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.     

Giardia lamblia: isolation and axenic cultivation.

Giardia lamblia trophozoites have been axenically cultured for more than a year. Initially, organisms were established in a complex liquid medium in the presence of the host's intestinal fungi; subcultures were made of these protozoa-fungus mixtures. G. lamblia trophozoites, free of yeast, were obtained by inoculating a protozoafungus culture in one arm of a U-tube, then later removing, from the other arm of the tube, Giardia trophozoites that had migrated across the base. Medium was changed at 2- or 3-day intervals; numerous subcultures were made. Tests for the possible presence of other organisms in these axenic cultures were negative. Trophozoite cultures remained viable, after freezing in the presence of glycerol, for 14 months. This is the first reported axenic culture of this common human intestinal parasite and pathogen; its study in pure culture is now possible.     

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.     

Improved fermentative production of Monascus pigments in roller bottle culture

The quantities and qualities of Monascus pigments produced by the filamentous fungus Monascus anka in batch submerged, agar surface, and roller bottle cultures were compared. In roller bottles, the fungus became attached to the wall of the culture vessels and produced a larger quantity of both intracellular (1508 absorbance units g⁻¹ cell mass) and extracellular (27 absorbance units g⁻¹ cell mass) Monascus red pigments, a yield that was about 10-fold greater than that of batch submerged and agar surface cultures. The optimum time required for maximum pigment production was reduced from 7 days in batch submerged or agar surface cultures to 4 days in roller bottle culture. In the roller bottle culture, the ratio of red to yellow pigments was also greatly increased. The advantage of the rotating vessel might be due to a combination of factors, including better gas exchange, higher medium pH, efficient pigment secretion, solid support for mycelium, and retarded conidiation.     

Entamoeba histolytica: Nucleic acid precursors affecting axenic growth

Treatment of Panmede and Trypticase, in water, with activated carbon and subsequently combining the filtered solution with glucose, cysteine, salts, serum and vitamins results in a medium depleted in the nucleic acid precursors required to sustain a high level of growth of axenic Entamoeba histolytica. Additions to the depleted medium which stimulated sustained growth, were the following, in the order of increasing efficacy: adenosine, adenine, AMP, AMP + GMP, AMP + GMP + UMP + CMP, or yeast ribonucleic acid. The last three additions restored growth to the level of cultures in TP-S-1 medium. No stimulation of sustained growth was found with GMP, IMP, UMP, or CMP, singly.     

Entamoeba histolytica and Entamoeba invadens: Effects of temperature and oxygen tension on growth and survival

The temperature ranges for axenic growth of Entamoeba histolytica strain HM-1 and Entamoeba invadens strain 165 clone II in TYI-S-33 medium were: 32 to 40 C for E. histolytica with an optimum of 35.5 to 37 C; whereas the range for E. invadens 165 II was 20 to 30 C, optimum 24 to 27 C. Growth of strain HM-1 at 40 C was dependent upon the cell density of the culture used as a source of the inoculum. Clonal growth in agar was used to assay survival of Entamoeba spp. trophozoites after exposure to deleterious physical conditions. The lowest temperature for thermal killing of E. histolytica HM-1 was 41.5 C and for E. invadens 165, 35.5 C. Both were killed rapidly at 42 C, but slowly at 0 C. Killing of E. histolytica HM-1 trophozoites by exposure to increased oxygen tensions was a function of temperature and time. At 24 C and 35.5 C, there was little loss of viability during the first 7 hr exposure, then killing was quite rapid. The cells were killed sooner if the medium was preexposed to air. In contrast, at 0 C there was less killing by oxygen. E. invadens 165 II appeared to be more oxygen sensitive at 24 than at 0 C. Other E. histolytica strains tested were similar to HM-1 in their responses to temperature and air.     

