Asymptomatic cyst passers of Entamoeba histolytica but not Entamoeba dispar in institutions for the mentally retarded in Japan

Entamoeba histolytica/Entamoeba dispar was isolated from 50 asymptomatic amebic cyst passers in three institutions for the mentally retarded in Kanagawa Prefecture, Japan. To distinguish between E. histolytica and E. dispar, the isolates were analyzed by PCR, reactivity to monoclonal antibodies, and zymodemes. All isolates were identified as E. histolytica. The results lead us to conceive that, in Japan, E. histolytica is predominant even in asymptomatic cyst passers.     

A mouse model for Entamoeba histolytica infection

2 strains of Entamoeba histolytica, SAW 760 and SAW 408 (non-invasive zymodeme IX and invasive zymodeme II respectively, Sargeaunt & Williams 1979) were administered intra-intestinally to 15 inbred and 3 outbred strains of mouse. All were known to be free of the mouse amoeba E. muris. During the investigation, all animals were maintained in plastic film isolator units, under gnotobiotic conditions. Both zymodemes were found to be capable of infecting all 18 strains of mice for varying periods of time and both were capable of invading the gut mucosa in genetically susceptible strains. Mortality was high in some of the immunologically deprived strains, particularly with the more invasive strain of amoeba. 4 mouse strains: DBA/2, MRL, NZB, C57BL/6 bg, were found to be particularly good hosts to SAW 760.     

Virulence of Entamoeba histolytica strains of human origin in Bombay to golden hamster

The virulence following intrahepatic inoculation to hamsters with 17 strains of Entamoeba histolytica isolated in cultures from clinical cases of amoebiasis and from asymptomatic carriers has been investigated. All 10 strains from clinical cases and four from asymptomatic carriers were found to be virulent for hamster liver, producing moderate or high grade of hepatic lesions, whereas three strains from asymptomatic carriers were found to possess low virulence with production of low grade of liver lesions. The strains from clinical cases produced marked decrease in the weight of the infected animal which was associated with high mortality. On the other hand, those from asymptomatic carriers produced minimal weight loss and low mortality.     

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.     

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.     

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.     

Prevalence of Escherichia coli strains in the canteens

The prevalence of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli strains in stool specimens from asymptomatic human carriers working in the canteens and also in the kitchen and sanitary facilities was evaluated. The E. coli genes coding for the following virulence markers: intimin (eae), enterohaemolysin (hlyA), and verotoxins type I and II (stx1 and stx2) were sought by multiplex PCR assay. E. coli isolates were obtained from 144 stool specimens, 295 swabs taken from kitchen hardware and surrounding facilities, and from 33 meat specimens. Only 66 (8.5%) of total 777 E. coli isolates belonged to O44, O18, O25, O127, O55, O114, O125, and O142 serogroups, the prevalent serogroups in Poland. None of the strains was classified as serogroup O157. The serogroups O44 and O18 were present most often among all typeable strains and their incidence was 51.5% and 25.8% respectively. Among 363 isolates assayed for the presence of the genes encoding virulence markers only 10 isolates (2.8%) carried eae gene. None of the isolates possessing eae gene belonged to the serogroups tested. The hlyA, stx1 and stx2 genes were absent in all E. coli isolates tested.     

Entamoeba histolytica zymodemes: exhibition of gamma and delta bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch content in the medium.

Entamoeba histolytica isolated from human can be associated with either symptomatic disease or with asymptomatic carriers. Pathogenic and nonpathogenic strains can be distinguished on the basis of differences in the electrophoretic patterns of four isoenzymes (zymodeme). With glucose phosphate isomerase and phosphoglucomutase, we observed variation of the expression of their gamma bands as a function of starch volume in culture. We cultured E. histolytica strains from different zymodemes in Robinson medium using both a low (2-4 mg/bottle) and a high (12-15 mg/bottle) content of rice starch as supplement. These cultures were monitored by electrophoresis of glucose phosphate isomerase and phosphoglucomutase. Strains having gamma or delta bands exhibited those bands when a high content of starch was used in culture, but did not do so with a low content. Contrarily, strains that never exhibited those bands did not express them when the amount of starch in the culture was increased. However, alpha and beta bands of the same isoenzymes were always present and never showed any variation. The results suggest that expression of gamma and delta bands of glucose phosphate isomerase and phosphoglucomutase are subject to culture conditions and that genes coding for those isoenzymes may be different from those coding for the alpha and beta bands.     

