of anopheline mosquitoes to blood fractions and artificial solutions

ABSTRACT. Anopheles stephensi Liston, Anopheles freeborni Aitken, Anopheles gambiae Giles and Anopheles dirus (Peyton & Harrison) fed equally well on whole blood, red blood cells, platelet‐rich plasma and platelet‐poor plasma. Similar feeding ability on 0.15 M NaCl containing 10‐2 M NaHCO3 was shown by the first three species, but An. dirus required an addition of albumin. The need for ATP as a phagostimulant could not be demonstrated in any of these species.

Plasmodium vinckei: Production of chloroquine-resistant strain

Abstract To date only two species of malarial parasites, Plasmodium falciparum in man and P. berghei in mice, have demonstrated the ability to develop a high degree of resistance to chloroquine. A strain of P. vinckei which is resistant to the maximum tolerated dose (MTD) of chloroquine in mice has been selected from a pyrimethamine-resistant parent strain. Groups of five or more mice were treated with varying doses of drug for 3–4 days starting the day after parasite inoculation. Subpassages were made from mice treated with the highest dose of drug that allowed for some development of parasites. Resistance developed gradually during 37 successive chloroquine-treated passages over a period of about 44 weeks. In subsequent passages, the parasite was resistant to the MTD of chloroquine for mice (200 mg/kg/day). The dose of chloroquine which suppressed the parent strain by 99.9% on Day 7 was 5 mg/kg/day for 4 days. Sixty percent of mice treated with 10 mg/kg/day of chloroquine and all mice treated with doses of 20 mg/kg/day and greater were cured. Associated with resistance to chloroquine were the parasites failure to produce detectable pigment in treated mice and the appearance of multiple vacuoles. The resistant strain in treated mice is characterized by slower developing, less fatal parasitemias while in untreated mice fulminating infections often result in death. This new species of chloroquine-resistant malaria may be of value as a laboratory model for the study of acquired drug resistance.

Plasmodium berghei: Development of resistance to clindamycin and minocycline in mice

Strains of Plasmodium berghei resistant to clindamycin or minocycline were selected by a procedure in which groups of infected mice were treated with increasing doses of drug during each of a series of subpassages. Groups of five mice, each infected by intravenous inoculation with 10 million parasitized erythrocytes, were treated orally with different doses of drug for four consecutive days beginning on the day of infection. Subpassages were routinely made by Day 7, using donor mice from the group that had been treated with the highest dose of drug that allowed for some development of parasitemia during the preceding passage. Drug doses were increased in each passage as dictated by the development of parasitemia during the previous treated passage. The rate of development of resistance to clindamycin or minocycline was much slower than to conventional antimalarials such as chloroquine, quinine, or pyrimethamine. P. berghei developed total resistance to the latter compounds in nine to 12 treated passages in mice over a period of 60 to 85 days. In contrast, development of total resistance to clindamycin required 42 treated passages over a period of 300 days. Total resistance to minocycline was not attained during 86 successive minocycline-treated passages in mice over a period of 600 days, but a sixfold increase in resistance to minocycline was observed. The clindamycin-resistant strain was normally sensitive to minocycline, chloroquine, quinine, and pyrimethamine. The strain partially resistant to minocycline was normally sensitive to clindamycin, chloroquine, quinine, and pyrimethamine. Resistance to clindamycin was stable during 51 drug-free passages in mice over a period of 1 year. Resistance to minocycline was unstable. During 16 drug-free passages in mice the strain reverted towards normal sensitivity to minocycline. Strains resistant to clindamycin or minocycline showed no difference in rate of development in mice as compared to the parent strain. Likewise, only minor morphological modifications were seen in Giemsa-stained blood smears between the two resistant strains and the parent strain. These results suggest that other species of malaria may develop resistance to clindamycin or minocycline. Should resistance to one of these compounds appear, however, it should not invalidate the use of the other in the treatment of malaria.

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.

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 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.

Plasmodium berghei: Uptake of clindamycin and its metabolites by mouse erythrocytes with clindamycin-sensitive and clindamycin-resistant parasites

Abstract Tritiated Clindamycin was used to compare the uptake of Clindamycin in plasma and red cells of mice infected with clindamycin-sensitive or clindamycin-resistant Plasmodium berghei and in uninfected mice. Red cells infected with either sensitive or resistant parasites have a higher concentration of [3H]clindamycin and its active metabolites 1 hr after drug administration than uninfected red blood cells. There was no significant difference in uptake of Clindamycin by red blood cells parasitized by sensitive or resistant parasites. Levels of Clindamycin and its metabolites were consistently higher in red cells than in plasma, both in infected and uninfected mice, but the drug was readily removed by washing red cells with phosphate buffered saline in either case. It is concluded that resistance to Clindamycin is not due to an impaired uptake of the drug by the parasitized red cell as has been shown for chloroquine resistance in P. falciparum and P. berghei.