Tritrichomonas foetus: Stable anaerobic resistance to metronidazole in vitro

Stable anaerobic resistance of Tritrichomonas foetus to metronidazole was induced in vitro by cultivation of trichomonads in the Diamond's TYM medium with metronidazole in concentrations sublethal to the parasites. Nine metronidazole-resistant strains were derived from four drug-susceptible clones of the T. foetus strain KV-1. Subculturing the parasites at both increasing and constant pressure of the drug resulted in development of resistance if the medium contained at least 3 μg ml^?1 of metronidazole and the organisms were exposed to the drug for 3 to 8 months. The development of resistance was gradual and in all clones investigated proceeded through similar sequence of stages: (1) Survival without growth and subsequent reproduction at low metronidazole concentrations (1 to 5 μg ml^?1. (2) Survival and reproduction at moderate concentrations of the drug (10 to 15 μg ml^?1. (3) Resistance to 100 μg ml^?1 metronidazole, unstable in absence of selective pressure of the drug. (4) Resistance to high concentrations of metronidazole, stable when the organisms were maintained under nonselective conditions. The trichomonads with fully developed resistance were able to grow in anaerobic culture at 100 μg ml^?1 metronidazole and could be maintained indefinitely under these conditions. The minimal lethal concentrations for metronidazole obtained with these strains in an anaerobic in vitro assay were, at 48 h, 500 to 1000 μg ml^?1. This is 100 to 400 times higher than those obtained with the parent clones. The fully developed resistance was stable in organisms maintained in the absence of the drug over 2 years. The substrains with unstable resistance regained the susceptibility to high concentrations of metronidazole after 80 to 100 transfers in drug-free media. These strains, however, retained their resistance to moderate doses of metronidazole and full resistance could be restored by subculture in the presence of 10 μ ml^?1 metronidazole.     

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.     

Trypanosoma cruzi: Nucleotide and vitamin requirements of growing epimastigotes

Using a defined culture medium it was shown that Trypanosoma cruzi epimastigotes (strains Y, Ma, and F1) do not require exogeneous nucleotides for continuous cultivation. Biochemical determinations carried out on parasites grown in the presence or absence of exogenous nucleotides revealed no differences in intracellular nucleotide concentrations. This suggests that T. cruzi epimastigotes have the capacity for de novo nucleotide synthesis. Choline and folic acid were necessary only for high yields of T. cruzi, suggesting that epimastigotes can partially satisfy their vitamin requirements.     

Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium

A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 35 C at 4- to 10-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 × 10⁷/ml after 10 days of incubation at 33 C, and 5 × 10⁷/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 × 10⁷/ml after 12 days of incubation.     

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.     

Giardia lamblia: autoradiographic analysis of nuclear replication

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.     

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.     

Tritrichomonas foetus: Partly defined cultivation medium for study of the purine and pyrimidine metabolism

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose—the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.     

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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.     

Entamoeba histolytica: uptake of purine bases and nucleosides during axenic growth.

A comparison was made of the uptake mechanisms of selected purine bases and nucleosides by axenically grown Entamoeba histolytica. Adenine, adenosine, and guanosine were taken up, in part, by a “carrier”-mediated system. Guanine, hypoxanthine, and inosine entered amoebas via diffusion. Inhibitor studies support the presence of individual transport sites for adenine-adenosine and adenosine-guanosine. Additional sites for transport of adenine, adenosine, and guanosine are implied by “non-productive binding” involving guanine, hypoxanthine, and inosine. Uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N-ethylmaleimide. Ribose failed to inhibit uptake of purine nucleosides.     

Giardia muris: Correlation between oral dosage,course of infection,and trophozoite distribution in the mouse small intestine

Oral inoculations of Giardia muris cysts to CD-1 Swiss mice resulted in a reproducible pattern of infection measured by cyst excretion and the number of trophozoites in the small intestine. Housing of animals together or individually did not alter the pattern of cyst release for the first 30 days of infection. Administration of 10 to 10⁵ cysts/mouse resulted in a similar rate of cyst excretion from Day 10 to the end of the infection period. There was a direct correlation between the size of inoculum and the length of the latent period. Mice which received larger doses (10³, 10⁴, 10⁵) had significantly higher numbers of cysts in feces during the first week of infection, compared to those which received lower doses (10 or 100). The extent of trophozoite colonization and their distribution in the small intestine was not related to the size of inoculum. The location of trophozoites in the small intestine varied during the infection. In the first 3 to 8 days of infection the trophozoites were situated in the upper 25% of the small intestine, but moved posteriorly (upper 40%) during the period of peak cyst output. The number of trophozoites in the small intestine and cyst excretion were found to be proportional at all stages of infection.     

Plasmodium falciparum: Comparative analysis of erythrocyte stage-dependent protein antigens

Proteins of erythrocytic stages of Plasmodium falciparum were biosynthetically labeled at different times during the first cycle of in vitro synchronous cultivation after collection from patients in the Madang region of Papua New Guinea. Proteins were immunoprecipitated with a pool of hyperimmune serum collected in the region then analyzed by sodium dodecyl sulfate-gel electrophoresis. Antigens were recognized in all life cycle stages but the majority of antigens, particularly those of high molecular weight, were present in the mature forms of the parasite.