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.     

Virulence and fitness of the fungal pathogen Entomophaga maimaiga in its host Lymantria dispar, for pathogen and host strains originating from Asia, Europe, and North America

In this study, we tested (1) whether non-North American gypsy moth strains are susceptible to North American isolates of Entomophaga maimaiga and (2) the potential for erosion in the efficacy of E. maimaiga in controlling gypsy moth. We used bioassays to assess the variability in virulence (measured as time to death) as well as fitness of the pathogen (measured as spore production) in four gypsy strains challenged with six E. maimaiga isolates, using host and pathogen strains originating from Asia, Europe, and North America. We found that all E. maimaiga isolates tested were pathogenic to all strains of Lymantria dispar, regardless of the geographical origin of the fungal isolate, with at least 86% mortality for all combinations of fungal isolate and gypsy moth strain. We therefore conclude that Asian gypsy moths are susceptible to North American strains of E. maimaiga. No significant interactions between fungal isolates and gypsy moth strains with regard to time to death were found, indicating that each fungal isolate had the same overall effect on all the gypsy moth strains tested. However, fungal isolates differed significantly with regard to virulence, with a Russian isolate being the slowest to kill gypsy moth (5.1+/-0.1 days) and a Japanese isolate being the overall fastest to kill its host (4.0+/-0.1 days). Fungal isolates also differed in fitness, with variability in types of spores produced. These differences in virulence and fitness were, however, not correlated with geographical origin of the fungal isolate. Gypsy moth strains had no or only little effect on fungal virulence and fitness. Based on our studies with laboratory-reared gypsy moth strains, erosion of successful control of gypsy moth by E. maimaiga seems unlikely.     

Outcome of untreated infection with Entamoeba histolytica in homosexual men with and without HIV antibody.

Among homosexual men the prevalence of infection with Entamoeba histolytica is high. To determine the clinical importance of this infection 55 homosexual men carrying the parasite were investigated in detail. No clinical, serological, or histological evidence of invasive amoebiasis was found in any of them. The patients were not treated and were followed up for 12 to 29 months (mean 21.6 months), during which period none developed symptoms that could be attributed to E histolytica. Spontaneous loss of the parasite occurred in 17 patients, some of whom later became reinfected. Sixteen patients had antibody to human immunodeficiency virus, and infection with E histolytica showed the same benign course in them as in the patients who did not have antibody. Throughout the study classification of the isolates of E histolytica consistently showed that they belonged only to non-pathogenic zymodemes. The findings provide further evidence that E histolytica in homosexual men is a commensal organism.     

Molecular differentiation of Entamoeba histolytica and Entamoeba dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy

The purpose of the study was to obtain more reliable epidemiological data concerning Entamoeba (E.) histolytica infection in Tunisian food handlers using established molecular tools able to differentiate E. histolytica from E. dispar. From 2002 to 2005, 4,266 fresh stools specimens received in the setting of the National program of food handlers' control were analysed by optical microscopy. Twelve (2.8 per thousand) were positive for the presence of four nuclei cysts identified as E. histolytica/E. dispar. Extraction of DNA from the 12 samples, followed by specific amplifications of E. histolytica and E. dispar SSU rDNA, showed that 11 samples (92%) were positive for E. dispar and negative for E. histolytica. Sequencing analysis of 8 PCR products permitted to verify the results obtained with conventional PCR. The remaining sample was negative by PCR amplifying E. histolytica DNA or E. dispar DNA specifically, although it did not show any inhibition. It probably contains protozoan cysts genetically distinct from these two species but morphological similar. Estimation of relative proportions between E. histolytica and E. dispar in cyst carriers showed that all explored individuals harboured the non pathogenic E. dispar strains. This result highlights the need of use in this population of complementary tests that allow specific diagnosis and obviate unnecessary chemotherapy.     

Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment

The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome ('pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. coli.     

