Entamoeba histolytica cysts with a defective wall formed under axenic conditions

Axenic HK9Entamoeba histolytica strain amoebae, maintained in PEHS medium, displayed several cystic characteristics that involve an active process of cystic wall formation, cellular volume and density diminution, and one or two nuclear divisions. The differentiation process was asynchronic, beginning after the logarithmic growth phase. The axenic cysts, which were maintained in a 50 mOsm/kg medium at 4°C for 72 h, produced growing trophozoites within 1–7 days of incubation at 36°C in fresh medium. Negative results were obtained with trophozoites submitted to the above treatment, and with axenic cysts maintained in double-distilled water at 4°C for 24 h, or in 0.1% sarkosyl, for 10 min at room temperature instead of 55 mosmol/kg medium. Thus, the HK9E. histolytica strain, cultured in PEHPS, produced under axenic conditions a small proportion of mature, metabolically active cysts, but with an immature or abnormal wall.     

Increase of DNA content in tissue stages ofEntamoeba histolytica strain SFL 3

The DNA content of culture forms and tissue stages of pathogenicE. histolytica strain SFL 3 were measured photometrically after the nuclei had been stained with the fluorochrome BAO. As a control, the DNA guartity ofE. histolytica strain HK 9 andE. invadens were determined by the same method and compared with reference values. Tissue stages were obtained from hamsters experimentally infected by intrahepatic injection of SFL 3 amoebae. Further studies concerning possible changes in the DNA content of tissue stages involved the following methods: (a) isolation of tissue stages from the liver, followed by distinct suspension periods. (b) Infected liver pieces were directly transferred into culture medium; amoebae emigrating therefrom were cultivated. The study demonstrated that tissue stages contained up to 4 times more DNA than did culture forms. After 3 h cultivation, the DNA content of tissue stages decreased to the level of culture forms. Possible reasons for this change are discussed.Supported by Deutsche Forschungsgemeinschaft (DFG)     

Auxotrophy to lipoproteins of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> cultivated under axenic conditions

Entamoeba histolytica grows in media without serum but with a mixture of aminoacids, vitamins, lipoproteins, free cholesterol, phospholipids and fatty acids called PACSR. The ability of lipoproteins and free lipids to support growth of three E. histolytica strains (HK9, HMI:IMSS and HM3:IMSS) was analysed. Tubes containing 5 ml culture medium, amino acids, vitamins and either 120–1,200 μg lipoproteins/ml or 0.017–0.10 mg free lipids/ml (predissolved in absolute ethanol) were inoculated with 1 × 10⁴ trophozoites/ml and incubated at 37 °C for 72 h. Amoebae died within 12 h in the presence of any free lipid combination, while those having 240–480 mg lipoproteins/ml reached densities similar to or higher than those of controls (depending on strain). The addition of ethanol (0.1%) to the media produced stable lipid solutions and did not show significant adverse effects. Accordingly, E. histolytica is auxotrophic to lipoproteins and unable to use free cholesterol, phospholipids or fatty acids. Received: 9 November 1999 / Accepted: 9 June 2000     

Trophozoite and nuclear size,DNA base composition,and nucleotide sequence homology of several Entamoeba strains in axenic culture

We carried out a comparative study of nuclear and trophozoite diameters and of DNA thermal denaturation in eight Entamoeba strains cultured axenically (four of them E. histolytica, two initially designated as E. invadens, one E. moshkovskii, and one E. histolytica-like), as well as an analysis of the overall DNA sequence homology of the non-E. histolytica strains. The average nuclear (N) and trophozoite (T) diameters (in m) were, respectively: global averages ±SD for E. histolytica strains, 6.5±2.5 and 28.8±3.7; E. invadens IP101, 5.8 and 27.5, and PZ, 7.8 and 33.6; E. moshkovskii FIC, 4.1 and 12.9; E. histolytica- like Laredo strain, 5.0 and 20.6. The GC content of DNA, estimated by thermal elution in hydroxyapatite, was around 23% in HK9 and its clone HK9-1 and around 27% in the HM2 and HM3 E. histolytica strains; it was 37% in the Laredo strain, 26% in IP101, 35% in PZ, and 33% in FIC. The reassociation kinetics of PZ strain DNA showed that it consists of 40% repeated sequences and 60% unique sequences. By means of DNA association experiments in which one of each pair of DNAs tested had been labeled in vitro with ¹²⁵I, we found the following overall sequence homology among the strains tested: PZ-FIC, 38%; IP101-Laredo, 38%; IP101-FIC, 47%; PZ-Laredo, 49%; Laredo-FIC, 69%; and IP101-PZ, 83%. We conclude that trophozoites of different E. histolytica strains have similar nuclear size and GC content, whereas these parameters and the nucleotide sequences are clearly different in every other Entamoeba species. Our data also suggest that PZ and IP101 strains do not belong to the same species.     

