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.

Immunologic response to infection with Giardia lamblia in children: effect of different clinical settings.

Infection with Giardia lamblia varies in both its severity and duration. A high incidence of giardiasis in immunoglobulin-deficient individuals suggests a role for the humoral immune response in resistance to Giardia infection. Levels of specific anti-Giardia antibodies were determined in three populations of children infected with the parasite: in children attending a day-care centre in which strict hygiene measures were practised and in whom all Giardia infections were asymptomatic; in a rural population residing under poor hygienic conditions in close proximity to farm animals in which children with Giardia-associated diarrhoeal episodes were studied; and in Bedouin infants followed from birth and in whom a previous study has shown that Giardia infection is almost universal by the age of 2 years. In day-care children, infection was accompanied by a significant increase in anti-Giardia IgM levels, compatible with an initial exposure to the parasite. In populations in which exposure to the parasite occurs at an early age and the prevalence of infestation is high, the pattern of specific antibodies to the parasite is rather uniform and cannot differentiate between current infection and previous exposure. Thus, other immune parameters such as salivary or urinary secretory IgA, which reflect the intestinal IgA response, should be studied in order to delineate further the humoral immune response to Giardia.

Salivary IgA antibodies to Giardia lamblia in day care center children.

An enzyme-linked immunosorbent assay was developed for the detection of specific salivary IgA antibodies to Giardia lamblia. Among 73 infants and children in a day care center 9 asymptomatic subjects had stools positive for G. lamblia. Salivary antigiardia IgA concentrations, expressed as OD units, were higher in the 2- to 4-year-old group: 0.899 +/- 0.03 vs. 0.660 +/- 0.03 in the < 2-year old group (P < 0.001). In both groups values were higher in the infected children (1.099 +/- 0.04 vs. 0.629 +/- 0.09 in the < 2-year-old group and 1.053 +/- 0.07 vs. 0.859 +/- 0.03 in the 2- to 4-year-old group). In children infected throughout the study period, salivary antigiardia antibodies remained consistently high and in 2 children whose stools were initially negative a significant rise in OD value was observed after stools tested positive. Total salivary IgA did not differ between the two age groups and did not correlate with specific salivary antigiardia antibodies in individual subjects. The enzyme-linked immunosorbent assay for detection of specific salivary antibodies to G. lamblia can be used in the study of the mucosal immune response to the parasite, and may serve as an screening tool in monitoring the exposure of various populations to G. lamblia.

The antimalarial effect of iron chelators: studies in animal models and in humans with mild falciparum malaria.

In this study we explore the antimalarial effects of 3-hydroxypyridin-4-ones (CP compounds), a family of bidentate orally effective iron chelators in experimental animal systems in vivo and in vitro, and examine whether the iron chelator deferoxamine (DF) is active against human infection with P. falciparum. There was direct relation between lipid solubility of the CP compounds, which would facilitate membrane transit, and their in vivo antimalarial action, suggesting direct intracellular iron chelation as the most likely explantation for the antimalarial effect of iron chelators. Results of the double-blind, placebo controlled trial of DF in humans with asymptomatic parasitemia provided unequivocal evidence that this iron-chelating agent has antimalarial activity. Depriving the parasite of a metabolically important source of iron may represent a novel approach to antimalarial drug development. DF is a relatively ineffective intraerythrocytic chelator, and our data indicate that other orally effective iron chelators may have superior antimalarial activity in vivo. A systematic screening of available iron chelating drugs may result in the identification of potentially useful antimalarial compounds.

The effect of N-alkyl modification on the antimalarial activity of 3-hydroxypyridin-4-one oral iron chelators.

