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PubMed2003

Entamoeba histolytica infections in captive primates.

A group based survey on the presence of Entamoeba histolytica and Entamoeba dispar using real-time PCR among 20 species of captive non-human primates was performed after diagnosis of E. histolytica dysentery in a spider monkey ( Ateles belzebuth hybridus). E. histolytica DNA was detected in three species of New World primates and in three species of Old World primates. In five of six E. histolytica isolates, it was possible to amplify the SREHP gene. They all revealed the same pattern after AluI digestion, indicating a common source of infection. E. dispar DNA was detected in two species of New World monkeys and three species of Old World monkeys. The results demonstrate that E. histolytica is capable of causing symptomatic and non-symptomatic infections in Old World and New World non-human primates. To our knowledge, this is the first report of E. histolytica sensu stricto in non-human primates after the redescription separating it from E. dispar in 1993.

Animals↗
PubMed2000

Entamoeba dispar, but not E. histolytica, detected in a colony of chimpanzees in Japan.

Chimpanzees (Pan troglodytes) residing in the Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho, were surveyed for the presence of intestinal parasites. Stool samples from 107 chimpanzees were examined by microscopy after formalin-ether sedimentation. Of these animals, 100 were infected with at least 1 species of ameba. The positivity rates recorded were as follows: Entamoeba coli, 88%; E. histolytica/E. dispar, 48%; E. hartmanni, 15%; Iodamoeba buetschlii, 8%; Endolimax nana, 4%; and Entamoeba chattoni, 2%. Polymerase chain reaction (PCR) analysis to distinguish between E. histolytica and E. dispar was performed on these samples. E. dispar DNA was detected in 60 of 107 samples (56%), including 9 that had been microscopically determined to be negative for E. histolytica/ E. dispar. In contrast, no E. histolytica DNA was detected in the 107 samples. Zymodeme analysis indicated that 10 isolates were E. dispar. When 104 chimpanzees were examined serologically for E. histolytica infection, 1 sample was scored as positive by indirect hemagglutination and another was found to be positive by an indirect fluorescent antibody test. However, both specimens were borderline-positive and were clearly negative in other tests, suggesting that they might be false-positives. These results demonstrate that the pathogenic E. histolytica was absent in this colony, regardless of the high degree of prevalence of other amebas. For an accurate diagnosis, PCR analysis is recommended in addition to microscopic examination.

Animals↗
PubMed1983

Entamoeba invadens and E. histolytica: separation and purification of precysts and cysts by centrifugation on discontinuous density gradients of Percoll.

The different cell forms in the life cycle of Entamoeba invadens (trophozoites, precysts, and cysts) were rapidly and quantitatively separated on density step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll). With this method, the gradual process of encystation by E. invadens trophozoites could be monitored. Percoll gradients were also efficient in separating trophozoites of Entamoeba histolytica and bacteria. After purification on Percoll, trophozoites display no evidence of damage when examined by light microscopy and no loss in viability as judged by their ability to multiply.

Centrifugation, Density Gradient↗
PubMed2001

Characterization of a retrotransposon-like element from Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica is the causative agent of amoebiasis. The genome organization of this organism is not well understood. We had earlier reported the presence of a multicopy sequence, HMc, in E. histolytica. Subsequent analysis showed that HMc is a member of a retrotransposon family that we have named the E. histolytica retrotransposon-like element (EhRLE). Four other members of this family have been characterized. The EhRLE family is distributed across all chromosomes of the parasite. There are 140 copies, which show minor sequence variation with respect to one another (2--4% from the consensus sequence). From a sequence analysis of five members of the EhRLE family, the complete EhRLE unit is estimated to be 4086 bp in length. It has a 27-mer inverted repeat at its ends. A pairwise comparison with sequences in the database showed a highly significant match of a part of EhRLE with reverse transcriptases (RT), especially those encoded by non-long terminal repeat retrotransposons. There are stop codons in all the five EhRLEs, but a continuous open reading frame of 464 amino acids could be reconstructed by comparing the sequences of several EhRLEs. The reconstructed sequence showed a much better identity with RT as compared with any of the original EhRLE sequences. The non-pathogenic species, Entamoeba dispar, also contains this element, with 85% sequence identity with EhRLE. The data suggest that EhRLE may be a retrotransposon, but many of its members are probably nonfunctional due to the accumulation of mutations.