Labeling of sporozoites of Plasmodium berghei with tritiated purines.

Sporozoites were labeled in vivo. Mosquitoes (Anopheles stephensi) infected with the NK-65 strain of P. berghei were allowed to feed ad lib. for 14 days subsequent to an infective blood meal on 5% Karo syrup solution containing different levels of tritium-labled purines and pyrimidines. Sporozoites were harvested on day 16 by dissection of mosquito salivary glands. Isotope incorporation in sporozoites was determined by autoradiography. Each of the purines tested, including adenine-8-3H, adenosine-3H (G), deoxyadenosine-3H (G), and deoxyguanosine-3H (G), was incorporated into both nuclear and cytoplasmic regions of sporozoites when fed to mosquitoes at 5 to 100 ,uCi/ml. Similar levels of the pyrimidines tested, including thymine-methyl-3H, thymidine-methyl-3H, or orotic-5-3H acid, failed to label sporozoites. The incorporation of preformed purines, but not pyrimidines, by developing sporozoites suggests that the parasite may require exogenous sources of purine during its development in the mosquito but relies on de novo synthesis of pyrimidines. The adenine8-3H-labeled sporozoites were shown to be infective by intravenous inoculation into A/J mice. These results demonstrate the feasibility of labeling developing sporozoites and should provide a means for further studies on the developmental cycle of malaria in the vertebrate host. After the discovery of exoerythrocytic bodies of malaria in the liver of monkeys and man (Shortt and Garnham, 1948) it was generally assumed that relapses were caused by a continual cycling of histiotrophic merozoites in the liver. More recently, Garnham (1967) has proposed that relapses may be caused by sporozoites which remain latent in parenchymal cells of the liver for various periods of time before undergoing development. To date, these latent forms have not been demonstrated, possibly because of their small size and similarity to the cytoplasm of liver cells in stained preparations. Radionuclide labeling of sporozoites would provide a means for recognizing these latent forms in tissue if, indeed, they exist. In the present study a method for labeling sporozoites in vivo with tritiated purines was developed. In addition, the labeled sporozoites were shown to retain their infectivity when inoculated into susceptible hosts. MATERIALS AND METHODS Anopheles stephensi mosquitoes were allowed to feed on hamsters infected with the NK-65 strain of Plasmodium berghei and maintained at Received for publication 18 September 1973. * This work was presented in part at the 57th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey (1973, Fed. Proc. 32: 704). 21 C as described by Yoeli et al. (1965). Subsequent to the infective blood meal, mosquitoes were allowed to feed from the mouth of a plastic dropping pipette that contained tritium-labeled purines or pyrimidines in 5% Karo syrup solution. The mouth of the pipette was mounted in contact with the mesh on top of the cage. Fresh isotope solution was supplied every other day for a period of 16 days. Sporozoites were harvested on day 18 by dissection of mosquito salivary glands into a 1:1 mixture of saline and mouse plasma. The glands were gently homogenized in a 3-ml tissue grinder fitted with a Teflon pestle in an ice bath. Sporozoite yield was estimated from counts in a Neubauer hemocytometer. Viability of adenine-8-3H-labeled sporozoites was evaluated by the intravenous inoculation of approximately 10,000 sporozoites into 6to 8-weekold A/J mice (Jackson Laboratories, Bar Harbor, Maine) within 1 hr after dissection of salivary glands. Infectivity and prepatent period were determined by daily Giemsa-stained blood films. Isotope incorporation by sporozoites was determined by autoradiography using Kodak NTB-2 nuclear track emulsion (Eastman Kodak Co.). Infected salivary glands were crushed with a glass cover slip on slides previously coated by dipping in 1% bovine serum albumin. Sporozoites were then fixed with methanol and allowed to air-dry. Slides were coated by dipping into undiluted photographic emulsion at 40 C. After exposure to the emulsion for various intervals at 4 C, slides were processed with Kodak D-19 developer and fixer. The preparation was then stained through the emulsion with 7.5% Giemsa for 1 hr at pH 7.2. The degree of labeling was determined microscopically under oil immersion by counting silver grains associated with individual sporozoites.