Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.     

Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.     

Pathogenic and Nonpathogenic Acanthamoeba spp. in Thermally Polluted Discharges and Surface Waters1

During spring and autumn, the total number of amoebae and the number of Acanthamoeba species able to grow at 37°C were determined in six thermally polluted factory discharges and the surrounding surface waters. The isolated Acanthamoeba strains were studied for growth in axenic medium, cytopathic effect in Vero cell cultures, and virulence in mice. Although more amoebae were isolated in autumn, the number of Acanthamoeba species was lower than in spring, when the percent of pathogenic strains among the isolates was highest. Higher concentrations of amoebae were found in warm discharges, and more virulent strains occurred in thermal discharges than in surface waters.     

Entamoeba dispar: molecular characterization of the galactose/N-acetyl-d-galactosamine lectin

Amebiasis contributes to approximately 50 million cases of life-threatening dysentery worldwide. Comparison of the lectins from Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic) was undertaken to elucidate the differential roles of this molecule in invasion versus colonization. Surface lectin was less abundant on axenic E. dispar than on axenic E. histolytica, commensurate with differences in lectin (heavy and light subunits) RNA when assessed by semiquantitative RT-PCR. The 1G7 epitope, which falls within the immunodominant and immunoprotective cysteine-rich region (480-900), was absent on axenic E. dispar. Indirect immunofluorescence, transient transection of COS7, and immunoprecipitation demonstrated that the 1G7 epitope was conserved in the nonpathogenic lectin homologue but not exposed on live E. dispar trophozoites. Hgl2 (E. histolytica) and Dhgl2 (E. dispar) lectin homologues demonstrated comparable high-affinity binding to multivalent GalNAc(19) BSA. These data provide evidence for relative gene and conformational regulation of the E.dispar lectin.     

Entamoeba dispar: infrastructure, Surface Properties and Cytopathic Effect

The cytological features of Entamoeba dispar , recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoeba histolytica. When cultured under axenic conditions, living trophozoites of E. dispar strain SAW 760RR clone A were more elongated in form, had a single frontal pseudopodium, and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition, E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histolytica phagocytosis of epithelial cells plays an important role in its cytopathic effect, E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica , both at the light microscopical and the ultrastructural levels. In addition, they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelial cell monolayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.     

Amoeba-bacteria relationship: Factors in the bacterial lipids supporting the growth of axenicEntamoeba histolytica

Live bacteria in modifiedDiamond’s axenic medium did not support growth ofEntamoeba histolytica. Cysteine hydrochloride, required for the multiplication of amoeba, was broken down by live bacteria and toxic substances were produced which were lethal for amoebae. Monoxenic and xenic cultures ofaxenically grownE. histolytica could be established in Boeck and Drbohlav medium with bacteria and rice starch. Bacterial lipids prepared from 15 human intestinal bacteria supported growth and multiplication ofE. histolytica in axenic medium. In a pilot experiment using lipids ofStreptococcus faecalis, free fatty acids did stimulate the multiplication of amoebae. When total lipids of this bacteria were fractionated into neutral lipids and phospholipids by chromatography and used, neither fraction was found to stimulate growth. Free fatty acids prepared by chemical hydrolysis of the total lipids, neutral lipids and phospholipids stimulated growth ofE. histolytica, The sterols present in the bacterial lipids (neutral lipids or non-saponifiable fractions) stimulated growth of amoebae. It was found thatE. histolytica is incapable of liberating fatty acids from di- or triglyceridesof phospholipids and the multiplication of the organism is stimulated by the presence of free fatty acids and sterols (cholesterol).     

Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates.

Growth in an axenic medium composed by Chang (3rd Int. Congr. Parasitol. Munich Abstr. ICPIII 1:187-188, 1974) allowed separation of pathogenic from nonpathogenic Naegleria fowleri strains, since only the former show luxuriant growth in this medium. On the basis of these results, this medium was used in early screening for virulent Naegleria isolates. During an extensive ecological study, data were obtained on 102 Naegleria strains. Twenty of these strains grew luxuriantly in this liquid medium. Seventeen of them were tested by intranasal instillation in mice, and all proved to be highly pathogenic. Strains showing only moderate growth or no growth at all in this axenic medium were found to be nonpathogenic for mice. Moreover, it was found that using this medium in the early stage of Naegleria sampling favors isolation of pathogenic strains in mixtures of Naegleria. During these experiments, further evidence was obtained that thermal polluted waters are the main origin of N. fowleri in the environment.     