Amino Acid Sequence↗
PubMed1996

A homologue of the cysteine proteinase gene (ACP1 or Eh-CPp3) of pathogenic Entamoeba histolytica is present in non-pathogenic E. dispar strains.

One of the three cysteine proteinase genes, ACP1 (or CP 3), has been reported to be missing in non-pathogenic strains of Entamoeba histolytica (or Entamoeba dispar as recently labeled). Unexpectedly, a gene fragment very similar in its sequence (95% homology) to ACP1 of pathogenic strains was obtained by use of the polymerase chain reaction from genomic DNA and cDNA of various cloned non-pathogenic strains as well as in 23 clinical isolates from asymptomatic carriers. The finding of the ACP1 homologue in non-pathogenic or E. dispar strains rules out the proposed use of its absence for diagnostic purposes.

Amino Acid Sequence↗
PubMed2000

Absence of lipophosphoglycan-like glycoconjugates in Entamoeba dispar.

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl-Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.

Animals↗
PubMed1999

Pore-forming peptides of Entamoeba dispar. Similarity and divergence to amoebapores in structure, expression and activity.

Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.

Amino Acid Sequence↗
PubMed1980

Surface redistribution and release of antibody-induced caps in entamoebae.

Polyspecific antibodies bound to Entamoeba induced surface redistribution of membrane components toward the uroid region. Capping of surface antigens was obtained with a single layer of antibodies in E. histolytica and E. invadens. This surface segregation progressed to a large accumulation of folded plasma membrane that extruded as a defined vesicular cap. A spontaneous release of the cap at the end of the capping process took place. These released caps contained most of the antibodies that originally bound to the whole cell surface. Two-thirds of radiolabeled antibodies bound to the surface of E. histolytica were released into the medium in 2 h. Successive capping induced by repeated exposure of E. invadens to antibodies produced conglomerates of folded surface membrane, visualized as stacked caps, in proportion to the number of antibody exposures. These results indicate the remarkable ability of Entamoeba to rapidly regenerate substantial amounts of plasma membbrane. The properties of surface redistribution, liberation of caps, and plasma membrane regeneration, may contribute to the survival of the parasite in the host during infection.

Animals↗
PubMed1985

Incorporation and toxicity of 32P-orthophosphate and occurrence of polyphosphate in Entamoeba trophozoites.

We explored the requirements of inorganic phosphate (Pi), the incorporation of 32P-orthophosphate (32Pi), and the occurrence of inorganic polyphosphate (polyP) in axenic Entamoeba cultures. Maximal population densities and growth rates of Entamoeba histolytica trophozoites were attained in complete TP-S-1 medium. As 32Pi concentration was increased in the medium, its own incorporation and the culture growth rate were progressively inhibited, especially in Pi-deficient medium. PolyP grains were found in the cytoplasm and occasionally in the nuclear membrane of E. histolytica-like, E. invadens, and E. moshkovskii trophozoites.

Animals↗
PubMedDate not supplied

Elucidation of the DNA synthetic cycle of Entamoeba spp. using flow cytometry and mathematical modeling.

We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G1, S, and G2 phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G1, S, and G2 proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 x 10(-14) g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 x 10(-14) g DNA/cell.

Animals↗
PubMedDate not supplied

Entamoeba dispar: ultrastructure, 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 76ORR 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 epithelia] cell monoLayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.

Agglutination↗
PubMed2003

Intestinal antilectin immunoglobulin A antibody response and immunity to Entamoeba dispar infection following cure of amebic liver abscess.

We followed 93 subjects with amebic liver abscess (ALA) and 963 close associate controls at 3-month intervals for 36 months to characterize intestinal and humoral antibody responses to the amebic galactose-inhibitable lectin and to determine whether immunity developed to Entamoeba histolytica or Entamoeba dispar infection following cure of ALA. We found that ALA subjects had a higher prevalence and level of intestinal antilectin immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant lectin protein) IgA and IgG antibodies, P < 0.01 and P < 0.05, respectively, compared to controls. The intestinal antilectin IgA antibody response was sustained over a longer time period in ALA subjects (71.8% remained positive at 18 months and 52.6% at 36 months, P < 0.001 compared to 17.6% and 10.3% of controls, respectively). ALA subjects were highly immune to E. dispar infection throughout the study (0% infected at 6 and 36 months, compared to 6.5% and 4.9% of control subjects, respectively, P < 0.05). Upon entry into the study, 6.3% of ALA subjects were infected with E. histolytica; the incidence of new E. histolytica infections in controls (as determined by culture) was too low (1.4%) to determine whether ALA subjects exhibited immunity to new infections. We found that stool cultures every 3 months markedly underestimated the occurrence of new E. histolytica infections, as 15.3% of controls seroconverted after 12 months of follow-up. Unfortunately, under the field conditions present in Durban, South Africa, enzyme-linked immunosorbent assay for detection of lectin antigen in stool yielded unreliable results. In summary, subjects cured of ALA exhibited sustained mucosal IgA antibody responses to the amebic galactose-inhibitable lectin and a high level of immunity to E. dispar infection. Determination of immunity to E. histolytica following cure of ALA will require the use of more sensitive and reliable diagnostic methods.