The antimalaria effect of iron chelators is attributed to their interaction with a labile iron pool within parasitised erythrocytes, and it was postulated that increased affinity to iron as well as increased lipophilicity may improve antimalarial activity. In the present study we have examined the antimalarial effect of 3-hydroxypyridin-4-ones, a family of bidentate orally effective iron chelators whose lipophilicity may be modified by altering the length of the R2 substituent on the ring nitrogen. A significant dose-related suppression of Plasmodium falciparum cultures was observed with all drugs tested in vitro at concentrations of 5 mumol/L or higher. In contrast, there was a clear segregation of the in vivo effect on P berghei in rats (300 mg/kg/d subcutaneous) into two categories: compounds CP20, 38, and 40 failed to suppress malaria, whereas CP51, 94, and 96 had a strong antimalarial effect, similar or better than deferoxamine. There was a close linear correlation between the suppression of peak parasite counts and the reduction in hepatic nonheme iron induced by the various drugs tested (r = .9837). The most lipophilic compounds were also the most effective in suppressing malaria and in depleting hepatic iron stores. These data indicate that 3-hydroxypyrydin-4-ones are able to suppress malaria in vivo and in vitro. Because lipid solubility is an important determinant of antimalarial action, our study provides useful information regarding the selection of orally effective iron-chelating compounds that may be suitable for clinical application as antimalarial agents.

Fe (II)-chelates based on redox-active pyridoxal-betaines as C-centered radicals causing single- and double-strand scissions to DNA.

The ability of 1-[N-Ethoxycarbonylmethylpridoxylidenium]-2-[2'- pyridyl]hydrazine bromide code name - [L2.9 = L+,X-]-FE(II) chelate [L2-9-Fe(II)] to induce breaks both in the 43kb linear double-strand lambda phage DNA, and in the 4363 base pair supercoiled pBR322 plasmid DNA is herein described. Neither the free ligand nor FE(II) alone demonstrated any effect on the DNA. The cleaving ability is shown to occur instantaneously under strictly anaerobic conditions, either in the presence or absence of the enzyme catalase. It is also shown to be dose dependent. Thus, at lambda DNA:L2-9-Fe(II) molar ratio of 3.7:1.0, the linear DNA is randomly cleaved into fragments ranging from 23.1kb to 4.3kb, whereas at approximately 1:1 molar ratio, the range extends down to 2.5kb fragments. By contrast, at 1:2.7 [plasmid DNA]: chelate-Fe(II) molar ratio, a single-strand nick was observed, and a double strand break was noted at a 1:50 ratio [( plasmid DNA]: chelate-Fe(II). A multi-stage redox cycling involving a carbon-centered (L,X-)-Fe(III) radical capable of transferring an electron to the DNA to form high unstable [DNA].- anion-radical is invoked to explain the degradation of the chain macromolecule. Possible modes for regeneration of the chelate-Fe(III) radical both at the cell-free and at the cell levels are proposed.

Growth inhibition of Plasmodium falciparum involving carbon centered iron-chelate radical (L., X-)-Fe(III) based on pyridoxal-betaine. A novel type of antimalarials active against chloroquine-resistant parasites.

Malaria parasites have been shown to be more susceptible to oxidative stress than their host erythrocytes. In the present work, a chloroquine resistant malaria parasite, Plasmodium falciparum (FCR-3) was found to be susceptible in vitro to a pyridoxal based iron chelator--(1-[N-ethoxycarbonylmethylpyridoxlidenium]-2-[2'-pyridyl ] hydrazine bromide--(code named L2-9). 2h exposure to 20 microM L2-9 was sufficient to irreversibly inhibit parasite growth. Desferrioxamine blocked the drug effect, indicating the requirement for iron. Oxygen however, was not essential. Spectrophotometric analysis showed that under anoxic conditions, L2-9-Fe(II) chelate undergoes an intramolecular redox reaction which presumably involves a one electron transfer and is expected to result in the formation of free radical. Spin trapping coupled to electron spin resonance (ESR) studies of L2-9-iron chelate showed that L2-9-Fe(II) produced free radicals both in the presence and absence of cells, while L2-9-Fe(III) produced free radicals only in the presence of actively metabolising cells.

Plasmodium falciparum: properties of an alpha-like DNA polymerase, the key enzyme in DNA synthesis.