Induction of permeability changes and death of vertebrate cells is modulated by the virulence of Entamoeba spp. isolates

Although Entamoeba histolytica is capable of inducing an apoptotic response in vertebrate cells in vitro (Cell. Microbiol. 2 (2000) 617), it is not known whether vertebrate cell death requires direct amoeba-vertebrate cell contact or simply the presence of amoebae in the area of the vertebrate cells. In addition, Entamoeba spp. vary in their virulence and pathogenicity. The potential effects of these critical parameters also have not been elucidated. We tested the virulent HM-1:IMSS isolate and the non-virulent Rahman isolate of E. histolytica, and the non-virulent E. dispar CYNO16:TPC isolate against two vertebrate cell lines, HeLa and Chinese hamster ovary cells in vitro using ethidium homodimer as a fluorescent indicator of changes in vertebrate cell permeability. Fluorescence appeared in vertebrate cell nuclei within approximately 2-3 min of contact between HM-1 amoebae and vertebrate cells independent of vertebrate cell type. However, vertebrate cells in the immediate vicinity of but not contacted by HM-1 amoebae were not affected. In contrast, although both E. histolytica Rahman and E. dispar CYNO16 amoebae moved freely among and contacted vertebrate cells, the nuclei of the vertebrate cells never fluoresced implying that the cells remained alive and impermeant to the ethidium homodimer. This is the first demonstration that direct contact between virulent amoebae and vertebrate cells is required to kill vertebrate cells and that the process is restricted to virulent Entamoeba isolates. An understanding at the molecular level of the processes involved could help to reduce the pathology associated with this parasite.     

Modulation of Complement Resistance and Virulence of Naegleria fowleri Amoebae by Alterations in Growth Media

ABSTRACT. Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant. The present study evaluates the effect of complement on N. fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media. Pathogenic N. fowleri maintained in "enriched" Cline medium are virulent for mice and resistant to complement lysis. A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N. fowleri are grown in Nelson medium lacking hemin. N. fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement. Cycloheximide treatment of N. fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium. Proteins were radiolabeled with [³⁵S] during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement.     

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia , and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica . In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.     

Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.     

Virulence of bacteria-associated, Crithidia-associated, and axenic Entamoeba histolytica: experimental hamster liver infections with strains from patients and carriers.

Experimental infections of the hamster liver were carried out with five strains from patients with clinical amoebiasis and ten strains from asymptomatic carriers. Inocula of comparatively small size (12000-36000 amoebae) were injected under the liver capsule. 1. The virulence of the patient strains varied from 21-96% (see article) and declined sharply within 7-15 weeks after elimination of the associated bacterial flora. The virulence of the carrier strains varied from 0-100%, probably fluctuating with changes in the concomitant bacterial flora (Table 1). 2. The interrelation between size of inoculum, period of bacteria-free growth, and virulence was demonstrated with a Crithidia-associated patient strains (Table 2). 3. A patient strain showed a faster decrease of virulence during axenic than in Crithidia-associated cultivation (Table 3). 4. Two successive passages through hamster liver resulted in a marked increase of virulence of two bacteria-free strains, lasting for several months (Table 4). 5. A significant enhancement of virulence of Crithidia-associated and axenic amoebae by reassociation with a mixed bacterial flora during two weeks, followed by elimination of the bacteria, was demonstrated with two strains. The restored virulence was lost again within a few weeks (Table 5). 6. The virulence of an attenuated patient strain did not become manifest by adding large numbers of dead amoebae to the inoculum (Table 6). 7. The pathology of the different lesions caused in the hamster liver by the amoebae is described, including one of a granulomatous type, frequently found after inoculation with bacteria-free amoebae. 8. In an attempt to explain the occurrence of strains differing in pathogenicity an hypothesis is put forward based on the idea of selection of virulence and avirulent amoebae.     

Changes in the DNA content of amoebae of Dictyostelium discoideum during growth and development.