Entamoeba histolytica: changes in the zymodeme of cloned nonpathogenic trophozoites cultured under different conditions

In this paper we report the occurrence of changes in the migration of certain isoenzymes of a cloned strain (MAV-CINVESTAV) ofEntamoeba histolytica. This strain was isolated from an asymptomatic carrier and showed an initial nonpathogenic zymodeme I. The transfer of polyxenic trophozoites from Robinson's medium (7% serum) to Jones' medium (30% serum) provoked changes in isoenzyme expression, resulting in the conversion of zymodeme I to a zymodeme that was similar to the XVII zymodeme except for two slow-running bands and a single fast-running band that were detected for hexokinase (HK). This XVII-like zymodeme reverted to zymodeme I when trophozoites were cultured under monoxenic conditions in TY1-S-33 medium (10% serum). When we cultured cloned strain MAV-CINVESTAV under axenic conditions in TY1-S-33 medium, trophozoites expressed a pathogenic zymodeme with two fast-running HK bands and a betaphosphoglucomutase band. In addition, phagocytosis and the ability of the trophozoites to destroy cell-culture monolayers were expressed only in trophozoites cultured under axenic conditions. The axenization procedure required the presence of liveFusobacterium symbiosum and is also described herein.Abbreviations HK hexokinase - ME l-malate: NADP⁺ oxidoreductase - GPI glucose phosphate isomerase - PGM phosphoglucomutase - P pathogenic - NP nonpathogenic - px polyxenic - mx monoxenic - ax axenic     

Cytopathogenicity ofEntamoeba histolytica: Trophozoite homogenates modulate DNA synthesis in a mammalian cell line

We examined the effect of total trophozoite homogenates from four axenized strains ofEntamoeba histolytica (HK9, HM1, HM2, and HM3) on the DNA synthesis of subconfluent cultures of Chinese hamster ovary (CHO) cells incubated at low (0.1%) serum concentration. HM1, HM2, and HM3 extracts increased [³H]thymidine incorporation to acidinsoluble material in CHO cells up to a maximum of 2.5, 1.5, and 1.5 times respectively, at doses of amebal protein ranging from 16 to 125 g/ml. HM1 and HM2 extracts at doses higher than those causing maximal stimulation, and HM3 and HK9 extracts above 250 g protein per ml, progressively inhibited [³H]thymidine incorporation by CHO cells at a strain-specific rate. The extracts with both the most potent stimulatory and inhibitory effects were those from HM1 and HM2, also the most virulent strains. This strain-specific ability of amebal products to modulate cell DNA synthesis may play a significant role in amebal virulence.     

Adaption von amoeben-crithidia-kulturen (Entamoeba histolytica) an axenisches Wachstum in TP-S-1-Medium nach diamond (1968)

Zusammenfassung Ausgehend von Amoeben-Crithidia (A-C)-Kulturen wurde mit mehreren E. histolytica-Stämmen (F, A, Q, SFL₃) versucht, nach der von Diamond (1968) entwickelten und modifizierten Technik axenische Stammvarianten im TP-S-1-Medium zu züchten. Nur Stamm F (Hoe) konnte durch Anwendung verschiedener modifizierter Diamond'scher Techniken an axenische Haltungs-bedingungen adaptiert werden; die übrigen Stämme waren unter gleichen Versuchsbedingungen bereits nach wenigen Subkulturen im TP-S-1-Medium nicht mehr vermehrungsfähig. Vorteilhaft erwies sich Rinderserum im TP-S-1-Medium, das bei Stamm F und auch älteren axenischen Stämmen, wie HK-9 oder 200:NIH, ein gleichmäßigeres Wachstum der Amoebenkulturen hervorrief als Nährmedium mit Pferdeserum als Zusatz. Nach den vorliegenden Untersuchungen nimmt die Qualität des verwendeten Serums sowie die Species der Spendertiere einen entscheidenden Einfluß auf eine erfolgreiche axenische Züchtung.

