Dan T. Spira

Plasmodium falciparum: Inhibition in vitro with lactoferrin, desferriferrithiocin, and desferricrocin

The microbial iron chelators desferriferrithiocin and desferricrocin as well as human lactoferrin were tested in vitro against Plasmodium falciparum. The microbial chelators inhibit the growth of P. falciparum in a dose dependent way. Parasite multiplication is stopped at 25-30 microM desferriferrithiocin, whereas 60-90 microM desferricrocin are needed to exhibit the same effect. After iron saturation, the microbial chelators are ineffective. Human lactoferrin (30 microM), both iron free and iron saturated, inhibits P. falciparum. A 3-day preincubation of host erythrocytes with iron free and iron saturated lactoferrin prior to infection enhances this effect, which is therefore attributed to lactoferrin bound iron. It has been suggested that the lactoferrin/iron complex generates oxygen free radicals, which may cause membrane damage of both erythrocyte and parasite. This process can be considered to lead to growth inhibition of the parasite.

Leishmania major: Excreted factor, calcium ions, and the survival of amastigotes

Mouse macrophages infected with amastigotes of Leishmania major contain about 40% more intracellular exchangeable calcium than control macrophages. Similar elevation of intracellular exchangeable calcium was observed in macrophages engulfing red blood cells coated with purified excreted factor from L. major. The rate of cytolysis of red blood cells coated with excreted factor was significantly lower than that of uncoated controls. Excreted factor strongly binds calcium; thus, the possible role of a microenvironment rich in calcium bound to excreted factor within the phagolysosome in protecting the amastigotes may be considered.

Plasmodium falciparum: Thiol status and growth in normal and glucose-6-phosphate dehydrogenase deficient human erythrocytes

Thiol status and growth in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Experimental Parasitology 57, 239-247. The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamide-treated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria.

Giardia lamblia: Identification of different strains from man

Four axenically cultured humanGiardia lamblia isolates from Jerusalem (KC-1, 2, 3 and 4) and one from Bethesda (WB) were compared. Three distinct groups were defined by agglutination response to rabbit anti-G. lamblia sera viz. WB; KC-3; and KC-1, 2 and 4. The same major groups were identified by isoenzyme analysis using thin-layer starch-gel electrophoresis, each group differing from the others in three or more of five enzymes studied. In addition, a single enzyme difference distinguished KC-2 from KC-1 and 4. These findings reveal significant heterogeneity inG. lamblia isolates both from widely separated areas and within a single region. Immunoassays for diagnosis of giardiasis should take into account the differences between strains. Heterogeneity amongG. lamblia strains may explain the variable clinical manifestations, host response and treatment efficacy characteristic of human giardiasis.

Plasmodium vinckei: suppression of mouse infections with desferrioxamine B

Plasmodium vinckei kills NMRI mice within 6 days after infection. Treatment of infected animals with desferrioxamine B for 5 days was found to suppress the parasitemia in a dose-dependent manner. The desferrioxamine B-iron complex (DFO/Fe3+) was ineffective, which suggests that the iron-chelating capacity of free desferrioxamine B is the antimalarial principle. All mice survived when they were given 0.3 mg desferrioxamine B/g every 12 hr for 14 days after infection. In addition, they were resistant to reinfection for at least 8 weeks. Eight months after desferrioxamine B treatment, all mice had lost their induced immunity and were as susceptible to malaria as controls. These results illustrate the dependence of the malarial parasite on ionic iron and suggests new methods for the therapy of parasitic diseases.

Cytostatic effect of DL-?-difluoromethylornithine against Plasmodium falciparum and its reversal by diamines and spermidine

DL-α-difluoromethylornithine (DFMO) inhibited ornithine decarboxylase (ODC) activity and arrested the growth of Plasmodium falciparum at the early trophozoite stage. The inhibition of ODC activity did not result in the formation of an alternative diamine such as cadaverine. When putrescine or spermidine were added to the parasites grown in culture, the arrest was reversed, and normal schizogony was completed even in the presence of DFMO. Some reversal of the inhibition was achieved with cadaverine at high concentrations, while 1,3-diaminopropane and spermine failed to restore the development. Resumption of growth could be detected when putrescine was added even after 67 h of DFMO treatment. Electron microscopy did not reveal any changes in the morphology of parasites treated for 47 h, while 73 h of treatment with DFMO induced massive accumulation of pigment. Death was observed a few hours later. These results suggest that DFMO acts as a cytostatic rather than as a cytocidal agent. The four carbon diamine restored cell growth while the shorter or the longer homologous compounds showed little activity.

Antigenic Structure of Plasmodium vinckei

An erythrocyte-free preparation of the erythrocytic stages of Plasmodium vinckei was made from infected mouse blood and disintegrated in a Hughes press. Rabbit antisera yielded a series of precipitation arcs against plasmodial extract when examined by immunoelectrophoresis. No arcs developed when uninfected mouse erythrocytes or stromata were tested against the same antiserum.

A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites.

Rat erythrocytes infected withPlasmodium berghei were disrupted by gentle passage of Coneanavalin A (ConA) agglutinated cells through a 100 mesh stainless steel grid. The free parasites were separated from cell debris, unbroken infected cells, and from uninfected rat erythrocytes on a Percoll gradient. The free parasites remained morphologically intact, metabolically active, and infective to mice.The parasites were observed by light and electron microscopy. The incorporation of3H-isoleucine and3H-hypoxanthine was compared in intact and infected cells, and the infectivity was measured by the injection of parasites into susceptible mice. It seems that the combination of the two techniques used, produces a high yield of intact free parasites suitable for physiological or immunological studies.

Growth of Leishmania amastigotes in macrophages from normal and immune mice

SummaryAn in vitro system of prolonged culture of Leishmania tropica amastigotes in mouse macrophages is presented. The division rate of parasites was monitored by microscopic observations and by 3H-thymidine incorporation. The dynamics of macrophage infection and parasite division are influenced by the initial rate of promastigotes per cells in culture. Parasites multiply, gradually infect and finally destroy all available macrophages from normal mice releasing large numbers of viable amastigotes. Macrophages from immune donors were inferior in their ability to support parasite multiplication and did not survive long periods in culture.

An outbreak of Toxoplasmosis amongst squirrel monkeys in an Israeli monkey colony

An outbreak of Toxoplasmosis in a colony of squirrel monkeys (Saimiri sciureus) in Israel is described. Serological, pathological, and molecular findings of monkeys, as well as rodents and pigeons from the vicinity are summarized. Seventy-nine percent (19/24) of monkeys were T. gondii seropositive at titer 1:16 whilst 4% (1/24) were also seropositive at titer 1:64 using the Modified Agglutination Test (MAT). Eighty four percent (21/25) of rats were positive at titer 1:16 and 8% (2/25) of rats were positive at titer 1:32. DNA amplification of a 529bp repeated sequence of T. gondii was detected in the liver and lungs of all monkeys tested, 6/7 in myocardial extractions and 5/6 in brain extractions. Sequence analysis of the SAG2 locus disclosed that T. gondii detected was of Type III genotype. The source of disease was thought to be contamination of feed with infective feline oocysts. As a result of this study, the implementation of a program to capture and remove resident feral cats, to discontinue the feeding of stray cats, and to control rodent populations in the park was introduced.