A fluorimetric assay has been used to determine the DNA content of amoebae of Dictyostelium discoideum during growth and development. Amoebae grown in axenic culture tended to be multinucleate and had a greater DNA content than amoebae grown with a bacterial substrate, which were mononucleate. During the first 10 h of development there was little change in the DNA content of amoebae grown with a bacterial substrate, but the average DNA content per cell in amoebae grown axenically decreased as the amoebae became virtually mononucleate. Amoebae at 10 h development that had been harvested during exponential axenic growth were divided into two populations by countercurrent distribution in a polymer two-phase system. DNA content indicated that one population was largely in the G2-phase of the cell cycle, whereas the other population was largely in the G1-phase. Similar results were obtained at 10 h development with amoebae harvested during the stationary phase of axenic growth, although these amoebae start development all in the G2-phase of the cell cycle. Spores had a low DNA content, indicating that they were in G1-phase. It is proposed that all amoebae in G2-phase after early development differentiate, after mitosis, into spores and that stalk cells are formed from amoebae that remain in G1-phase after 10 h development.     

Entamoeba histolytica: inhibition in vitro by bithionol of respiratory activity and growth

Endogenous and 2-propanol-supported respiration of intact trophozoites of of Entamoeba histolytica (stain HM-1:IMSS) were inhibited by bithionol, an effective chemotherapeutic agent for some trematode and cestode infections in humans. Dichlorophene and hexachlorophene also inhibited 2-propanol-supported respiration of the parasite. In contrast, ethanol formation by E. histolytica extract in the presence of N2 was scarcely inhibited by bithionol. The compound also inhibited in vitro growth of axenic (HM-1 strain) and polyxenic (strain HJ-1:KEIO) amoebae in culture. It took less than 24 hr to kill and disrupt virtually all amoebae of either strain with 0.28 mM bithionol. Omission of bovine serum from BI-S-33 medium resulted in considerably less disruption of HM-1 strain amoebae by the compound. However, organisms that looked undisrupted were strained with trypan blue. Moreover, the number of amoebae incubated for 10 min in the serum-free BI-S-33 medium containing 0.14 mM bithionol did not increase, even after incubation for 24 hr following replacement of the experimental culture fluid with fresh complete BI-S-33 medium free of the compound. These findings suggest that, although serum appears to diminish the antiamoebic action, some halogenated bisphenols (in particular bithionol) may be useful for treatment of amoebiasis.     

Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

Growth-promoting effect on iron-sulfur proteins on axenic cultures of Entamoeba dispar

A growth-promoting factor (GPF) that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt), hydrogenosomes (Hg) and chloroplasts (Cp) obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L.) in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600). We also found that on treatment with 0.6% complexes of 2-mercapthoethanol (2ME), complexes of chlorophyll-a with iron-sulphur (Fe-S) proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum) and noncomplex rubredoxin (Rd) from C. posteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.     

Differentiation of Entamoeba: a new medium and optimal conditions for axenic encystation of E. invadens.

Nutritional and culturing requirements for efficient axenic encystation of Entamoeba invadens have been studied. A simple and reliable axenic encystation medium has been developed. It contains 0.5% tryptic digest of casein, 0.5% yeast extract, and 5% dialyzed serum in 5 mM potassium phosphate, pH 7.0. Mass encystation (avg 70%) occurred within 30 hr when axenically growing trophozoites of E. invadens IP-l were transferred to this medium before they entered stationary growth phase. Mass encystation of E. invadens PZ occurred similarly, but less reproducibly. Two E. histolytica strains did not encyst. Experiments established that differentiation did not depend upon changes in the external environment after amebase were transferred to encystation medium and, therefore, was initiated by the shift from growth to encystation medium.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.     

Photoinhibition of Growth in Acanthamoeba castellanii Cultures

Light from 350 to 680 nm at intensities up to 1.62 × 10⁵ ergs per sec per cm² slowed exponential growth and lowered the maximum yield in axenic cultures of Acanthamoeba castellanii. Photoinhibition was a linear function of light intensity up to 1.25 × 10⁵ ergs per sec per cm². At higher intensities, growth was too slow to be measured accurately. A photochemical change occurring in the growth medium on irradiation was a function of light dosage and not intensity per se. Light in dosages which appreciably changed the growth-supporting properties of the medium exceeded the dosages received by exponentially growing cultures during irradiation. Consequently, photoinhibition of growth was attributed to a direct effect of light on the amoebae, not to photodegradation of the medium. The growth-supporting properties of irradiated media could be restored by the addition of yeast extract and Proteose peptone. The reduced growth rate in the light was not due to cyst formation or induction of multinuclearity. Light affected the amoebae either through absorption by intracellular pigment(s) or through binding to the amoebae of a photosensitizing compound in the medium.