An alpha-like DNA polymerase has been identified and characterized in the extracts from the malarial parasite Plasmodium falciparum. The enzyme is sensitive to the specific inhibitors of alpha-DNA polymerase, N-ethylmaleimide and aphidicolin, and is cell-cycle specific. High activity has been found in the schizont, is lower in trophozoites, and has only negligible activity in the ring form. The enzyme has a molecular weight of about Mr 100,000-103,000 estimated by detecting activity in SDS-polyacrylamide electrophoresis and by Bio-Gel filtration. Another active band of a molecular Mr 68,000 was detected by SDS electrophoresis when the enzyme was stored for 2 months at -20 degrees C. The catalytic activity of parasite enzyme was optimal between pH 8 and pH 9. The apparent Michaelis constant for dTTP was 4.3 microM.

Inhibition of Plasmodium falciparum growth by a synthetic iron chelator.

The susceptibility of the chloroquine-resistant malaria parasite Plasmodium falciparum (FCR-3) to a pyridoxal-based iron chelator was tested. 10 microM of the chelator 1[N-ethoxycarbonylmethyl-pyridoxy-lidenium]-2-[2'-pyri dyl] hydrazine bromide (code name L2-9) effectively inhibited growth in vitro of the parasites. Presaturation of the chelator with either ferric or ferrous iron partially blocked the inhibitory effect. Two hours' exposure of parasites to 20 microM L2-9 was sufficient to inhibit their growth irreversibly. Desferrioxamine blocked the inhibitory effect of L2-9. It is suggested that the chelator may be acting by generating free radicals in complexing intracellular iron.

Routine in vitro cultivation of Giardia lamblia by using the string test.

In vitro axenic cultures of Giardia lamblia were successfully established in 14 successive subjects by a method employing the string test (Entero-Test) for obtaining samples of fluid from the proximal small intestine. This method is applicable to isolating G. lamblia from unselected subjects, including asymptomatic carriers, children, and individuals in diverse or remote geographic regions.

Antimalarial effect of HBED and other phenolic and catecholic iron chelators.

Previous studies showed that deferoxamine inhibits malaria by interacting with a labile iron pool within parasitized erythrocytes. Consequently, we studied the antimalarial properties of other iron-chelating drugs such as 2,3-dihydroxybenzoic acid (2,3-DHB) and its methyl ester as well as two polyanionic amines, N.N'-bis (o-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED) and N,N'-ethylenebis(o-hydroxyphenylglycine) (EHPG) in rats infected with Plasmodium berghei. All drugs were delivered by subcutaneous injection at 8-hour intervals, 40 mg per animal per day. All animals receiving N,N'-ethylenebis(o-hydroxyphenylglycine) died of drug toxicity between days 6 and 11. Peak parasitemia on day 11 of infection was 32.8% in control animals; 25.3% with methyl 2,3-DHB, 15.5% with 2,3-DHB, 8.0% with deferoxamine, and 0.9% with HBED. Subsequent studies of HBED and deferoxamine in P falciparum cultures in human erythrocytes showed a marked suppression of parasite counts by both drugs at concentrations of greater than 5 mumol/L. At all concentrations tested, HBED was four to five times more effective than deferoxamine in suppressing parasite counts. The superior antimalarial activity of HBED is attributed to its increased lipophilicity and higher affinity to ferric iron. These findings indicate that selective iron deprivation by interaction with an intracellular chelator may represent a novel approach to antimalarial drug development, and that systematic screening of available iron-chelating drugs may result in identification of potentially useful antimalarial compounds.

Inhibition of the intraerythrocytic development of Plasmodium falciparum in glucose-6-phosphate dehydrogenase deficient erythrocytes is enhanced by oxidants and by crisis form factor.