---

Adaption of amoebae-crithidia-cultures (Entamoeba histolytica) to axenic conditions of cultivation in TP-S-1-Medium of diamond (1968)

Summary Starting from amoebae/Crithidiae (A–C) cultures, an attempt was made with several E. histolytica strains (F, A, Q, SFL₃) to cultivate axenic strain variants in TP-S-1 medium, using the modified method of Diamond (1968). Solely strain F (Hoe) could be adapted to axenic conditions of cultivation by applying various modified techniques of Diamond; under the same trial conditions the other strains were no longer capable of replication after only a few subcultures in TP-S-1 medium. Using bovine serum in TP-S-1 medium proved to be to advantage, producing in the case of strain F and also older axenic strains, such as HK-9 or 200: NIH, a more uniform growth of the amoebic cultures than nutrient medium with added horse serum. It follows from the present investigations that the quality of the serum used as well as the species of the donor animals have a decisive bearing on successful axenic cultivation of organisms.

     

The anti-amoebic activity of some medicinal plants used by AIDS patients in southern Thailand

The anti-amoebic activities of chloroform, methanol and water extracts from 12 Thai medicinal plants (39 extracts) commonly used by AIDS patients in southern Thailand were screened, at a concentration of 1,000 μg/ml, against Entamoeba histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro. The extracts were incubated with 2×10⁵ E. histolytica trophozoites/ml of medium at 37°C under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and scored (1–4) according to the appearance and numbers of the trophozoites. The extracts that caused inhibition were selected and retested using the same conditions but with concentrations that ranged from 31.25 to 1,000 μg/ml using E. histolytica strain HM1:IMSS, and the IC₅₀ values for each extract were calculated. The chloroform extracts from Alpinia galanga (IC₅₀ 55.2 μg/ml), Barleria lupulina (IC₅₀ 78.5 μg/ml), Boesenbergia pandurata (IC₅₀ 45.8 μg/ml), Piper betle (IC₅₀ 91.1 μg/ml) and Piper chaba (IC₅₀ 71.4 μg/ml) and the methanol extract from B. pandurata (IC₅₀ 57.6 μg/ml) were all classified as “active”, i.e. with an IC₅₀ of less than 100 μg/ml, whereas those from Murraya paniculata (IC₅₀ 116.5 μg/ml) and Zingiber zerumbet (IC₅₀ 196.9 μg/ml) were classified as being “moderately active”. The IC₅₀ of a standard drug, metronidazole, was 1.1 μg/ml.     

Amebic liver abscess in a european patient: Zymodeme classification ofentamoeba histolytica

Summary This is a case report of a 36-year-old patient who developed an amebic liver abscess after a stay in the Sudan. He was first misdiagnosed as having pneumonia of the right lower lobe. Following establishment of the correct diagnosis, the patient recovered fully after metronidazole treatment. The fecal culture in Robinson's medium yielded extensive growth ofEntamoeba histolytica. Electrophoretic characterization proved it to be a zymodeme XIX, which is one of the zymodemes associated with pathogenicity in the host. This first report of a zymodeme classification ofE. histolytica in Germany should initiate further epidemiological studies.Abkürzungsverzeichnis E. histolytica Entamoeba histolytica - GPI glucose phosphate isomerase - ME malic enzyme - PGM phosphoglucomutase - HK hexokinase - MIFC Merthiolate-iodine-formaldehyde concentration technique     

Lectin-induced agglutination of trophozoites of different species and strains ofEntamoeba

In vitro agglutinability of trophozoites of threeEntamoeba histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains ofE. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains ofE. histolytica-like Laredo-type amebae, a strain ofE. terrapinae, and a strain ofE. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.     

