Plasmodium berghei: oxidant defense system

Glutathione oxidant defense system protects the erythrocyte from oxidative damage. This defense system was studied in mouse erythrocytes infected with Plasmodium berghei and in isolated parasites. The efficiency of this system was found to be increased in parasitized erythrocytes compared to the normal erythrocytes. The increase in the components of the oxidant defense system in the parasitized cells could result from parasitic addition to these components. This defense system present in the parasite may protect the parasite from oxidative damage and help the parasite in its growth and development.     

Eimeria tenella: stimulation of DNA synthesis in infected cultured animal cells.

An autoradiographic study was conducted to determine the influence of the intracellular coccidian parasite of chickens, Eimeria tenella, on the incorporation of tritium-labeled thymidine in kidney cell cultures. Evidence was obtained for a parasite-induced increase in thymidine incorporation in host cell cultures which is too great to be attributed to unscheduled DNA synthesis. The stimulatory effect became significant (P < 0.05) at 24 hr after inoculation and further increased at 48 and 72 hr postinoculation. Both parasitized and unparasitized cells in the infected cultures showed similar increases in thymidine incorporation. Furthermore, the increased incorporation of thymidine in the infected cultures was found to be independent of both the percentage of parasitized cells and development of the parasite.     

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.     

Purine Uptake and Utilization by the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae cannot carry out extensive de novo purine biosynthesis, and depends upon the host erythrocyte for a supply of preformed purines. Exogenous purines taken up by the parasitized erythrocyte may constitute a major source of preformed purines for parasite nucleotide biosynthesis. The uptake of exogenous radioactive purine compounds and their incorporation into nucleic acids by duck erythrocytes parasitized with P. lophurae, uninfected erythrocytes, and erythrocyte-free parasites were studied. P. lophurae was found to have a remarkable ability, both intracellularly and extracellularly, to take up and utilize certain exogenous purines such as adenosine, inosine, and hypoxanthine. Incorporation studies indicated that this species has a functional purine salvage pathway by which inosine, hypoxanthine, and adenosine can be converted to both adenine and guanine nucleotides.     

Relationship between partial inhibition of glycolysis and hemolysis after induction of gametocytogenesis in synchronous cultures of Plasmodium falciparum

In the present study, the low molecular-weight fraction of the culture supernatant of anti-Plasmodium falciparum antibody-producing hybridoma cells (HybSL) was used in synchronous culture with P. falciparum FVO strain. When synchronous cultures were treated with HybSL solution on day 5, gametocytogenesis was also induced. Gametocytes were consistently found from the third day after treatment and reached a peak on the fourth day. An increase in pH and hemoglobin concentrations and decrease in lactate concentrations were observed on the first day after treatment. These phenomena suggested that HybSL solution partially inhibited glycolysis of erythrocytes parasitized with schizonts and resulted in hemolysis of infected erythrocytes. On the other hand, the production of gametocytes did not increase in cultures treated with HybSL solution on day 4 of synchronous cultures in which ring forms were plentiful. Most ring forms were not killed by HybSL solution and quickly developed to trophozoites and schizonts rather than gametocytes. Consequently, it is assumed that ring forms on day 4 of synchronous cultures have finished differentiation into the asexual stage. The conversion of asexual parasites to gametocytes may be triggered only when late-stage trophozoites or early-stage schizonts are treated with HybSL solution.     

In vitro culture of erythrocytic stages, including gametocytes of Plasmodium berghei (NK 65 strain) using candle jar method and a newly designed continuous medium flow apparatus

Three methods have been described for cultivation of erythrocytic stages, specially gametocytes of P. berghei NK65 strain, (1) by using vial candle jar where the cultures were subcultured by addition of fresh erythrocytes, (2) a newly designed simple and compact continuous medium flow apparatus, where medium was continuously perfused but fresh erythrocytes were not added and (3) where the subcultures were also done using simple and compact continuous medium flow apparatus for comparison. The maximum percentage of parasitized erythrocytes obtained by these methods was 24.3, 27.1 and 26.4% respectively. Parasites in vials could survive for more than 10 days with 3 to 4 subcultures with maximum 1.96% gametocytaemia. However, the gametocytaemia in continuous medium flow apparatus, where subcultures were not made reached 2.2% compared to that of 2.7% in this apparatus where subcultures were done. The asexual as well as sexual stages of this parasite survived for about 16-18 days in compact continuous medium flow apparatus, where at least 7 subcultures were done.     

