Leishmania tropica: Biochemical aspects of promastigotes' attachment to macrophages in vitro

The in vitro attachment of promastigotes of Leishmania tropica to mouse peritoneal macrophages was studied under experimental conditions. Parasite binding by macrophages required an intact intracellular physiology as suggested by the action of several metabolic inhibitors. Parasite attachment was inhibited in the presence of azide, fluoride, iodoacetamide, and 2-deoxyglucose, but was affected very little by inhibitors of oxidative phosphorylation, by 2,4,-dinitrophenol and by cyanide. Parasite attachment was not prevented by cycloheximide, an inhibitor of protein synthesis. Successful parasite attachment required the simultaneous presence of magnesium, glucose, and a macromolecular component of fetal calf serum in the extracellular medium. Furthermore, glucose and serum supported parasite binding synergistically. The requirement for extracellular glucose could be replaced by mannose, suggesting that such a requirement is structural rather than metabolic. The active fraction of serum was nondialyzable, heat labile, and precipitable by ammonium sulfate. These various chemical ingredients of the culture medium were required mainly during the interaction of parasites with macrophages. The extracellular and metabolic requirements of successful parasite binding suggest that attachment of parasites to macrophages possess the characteristics of a biochemical reaction which is probably mediated by one or more enzymatic reactions.     

Eimeria meleagrimitis, E. adenoeides, and E. dispersa: Severity of infection and changes in the intestinal mucosa of the Turkey

Glucose and methionine were malabsorbed in some intestinal regions of turkeys infected with Eimeria meleagrimitis, E. adenoeides, or E. dispersa. The decrease in absorption was not always related to the numbers of parasites in the cells or the extent of damage to the mucosa. With E. adenoeides, malabsorption was found in the jejunum even though parasites were not present. Conversely, with E. dispersa, no malabsorption was observed in the duodenum even though light microscopy showed numerous parasites. In many intestinal regions, damage to the mucosal surface visible with scanning electron microscopy (SEM) was slight or absent, although malabsorption was marked. No changes were noted with SEM in the structure and orientation of the brush border in these regions. Villar height was significantly reduced in the regions of heaviest infection when intestinal damage was visible. Conversely, the crypts of Lieberkühn were often two or three times as deep in infected poults as in uninfected poults. In general, no differences were found in the thickness of the circular and longitudinal muscle layers between the infected and uninfected poults. The dry weight of the intestinal tissue was less from infected poults than from uninoculated controls and was related to both region of the intestine and severity of the infection.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Herpetomonas samuelpessoai: Changes in cell shape and induction of differentiation by local anesthetic

Lidocaine, a local anesthetic, induces changes in morphology and motility of Herpetomonas samuelpessoai when incubated under nonproliferative conditions. The cells become rounded and immotile. These effects are dependent on time and temperature of incubation, concentration of lidocaine, and pH. Divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, and Sr²⁺) reversed the effect of lidocaine. Lidocaine also induces the formation of membrane-bound cytoplasmic vacuoles as determined by morphometry applied to electron micrographs. Lidocaine had a dose-dependent effect on the growth of H. samuelpessoai in a chemically defined medium. At concentrations which did not interfere with cell growth, it induces the transformation of promastigote into opisthomastigote via paramastigote. It is suggested that lidocaine may be used as an inductor of differentiation in H. samuelpessoai opening the possibility of obtaining the three developmental stages of this trypanosomatid.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Trypanosoma vivax: Absence of host protein on the surface coat

The possible presence of host serum proteins on the surface of Trypanosoma vivax stock Zaria Y486 was studied. Intact washed bloodstream forms from mice were not lysed or neutralized by antisera against mouse serum proteins. Serum against T. vivax prepared in rabbits against an antigen which was a water-soluble trypanosome extract, failed to cross-react with mouse serum when tested by immunoelectrophoresis and immunodiffusion. The T. vivax antigen failed to cross-react with three different anti-mouse sera when tested by the same techniques.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of ¹²⁵I-surface-labeled parasites showed the presence of a cluster of proteins ranging in molecular weights between 57,000 and 45,000 daltons. None of these proteins was precipitated by anti-mouse serum protein sera. The serum against T. vivax precipitated a protein of 50,000 daltons molecular weight.     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Plasmodium falciparum: Attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10⁶ parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.     