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced byEntamoeba histolytica

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 ofEntamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (<1000 leukocytes/mm³), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains ofE. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulentE. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, andE. invadens) was generally proportional to their proteinase activity; however,E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential ofEntamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.     

Caerulomycin,an antifungal antibiotic with marked in vitro and in vivo activity againstEntamoeba histolytica

The anti-amoebic action of the bipyridyl antibiotic caerulomycin was assessed in vitro and in vivo using various strains ofEntamoeba histolytica from polyxenic, axenic and monoxenic cultures. Minimum inhibition concentrations of caerulomycin (metronidazole) were 7.5 (5), 15.6 (1.95) and 60 (2.5) g/ml against polyxenic, axenic and monoxenic cultures ofE. histolytica, respectively. The ED₅₀ values ascertained in golden hamsters (extraintestinal amoebiasis) and rats (intestinal amoebiasis) after the oral route were 136 and 199 mg/kg (×4), respectively. Metronidazole proved to be approximately four times more active against tissue forms ofE. histolytica than caerulomycin [ED₅₀ of metronidazole: <40 mg/kg (×4)]. The antibiotic was slightly superior to metronidazole in its action on lumen forms ofE. histolytica [ED₅₀ of metronidazole: 233 mg/kg (×4)]. The antibiotic was in some cases toxic to hamsters and rats within the therapeutic range.     

Delayed-type hypersensitivity toEntamoeba histolytica in mice and hamsters: a comparison

Delayed-type hypersensitivity (DTH) to live or fixedEntamoeba histolytica was induced and compared in mice and hamsters. Peak reactions were obtained 24 h post-challenge. In mice, challenge with 10⁵ amoebae produced maximal, specific footpad swelling; in hamsters, 5×10⁴ were required. Elicitation of DTH in mice was strongest 1 week after induction and remained comparatively high for 8 weeks. In hamsters, elicitability declined after 1–2 weeks. Cyclophosphamide increased footpad reactions in mice and hamsters when given 1 day prior to induction but not prior to challenge. Reactions were usually somewhat (but not significantly) stronger in mice than in hamsters, which was also evident from adoptive transfer experiments. Thus, differences in cell-mediated immunity as expressed by DTH in mice and hamsters do not explain the differential susceptibility of these animals to infection with this parasite. In hamsters, multiple footpad injections of live or fixed amoebae lowered the percentage of subsequent liver infections after i.p. injection of virulent amoebae.     

Subcellular distribution and stability of the major hemolytic activity ofEntamoeba histolytica trophozoites

Since the hemolytic activity of extracts fromEntamoeba histolytica trophozoites previously described by us might determine at least partially the necrotic lesions of amebiasis, we have continued its characterization in vitro. Using rat erythrocytes as target cells, we have found that cytolysis byE. histolytica trophozoite extracts was (1) dose dependent, (2) localized mainly in a vesicular fraction whose absolute and specific activities were respectively 1.9 and 4.0 times higher than those of total extracts, (3) maximal at pH 8 in the presence of 1 mM Ca⁺⁺, and (4) progressively lost by heating at 90°C or repeated freezing and thawing. From these results we infer that the major hemolytic factor ofE. histolytica may be a protein normally neutralized by an intracellular inhibitor or activated during fractionation.     

Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica

We examined the molecular mechanisms of the cytotoxicity of Entamoeba histolytica, using the loss of transepithelial electrical resistance (TER) of monolayers of Madin-Darby canine-kidney (MDCK) cells on their incubation with axenic trophozoites of the HM1-IMSS strain. Such loss of TER occurs very early (in 2–5 min) and is caused by the opening of tight junctions and the detachment of cells. We used specific inhibitors for three of the four molecules currently accepted as being responsible for cytotoxicity: galactose-specific adhesin(s), phospholipase A, and cysteine proteinases. We also used inhibitors of calcium channels. Axenic trophozoites of E. histolytica strain HM1-IMSS were preincubated with the different inhibitors for 1 h prior to their coincubation with MDCK-cell monolayers. The only inhibitor that effectively blocked the loss of TER caused by the parasite was galactose. We suggest that in this experimental model, galactose-specific adhesin(s) are essential for amebic cytotoxicity. Received: 8 June 1999 / Accepted: 21 July 1999     