Adult↗
PubMed1983

Polyacrylamide gel electrophoresis of isoenzymes from Entamoeba species.

In this preliminary report, we describe a polyacrylamide gel electrophoresis technique for the resolution of isoenzyme patterns of four isolates of Entamoeba histolytica and one isolate of Entamoeba coli. Our findings were similar to previous findings for three enzyme systems: maleic enzyme (malate dehydrogenase [EC 1.1.1.40]), hexokinase (EC 2.7.1.1), and phosphoglucomutase (EC 2.7.5.1). We found preliminary evidence that glucosephosphate isomerase (EC 5.3.1.9) may also differentiate invasive amoebae from noninvasive amoebae, when the isoenzymes are separated by polyacrylamide gel electrophoresis, whereas this differentiation is not evident with starch-gel electrophoresis. We used an Rf system to relate isoenzyme band mobility to the migration distance of a standard E. histolytica strain (HK-9). The numerical identification of isoenzyme bands can simplify the grouping of isolates into zymodemes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar.

A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.

Animals↗
PubMedDate not supplied

[Occurrence of Entamoeba histolytica/Entamoeba dispar in ambulatory patients of Recife, PE].

The objective this study was to determine the occurrence of the species Entamoeba histolytica/Entamoeba díspar in clinical samples of ambulatory patients in Pernambuco. A specific assay for Entamoeba histolytica was used in this study, which identified no positive fecal samples among the 213 patients. These results confirm that E. dispar is the dominant species in Pernambuco State.

Animals↗
PubMed2001

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.

Animals↗
PubMed1994

Short report: detection of Entamoeba histolytica and E. dispar directly in stool.

Current diagnosis of Entamoeba histolytica infection requires the direct microscopic identification of the parasite, a technique that is insensitive and cannot distinguish pathogenic E. histolytica from noninvasive E. dispar. Enzyme-linked immunosorbent assay (ELISA) antigen detection tests were developed to distinguish E. histolytica from E. dispar infection in stool specimens. The ELISA result for E. histolytica antigen was positive in 26 of 27 E. histolytica-positive stool specimens, three of 25 E. dispar-positive stools, and one of 30 stools with other or no intestinal parasites, giving a specificity and sensitivity for the detection of E. histolytica infection of 93% and 96%, respectively. The assay result used to detect both E. dispar and E. histolytica was positive in 26 of 27 E. histolytica-positive stools, 19 of 25 E. dispar-positive stools, and one of 30 stools negative by microscopy and culture for Entamoeba, giving a specificity and sensitivity of 97% and 87%, respectively. Because these ELISAs can be completed in several hours, they offer promise as rapid and sensitive means of detecting amebic infection.

Animals↗
PubMed2000

Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daoud, Egypt.

We performed a prospective field study in the village of Kafer Daoud in Menofia, Egypt to compare the fecal culture method with enzyme linked immuno assay (ELISA) for detection of 170 kDa lectin antigen in feces for diagnosis of asymptomatic Entamoeba histolytica and Entamoeba dispar infection. All subjects with E. histolytica or E. dispar infection detected by culture also had positive ELISA for amebic antigen in their feces and an additional 57 Entameoba infections missed by culture were detected by ELISA (P < 0.001 compared to culture). The presence of fecal anti-lectin IgA antibodies and serum anti-LC3 (recombinant cysteine-rich lectin protein) IgG antibodies were positive predictors for E. histolytica infection (P < 0.03). Of interest, infection with Trichomonas hominis but not Blastocystis hominis was positively associated with E. histolytica infection (P < 0.05). In conclusion, ELISA for detection of fecal lectin antigen is a more sensitive method than fecal culture for detecting asymptomatic E. histolytica infection.

Adolescent↗