In this study, we have examined P. falciparum development in untreated normal or G6PD deficient erythrocytes as well as in parasitized cells exposed to isouramil--a fava bean extract known to induce oxidant stress in G6PD deficient erythrocytes (a condition known as favism), to diamide--a thiol oxidizing agent and to crisis form factor (CFF)--a non-immunoglobulin product found in the sera of man immune to malaria. We observed a significant retardation in intraerythrocytic development of parasites cultured in the G6PD deficient cells in comparison with parasites grown in the normal ones. Plasmodia within the G6PD deficient erythrocytes were markedly more sensitive to the inhibitory activity of CFF or the oxidizing agents diamide and isouramil. Mature stages of the parasites in both normal and G6PD deficient erythrocytes were more vulnerable than young ring forms, to oxidizing agents. The overall results show that the genetic trait of the host cell and the degree of maturation of the plasmodia are both crucial factors in determining the sensitivity of the parasite to oxidant stress.

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis.

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.

Cryptosporidium, malnutrition, and chronic diarrhea in children.

Cryptosporidium was found in the stools of 13.5% of 221 children hospitalized with diarrhea. It was the single most prevalent pathogen isolated. Children with Cryptosporidium-positive stools were significantly more malnourished than children in whom Cryptosporidium was not detected. Children with more severe malnutrition (ie, less than 50% of their expected weight) and with Cryptosporidium in their stools had a significantly longer duration of diarrhea than similarly malnourished children without Cryptosporidium (63 vs 32 days, respectively). In 77 better-nourished outpatients with diarrhea, Cryptosporidium was found in only 5.2% of cases and was associated with less-severe illness. Our findings are consistent with the hypothesis that in less-developed areas, Cryptosporidium is a major pathogen, not only in acute but also in chronic childhood diarrhea, and may play an important role in the interaction between diarrhea and malnutrition.

The feasibility of a Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the diagnosis of Plasmodium falciparum antigens and antibodies.

An enzyme-linked immunosorbent assay for rapid detection of malarial antibodies and antigens was developed. Plasmodium falciparum antigen preparation, obtained from sonicated cultures of the parasite at a parasitemia of 10%-15%, was applied to cellulose filter discs in volumes of 0.1 microliter in 96-well microtiter plates. Antibodies were detected by successive incubations with: bovine serum albumin for blocking, tested serum at different dilutions, peroxidase-conjugated antihuman IgG, and the precipitable substrate 4-chloro-1-naphtol. Positive reactions appeared as blue dots on a white background which are easily read by eye. Pools of sera from patients with recent disease or from individuals with a history of malaria, contained antibodies detectable up to a dilution of 1:64,000. Negative results were obtained when normal RBC were used for dotting the filters. Normal sera showed no reaction at any antigen concentration. P. falciparum antigens were detected by their ability to inhibit the binding of antibody to the filters. RBC infected with P. falciparum in vitro can be detected at a level of 0.001% parasitemia. This report presents the feasibility of an assay for detecting malarial antibodies and antigens in blood samples which is easily applicable to field conditions.

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.

Effect of polyamine depletion on macromolecular synthesis of the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes.

DL-alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented the increases in putrescine and spermidine, but not in spermine, in human erythrocytes infected with the malarial parasite Plasmodium falciparum. The addition of putrescine to these polyamine-depleted cultures restored the normal concentrations of spermidine, whereas that of putrescine even exceeded that of the control cultures. DFMO also inhibited the incorporation of radioactive amino acids into the proteins of parasitized erythrocytes. Electrophoresis on polyacrylamide gels revealed that the synthesis of some proteins was completely blocked by DFMO, but the synthesis of others was not affected. DFMO also caused a partial inhibition of RNA synthesis, and DNA synthesis was completely blocked in polyamine-depleted parasitized erythrocytes. It has been suggested that putrescine and/or spermidine are required for the synthesis of certain proteins in parasitized erythrocytes and that at least one of those proteins is related to the synthesis of DNA of the malarial parasite. It appears that polyamines regulate the schizogony process of P. falciparum.

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

DL-alpha-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.

Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis.

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.