Long-term culture and cloning system forTrypanosoma brucei gambiense bloodstream forms in semi-defined medium in vitro

A new semi-defined medium extremely useful for the long-term cultivation and cloning ofTrypanosoma b. gambiense (Wellcome strain) bloodstream forms is described. Bloodstream forms could be continuously grown in 25 mM HEPES-buffered D-MEM supplemented with 10 M bathocuproine sulfonate (BCS), 100 M cysteine, and 20% heat-inactivated fetal calf serum at 37° C in vitro. Under these culture conditions,T. b. gambiense bloodstream forms increased in number up to 2–3×10⁶ trypanosomes/ml by day 3 after initiation of the culture. The trypanosomes maintained in this culture system for 200 days retained their infectivity for mice. Morphologically, they were long and slender, and a surface coat was evident on the cell surface and flagellar membrane. In vitro cloning with single bloodstream forms ofT. b. gambiense could be achieved with high efficiency.     

Cultivation of the Noguchi strain ofTreponema pallidum in a lipid medium in the absence of serum

Summary A lipid supplemented fluid medium supported good growth of the Noguchi strain ofTreponema pallidum in the absence of serum. It was composed as follows: casein hydrolysate, 3.0%, yeast extract, 0.5%, dextrose, 0.5%, NaCl, 0.25%,l-cysteineHCl monohydrate, 0.2%, TEM 4T, 0.0025%, cholesterol, 0.0002%, Tween 80, 0.0003%. The medium was sterilized by autoclaving.The maximum cell count in the absence of serum was reached in 3–4 days of incubation, and it amounted to 11.6×10⁷/ml (=±1.55×10⁷/ml;n=14). The final count of organisms was lower than the crop obtainable in a medium supplemented with rabbit serum, because of the toxic effect of lipid in the absence of serum and hence the impossibility to add lipid to a larger amount. The strain was carried through twenty-five successive subcultures in serum-free media.Study supported by Research Grant 2848/1/64 Federal Research Fund S.F.R.Y.     

Down-regulation of murine lymphocyte responsiveness to mitogens after treatment with antigens ofEntamoeba histolytica

Injection of mice with pathogenicEntamoeba histolytica (strain HM1-IMSS) antigens resulted in a decreased capacity of splenocytes to respond to mitogeninduced blastogenesis following a challenge with concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS), whereas no inhibition was observed in mice that had previously been injected with equivalent amounts of non-pathogenicE. histolytica-like Laredo antigens. Depletion of adherent cells in the splenocyte preparation indicated that these cells were not a major contributor to the observed immunosuppression. Quantification of splenic T-lymphocyte subsets demonstrated a significant decrease in Thy-1⁺ and Lyt-1⁺ cells, but Lyt-2⁺ cells were not affected. Splenocytes treated with pathogenic amoebic antigens in vitro affected the capacity of these cells to respond optimally to Con A-and LPS-induced blastogenesis but not to that induced by PHA. These findings demonstrate that amoebic antigens affect lymphocyte function and may be important co-factors in the immunoregulation and pathogenesis of amoebiasis.     

Adherence of pathogenic and non-pathogenicEntamoeba histolytica strains to neutrophils

The adherence of polymorphonuclear leukocytes (PMNs) to eight pathogenic and nine nonpathogenic strains ofEntamoeba histolytica was examined. No difference between pathogenic and nonpathogenic strains was found. The addition of different carbohydrates confirmed the importance of the 170-kDa lectin ofE. histolytica in binding to PMNs, corroborated by the finding that treatment of PMNs with galactosidase inhibited adherence. Inhibition of the microfilament system ofE. histolytica using cytochalasin B resulted in a loss of adherence to PMNs. Inhibition of the microtubule system using nocodazole did not affect adherence. Preincubation of the trophozoites with serum resulted in enbanced adherence, but the serum factor responsible for this effect could not be identified. Fibronectin, vitronectin, integrins (CD11/CD18 molecules), complement, and mannose-binding protein did not seem to mediate adherence betweenE. histolytica and PMNs. In summary, these results indicate that defective adherence mechanisms are not a common feature of nonpathogenicE. histolytica strains.     

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1–7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1–2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host. Received: 26 September 1999 / Accepted: 24 November 1999