Isolation and characteristics of sorbitol-fermenting Escherichia coli O157 strains from cattle

Cattle can be a reservoir of sorbitol-fermenting Escherichia coli O157 (SF E. coli O157) and a source of human diseases. In this study, six strains of SF E. coli O157 were isolated and characterized from cattle using an immunomagnetic separation procedure. PCR analysis of the SF E. coli O157 virulence markers showed that all six isolates tested positive for sfpA, rfbE and eaeA, and negative for terA, ureA, katP and espP. Two of the isolates contained the stx genes. Four isolates tested positive for enterohemorrhagic E. coli hlyA (EhlyA) by PCR but were nonhemolytic on the blood agar. Five isolates tested positive for the cdtA gene. The possession of these virulence factors was an indication of their pathogenic potential. The random amplified polymorphic DNA patterns, which were generated by the arbitrarily primed PCR of the SF E. coli O157 isolates from the cattle, were significantly different from those of the non-sorbitol-fermenting E. coli O157 (NSF E. coli O157) strains originating from cattle or humans. GelCompar analysis showed that the SF E. coli O157 isolates had only a 57% genetic similarity with the NSF E. coli strains. The minimal inhibitory concentration assay showed that imipenem inhibited the growth of the six isolates at a concentration of <4 microg/ml.     

Determination of taxonomic status of pathogenic and nonpathogenic Entamoeba histolytica zymodemes using isoenzyme analysis.

Entamoeba histolytica infection results in either asymptomatic colonization or invasion of host tissues leading generally to clinical symptoms. Zymodemes studies have demonstrated a correlation between isoenzyme profiles and clinical presentation. Thus, strains have been attributed to pathogenic or nonpathogenic groups according to their zymodeme. To determine the taxonomic relationship of these two groups, the isoenzyme profiles of 14 loci of 38 E. histolytica strains (pathogenic and nonpathogenic) and seven strains of other species of the same genus were analyzed. Genetic distance analysis clearly demonstrates the existence of two separate groups within the species E. histolytica.     

Determination of Taxonomic Status of Pathogenic and Nonpathogenic Entamoeba histolytica Zymodemes Using Isoenzyme Analysis

ABSTRACT Entamoeba histolytica infection results in either asymptomatic colonization or invasion of host tissues leading generally to clinical symptoms. Zymodeme studies have demonstrated a correlation between isoenzyme profiles and clinical presentation. Thus, strains have been attributed to pathogenic or nonpathogenic groups according to their zymodeme. To determine the taxonomic relationship of these two groups, the isoenzyme profiles of 14 loci of 38 E. histolytica strains (pathogenic and nonpathogenic) and seven strains of other species of the same genus were analyzed. Genetic distance analysis clearly demonstrates the existence of two separate groups within the species E. histolytica .     

A Detoxifying Oxygen Reductase in the Anaerobic Protozoan Entamoeba histolytica

We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ～5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.     

Entamoeba histolytica: genetic diversity of African strains based on the polymorphism of the serine-rich protein gene

The polymorphism of the serine-rich Entamoeba histolytica protein (SREHP) among isolates obtained from different geographic regions was analyzed by a nested PCR followed by restriction analysis. Thirteen different profiles were generated from 23 E. histolytica isolates from Cameroon, Zimbabwe and South Africa while 20 others were generated from 38 E. histolytica PCR positive stool samples from South Africa. One of the profiles was common to isolates from Cameroon, Zimbabwe and South Africa and constituted the most prevalent (26.1%) of all the profiles. However, profiles unique to each country were also observed amongst the samples. A non-significant difference was observed between isolates from diarrheic and non-diarrheic samples. Of interest, of the five HIV positive stool samples three had the same profile indicating the possibility that some E. histolytica strains might be more common/pathogenic in immuno-compromised individuals. The results obtained showed that African isolates of E. histolytica may possess extremely complex genetic structures independent of geographic location. This study indicates that certain profiles might be responsible for the presentation of intestinal amoebic symptoms. However, more extended studies need to be performed in order to confirm these observations.     

Primates as a source of Entamoeba histolytica, their zymodeme status and zoonotic potential

A survey of 17 species of non-human captive primates in Quebec, Canada, revealed that 40% of the animals were infected with the protozoan Entamoeba histolytica. Isoenzyme analysis of lysates prepared from cultured amebic isolates showed them to belong to either a zymodeme III or VIII, and therefore, characteristic of non-pathogenic strains of E. histolytica from humans. These isolates were also of low virulence in gerbil ceca. Serum samples from infected and uninfected primates had negative titres for E. histolytica and all the animals appeared to be healthy.