Trypanosoma congolense: Natural and acquired resistance in the bovine

A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.     

Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.     

Plasmodium berghei: The in Vitro immune response

Following primary in vitro Stimulation by Plasmodium berghei, IgM titers by the indirect fluorescent antibody test (IFAT) were negative in in vitro reconstituted syngeneic mouse spleen cultures containing T cells and macrophages, or B cells and macrophages, or macrophages alone, but IgM titers of 1:20 were obtained from cultures containing B cells, T cells, and macrophages. IFAT IgG titers were negative for cultures with T cells and macrophages together, or macrophages alone, but rose to 1:40 with cultures containing B cells and macrophages and 1,80 with cultures of B cells, T cells, and macrophages together. After a second in vitro challenge, IgM and IgG titers were similar to the stimulated cultures from immunized mice; the IgM titer reached only 1:20 but the IgG titer rose to 1:160. Total immunoglobulin was higher in the cultures from immunized mice than from in vitro primed cultures.     

Tritrichomonas foetus: Localization of filipin-sterol complexes in cell membranes

The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the freeze-fractured plasma membrane and the flagellar membranes of the pathogenic protozoa, Tritrichomonas foetus. A homogeneous distribution of filipin-sterol complexes was seen throughout the plasma membrane, and the membrane of the three anterior and the one recurrent flagella. No or very few filipin-sterol complexes were observed in some specialized regions such as the base of the flagella (necklace), the portion of the recurrent flagellum, and that part of the cell body to which the flagellum was attached. The density of filipin-sterol complexes varied from one cell to the other. In some cells, about 205 complexes/μm² were seen. A larger number of filipin-sterol complexes were observed on both faces of the membrane of cytoplasmic structures, probably corresponding to vacuoles. No complexes were seen in the nuclear membrane and in the membrane of the endoplasmic reticulum. Very few or no complexes were observed in the membrane of the hydrogenosomes. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Trypanosoma musculi: Passive hemagglutination technique to measure antibody in mice

A passive hemagglutination assay was developed to measure Trypanosoma musculi-specific antibody in mice. Indicator-erythrocyte donor mice received 550 rad ⁶⁰Co 24 hr before intraperitoneal injection of 3 × 10⁴T. musculi. T. musculi antigen-coated erythrocytes were obtained from these mice on Day 9 postinfection. T. musculi antigen-coated erythrocytes obtained in this manner were used as indicator erythrocytes in a passive hemagglutination procedure. Serum from hyperimmunized mice (three consecutive infections at 21-day intervals) gave titers as high as 1:1024. Titers of 1:256 and 1:512 were obtained from singly infected mice on Days 18 and 28 postinfection, respectively. In marked contrast, nude mice infected with T. musculi did not produce a detectable agglutinating antibody response. Erythrocytes obtained from either irradiated (550 rad ⁶⁰Co) uninfected mice, nonirradiated infected mice, or normal mice did not agglutinate when combined with any of the sera tested. These data suggest the usefulness of this passive hemagglutination assay for the measurement of antibody to T. musculi in the serum of infected mice.     

Entamoeba histolytica and Giardia lamblia: effects of cysteine and oxygen tension on trophozoite attachment to glass and survival in culture media

Attachment of Entamoeba histolytica and of Giardia lamblia trophozoites to glass was monitored during the culture cycle. Attachment of each parasite was greatest during the exponential phase of axenic growth. The effects of l-cysteine upon the kinetics of attachment of trophozoites to glass were determined quantitatively. Attachment in complex growth media required cysteine, even under N₂, atmosphere. With cysteine, the rates of attachment were greatest for the first 2 hr, then continued more slowly. The numbers of attached trophozoites decreased immediately upon exposure to medium without cysteine. The role of cysteine in protecting trophozoites of both species from the lethal effects of oxygen was assessed using clonal growth in agar or agarose medium to determine viability following exposure to varying oxygen tensions in liquid medium. Cysteine was required for viability of trophozoites. Without cysteine, decreasing the oxygen tension prolonged survival. Under increased oxygen tension, cysteine delayed the onset of exponential killing. Although it has no thiol reducing group, l-cystine similarly protected E. histolytica.     