Flow cytometry to evaluate Theileria sergenti parasitemia using the fluorescent nucleic acid stain, SYTO16

BACKGROUND: Although the infection of Theileria sergenti is demonstrated by intraerythrocytic localization of this parasite, much time and labor are necessary in order to determine this. We applied flow cytometry to evaluate T. sergenti parasitemia using the fluorescent nucleic acid stain method. METHODS: Peripheral blood samples from cattle infected with T. sergenti were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16, and hydroethidine (HE). Stained parasitized erythrocytes were measured by a flow cytometer equipped with a single argon laser operating at 488 nm. RESULTS: SYTO16-stained intraerythrocytic parasites were detected on the FL1 (525 nm) and parasitized cells were separated completely from unparasitized cells. However, HE-stained erythrocytes could not be divided clearly into parasitized and unparasitized cells. SYTO16-stained parasites were reproducibly detected at a percentage above 0.1%. Contaminating leukocytes, which were indicated by CD18-positive cells, were eliminated from the analysis by narrowing the light scatter gate of the erythrocyte fraction. A correlation (r = 0.983) between the percentage of SYTO16-positive cells and parasitemia in grazing cattle was observed. CONCLUSIONS: Flow cytometric detection using SYTO16 is a rapid and reliable method of monitoring parasitemia in T. sergenti-infected cattle.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Consequences of Interspecific Competition on the Virulence and Genetic Composition of a Nucleopolyhedrovirus in Spodoptera frugiperda Larvae Parasitized by Chelonus insularis

Nucleopolyhedroviruses ( Baculoviridae ) are virulent insect pathogens that generally show a high degree of host specificity and have recognized potential as biological insecticides. Whenever viruses are applied for pest control, a proportion of the infected insects will also be parasitized by hymenopteran or dipteran parasitoids and interspecific competition for host resources will occur; the severity of such competition is likely to be modulated to a large degree by the virulence of each type of parasite. We examined the impact of parasitism by the solitary egg-larval endoparasitoid Chelonus insularis (Hymenoptera: Braconidae) on the speed of kill of nucleopolyhedrovirus-infected Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae and the pattern of host growth and virus production in infected and/or parasitized hosts. We also examined the effect of parasitism on the virulence, infectivity and genetic composition of serially passaged virus. Both parasitism and viral infection resulted in a marked reduction in host growth. When third instar larvae were dually parasitized and virus-infected, the growth rate was even more severely affected compared to parasitized larvae. There was a significant increase in virus production in larvae infected at later instars. Interspecific competition resulted in a substantial decrease in pathogen production in parasitized larvae infected at the fourth instar, but not in parasitized larvae infected at earlier instars. The serial passage experiment resulted in the appearance of four distinct genetic isolates of the virus detected by restriction endonuclease analysis. Of the three isolates that appeared in nonparasitized larvae, two showed increased virulence, expressed by mean time to death, and for one of these the infectivity, expressed as LC 50 , was reduced. One isolate that appeared in parasitized larvae (isolate D) had increased virulence and infectivity. Southern blot analysis indicated that virus isolate D was most likely generated by point mutation of a restriction site or by alterations such as duplications, deletions or by recombination of two or more genotypic variants present in the wild-type nucleopolyhedrovirus isolate. Our study provides clear evidence of interspecific competition within the host, since, depending on the timing of inoculation, adverse effects were observed upon both the parasitoid and the virus.     

Human malaria parasite adenosine deaminase. Characterization in host enzyme-deficient erythrocyte culture

Human malaria infected erythrocytes show a dramatic increase in adenosine deaminase activity in vitro. Using recently developed culture techniques, adenosine deaminase-deficient human erythrocytes were infected in vitro with the major human pathogen Plasmodium falciparum. Adenosine deaminase activity was undetectable in the uninfected host red cells, but increased by 2-fold over normal levels in these cells with an 8% parasitemia. The enzyme in these cells appeared unique in that its activity was markedly elevated over that of other parasite purine enzymes, was not cross-reactive with antibody against human erythrocyte adenosine deaminase, and though inhibited competitively by deoxycoformycin was relatively insensitive to erythro-9-(2-hydroxy-3-nonyl) adenine. The use of adenosine deaminase-deficient erythrocytes for the in vitro cultivation of Plasmodium provides a unique system for the study of parasite enzyme and allows further insight into the purine metabolism of the intraerythrocytic malaria parasite.     