Leishmania enriettii: Immune induction of macrophage activation in an experimental model of immunoprophylaxis in the mouse

This study describes some of the parameters of the cellular immune response elicited in mice by inoculation of the nonpathogenic protozoan parasite, Leishmania enriettii. Incubation in vitro of leishmania-infected mouse peritoneal macrophages with spleen cells from syngeneic leishmania-immune animals resulted in activation of the phagocytes, leading to intracellular parasite destruction. Activation required interaction of sensitized lymphocytes with parasite antigen released or displayed by infected macrophages. The effect was dependent both on the dose of parasites used for in vivo priming and on the number of spleen cells cocultivated with parasitized macrophages. The activating capacity of lymphocytes was abrogated by anti-Thy-1 antiserum treatment and was retained in the effluent cells after nylon-wool separation. Activation was followed by lysis of part of the macrophage monolayer. Destruction of the phagocytes did not appear to result from the activation process per se and may represent a cytotoxic activity of sensitized lymphocytes for macrophages bearing parasite antigen on their surface.     

Leishmania mexicana and Leishmania tropica: Cross immunity in C57BL/6 mice

Leishmania mexicana and Leishmania tropica infection were comparatively studied in C57BL/6 mice. Infection with 10⁴ amastigotes of L. mexicana was followed by the appearance of a single lesion which ulcerated in 8 weeks and healed in 24 weeks. Mice infected with 10⁴ amastigotes of L. tropica developed less severe lesions which healed in 18 weeks. In both cases healing was accompanied by a delayed hypersensitivity response and an in vitro lymphocyte reactivity to leishmanial antigens. Mice recovered from a primary infection with L. mexicana or L. tropica were resistant to both homologous and heterologous challenge. In vitro and in vivo immunological tests indicated that L. mexicana and L. tropica share antigenic determinants which are involved in cell-mediated immune responses to these parasites.     

Plasmodium yoelii: Antibody and the maintenance of immunity in BALB/c mice

Subpatent persistence of parasitemia was detected for up to 7 weeks after infection of BALB/c mice with Plasmodium yoelii. Serum taken from recovered mice maintained parasitemias in recipient mice at a subpatent level when transferred repeatedly at 2-day intervals. Single doses of serum from convalescent donors delayed the course of infection in recipients. Small doses of transferred hyperimmune serum had the same effect, whereas large doses (>0.5 ml) totally suppressed parasitemia. Only a single secondary challenge of recovered mice was required in order to produce a maximally protective hyperimmune serum. Mice completely protected from a primary challenge with P. yoelii by transfer of hyperimmune serum were not at all resistant to a second challenge given some weeks later. After transfer of hyperimmune serum into mice with established P. yoelii infection, parasitemia fell to subpatent levels within 48 hr. During the first 21 hr after serum transfer, a progressive reduction in the proportion of ring forms present in blood smears was observed.     

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

Eimeria tenella: Cecal contraction in infected chickens

The muscle of ceca from chickens infected with Eimeria tenella had an increased amplitude of contraction when compared to the cecal muscle of uninfected control chickens. The increased amplitude was significant (P ? 0.05) at 5 days postinoculation (PI) and became nonsignificant at 7 days PI. The amplitude also increased with the severity of the infection. The sensitivity of the muscle from infected ceca to acetylcholine (ACH) was greater than the control. Infection by different strains of E. tenella also increased the sensitivity of the cecal muscle to ACH when compared to the uninfected control. The rate of spontaneous contractions was not different in any group or treatment. The wet weight of infected ceca increased with days PI and severity of infection.