Lipid peroxidation in Plasmodium falciparum-parasitized human erythrocytes.

cis-Parinaric acid (PnA) was used as a fluorescent probe to study lipid peroxidation in nonparasitized and Plasmodium falciparum-parasitized erythrocytes, upon challenge by cumene hydroperoxide and tert-butyl hydroperoxide. Parasitized erythrocytes were less susceptible toward lipid peroxidation than nonparasitized erythrocytes with which they had been cultured. Furthermore, nonparasitized erythrocytes cultured together with parasitized cells, and thereafter isolated on a Percoll gradient, were less susceptible toward lipid peroxidation than erythrocytes kept under the same experimental conditions but in the absence of parasitized cells. We concluded, therefore, that the intracellular development of the parasite leads to an increase in the resistance against oxidative stress, not only of the host cell membrane of the parasitized erythrocyte, but also in the plasma membrane of the neighboring cells. The erythrocyte cytosol of parasitized cells and/or the intraerythrocytic parasite was required for the increased protection of the host cell membrane, since ghosts prepared from parasitized erythrocytes were more susceptible to lipid peroxidation than those prepared from nonparasitized ones. Vitamin E content of parasitized erythrocytes was lower than that of nonparasitized cells. However, parasitized erythrocytes promoted extracellular reduction of ferricyanide at higher rates, which might be indicative of a larger cytosolic reductive capacity. It is suggested that the improved response of intact erythrocytes is due to an increased reduction potential of the host-erythrocyte cytosol. The role of vitamin C as a mediator of this process is discussed.     

Host cell apoptosis impairs Cryptosporidium parvum development in vitro

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.     

Culture of Plasmodium falciparum: the role of pH, glucose, and lactate

Yields of P. falciparum in intraerythrocytic in vitro cultures were maximized when extracellular pH was maintained between 7.2 and 7.45, and extracellular lactate was kept below 12 mM. Host erythrocytes metabolized 4.6 +/- 1.5 microM glucose/10(9) RBC/24 hr and produced 7.9 +/- 1.8 microM lactate/10(9) RBC/24 hr. Asynchronous parasite cultures used 122 +/- 34 microM glucose/10(9) parasitized RBC/24 hr and produced 143 +/- 47 microM lactate/10(9) parasitized RBC/24 hr. Synchronous cultures that were 80 to 100% ring forms after 24 hr in culture exhibited significantly lower glycolysis per 10(9) parasitized RBC than cultures that were 0 to 25% ring forms after 24 hr. The percent of glucose utilization accounted for by lactate production by parasites was significantly less than that of uninfected erythrocytes. These optimum ranges and metabolic rates can be used in the development of parasite culture techniques.     

Ultrastructure of erythrocytes parasitized by Haemobartonella felis.

Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.     

NMR studies of malaria 31P nuclear magnetic resonance of blood from mice infected with Plasmodium berghei

High resolution ³¹P-NMR has been used for the non-invasive observation of metabolites and metabolic rates in blood of normal mice and of mice infected with Plasmodium berghei, the causative agent of malaria. ³¹P-NMR was used to quantitate levels of 2,3-diphosphoglycerate in whole cells as a function of the degree of parasitemia and yielded good agreement with the results of enzymatic assays. The time-dependence of ³¹P metabolites was monitored in both normal and infected erythrocytes, greater rates of decay of 2,3-diphosphoglycerate being observed in malarial blood which correlate with the level of parasitemia. Very high metabolic rates of infected cells render measurement of intracellular pH unreliable on freshly drawn whole blood. When appropriate measures are taken to avoid this complication, no difference is observed in the intracellular pH of parasitized and non-parasitized erythrocytes from infected animals. In both normal and parasitized mice the intraerythrocytic pH is more acidic than that of the suspending medium by 0.15 pH unit at 25°C. Unlike free-living protozoa, the parasitic protozoan Plasmodium does not contain detectable levels of phosphonates or polyphosphates, in either whole cells or perchloric acid extracts thereof.     

Changes in fatty acids,amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grownIsochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.     

Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.     

NMR studies of malaria P nuclear magnetic resonance of blood from mice infected with Plasmodium berghei

High resolution ³¹P-NMR has been used for the non-invasive observation of metabolites and metabolic rates in blood of normal mice and of mice infected with Plasmodium berghei, the causative agent of malaria. ³¹P-NMR was used to quantitate levels of 2,3-diphosphoglycerate in whole cells as a function of the degree of parasitemia and yielded good agreement with the results of enzymatic assays. The time-dependence of ³¹P metabolites was monitored in both normal and infected erythrocytes, greater rates of decay of 2,3-diphosphoglycerate being observed in malarial blood which correlate with the level of parasitemia. Very high metabolic rates of infected cells render measurement of intracellular pH unreliable on freshly drawn whole blood. When appropriate measures are taken to avoid this complication, no difference is observed in the intracellular pH of parasitized and non-parasitized erythrocytes from infected animals. In both normal and parasitized mice the intraerythrocytic pH is more acidic than that of the suspending medium by 0.15 pH unit at 25°C. Unlike free-living protozoa, the parasitic protozoan Plasmodium does not contain detectable levels of phosphonates or polyphosphates, in either whole cells or perchloric acid extracts thereof.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.