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PubMed2006

Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis.

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

Acanthamoeba castellanii↗
PubMed2004

The identification of free-living environmental isolates of amoebae from Bulgaria.

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named "6" (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)--all exhibiting a highly efficient proliferation in axenic cultures--were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.

Acanthamoeba↗
PubMed2000

Vannella sp. harboring Microsporidia-like organisms isolated from the contact lens and inflamed eye of a female keratitis patient.

Viable Hartmannella sp. and two strains of Vannella sp.--but no Acanthamoebae--multiplied on NN-agar inoculated with pieces of the contact lens from a female keratitis patient. Within the cytoplasm of one Vannella isolate, intracellular parasites could be observed whose earliest stages were developing within the nucleus, resembling those Microsporidia-like parasites seen within Vannella isolated recently from a warm tapwater system. This assumption was also confirmed by electron microscopy. In swabs taken directly from the cornea, Pseudomonas aeruginosa were identified, but they did not yield any growth of amebas in culture. However, cocultivation of parasite-free Vannella strains with the above-mentioned swab matter resulted in infected amebas harboring the same intracellular parasites seen before. This infection could be established only if the corresponding spores were present as infective agents in the swab matter. The successful treatment of the patient with antibiotics supports the assumption that P. aeruginosa was the main cause of the corneal ulceration. The extent to which the Microsporidia-like organisms may have been involved in the development of keratitis remains a matter of discussion.

Adult↗
PubMed2002

Fasting induces cyanide-resistant respiration and oxidative stress in the amoeba Chaos carolinensis: implications for the cubic structural transition in mitochondrial membranes.

Large free-living amoeba (Chaos carolinensis) can survive in spring water without food intake for several weeks. Starvation is associated with a dramatic change in mitochondrial cristae from random tubular to ordered (paracrystalline) cubic morphology. Whole-cell polarography was used to monitor changes in respiratory activity during fasting. Basal respiration per cell decreased progressively during starvation, while the cyanide-resistant fraction increased. Spectrofluorometric assay of H2O2 and reactive oxygen species (ROS) in cell lysates (using the dye 2',7'-dichlorofluorescein diacetate) indicates greater H2O2 and ROS generation in starved than in fed cells. Fluorescence microscopy of intact cells incubated with the same dye demonstrates that H2O2 and ROS tend to accumulate in vacuoles. A remarkable generation of O2 observed with starved cells after addition of KCN may be explained by release of H2O2 from these compartments into the cytosol, where it can react with catalase. Together, these observations suggest that fasting increases oxidative stress in the amoeba and that this organism has several protective mechanisms to deal with it, including activation of a plantlike alternative oxidase. The hypothesis is forwarded that the cubic structural transition of the mitochondrial inner membrane represents another protective mechanism, reducing oxidative damage by enhancing the efflux of H2O2 and ROS and by reducing the susceptibility of membrane lipids to the oxidants.

Amoebida↗
PubMed2003

Cultivation and properties of Echinamoeba thermarum n. sp., an extremely thermophilic amoeba thriving in hot springs.

Here we describe a new, extremely thermophilic amoeba growing between 33 degrees C and 57 degrees C ( Topt.=50 degrees C). Isolates had been obtained from hot springs at Agnano Terme (Italy), Yellowstone National Park (USA), Kamchatka (Russia), and the Arenal Volcano (Costa Rica). They could be cultured monoxenically on a thermophilic alpha-proteobacterium. The morphology of the amoeba was studied using a microscope situated under a heatable polyacrylate hood. At 50 degrees C, the cells appeared flat with an irregular triangular or elongate shape, sometimes exhibiting fine spine-like subpseudopodia. On average, they were 22 microm long and 11 microm wide and had one nucleus with a central nucleolus. Based on morphology and on SSU rRNA comparisons, the amoeba belonged to the genus Echinamoeba, where it represents a new species. Referring to its extremely thermophilic lifestyle and its hydrothermal habitat, we name it E. thermarum.

Amoebida↗
PubMed1994

The origins of parasitism in the protists.

The origins of parasitism among the protists are, like the group itself, polyphyletic. Probably the majority of present-day parasitic forms evolved from free-living ancestors which were ingested as part of the food of their hosts, though origins from ectoparasitic forms and via a phase of facultative parasitism are possibilities, particularly among the ciliated protozoa and (for ectoparasitism) the Kinetoplasta. Sporozoan parasites most probably developed via a stage which was ingested and became adapted to life in the host's gut. Further developments in parasitism involved deeper penetration into the host's tissues and the adoption of more than one host in the life cycle, thus avoiding entirely the potentially hazardous phase of existence outside the host.

Amoebida↗
PubMed2006

Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA).

Sediment samples from rivers, canals and lakes in Arizona (USA) were cultured for free-living amoebae at three different incubation temperatures (22, 37 and 40 degrees C). Isolates belonging to the Vahlkampfiidae were identified by sequencing the PCR-amplified ITS1, 5.8S and ITS2 rDNA. With this molecular method three Naegleria spp. were identified, N. gruberi sensu stricto, N. australiensis and N. tihangensis. Also a strain each of Willaertia magna and Vahlkampfia avara were identified. Three samples yielded two new Tetramitus spp. of which the closest relative is T. ovis. Many Acanthamoeba strains were also isolated. The genotype of these strains was identified using Acanthamoeba-specific primers (JDP1 and JDP2) amplifying a part of the SSUrDNA and sequencing with an internal primer (892c). Five of the Acanthamoeba isolates belong to genotype T5 (A. lenticulata), while five are genotype T4.

Acanthamoeba↗
PubMed2004

Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals.

Knowledge that free-living amoebae are capable of causing human disease dates back some 50 years, prior to which time they were regarded as harmless soil organisms or, at most, commensals of mammals. First Naegleria fowleri, then Acanthamoeba spp. and Balamuthia mandrillaris, and finally Sappinia diploidea have been recognised as etiologic agents of encephalitis; Acanthamoeba spp. are also responsible for amoebic keratitis. Some of the infections are opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitides), while others are non-opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis, and cases of Balamuthia encephalitis occurring in immunocompetent humans). The amoebae have a cosmopolitan distribution in soil and water, providing multiple opportunities for contacts with humans and animals, as evidenced by antibody titers in surveyed human populations. Although, the numbers of infections caused by these amoebae are low in comparison to other protozoal parasitoses (trypanosomiasis, toxoplasmosis, malaria, etc.), the difficulty in diagnosing them, the challenge of finding optimal antimicrobial treatments and the morbidity and relatively high mortality associated with, in particular, the encephalitides have been a cause for concern for clinical and laboratory personnel and parasitologists. This review presents information about the individual amoebae: their morphologies and life-cycles, laboratory cultivation, ecology, epidemiology, nature of the infections and appropriate antimicrobial therapies, the immune response, and molecular diagnostic procedures that have been developed for identification of the amoebae in the environment and in clinical specimens.

Amebiasis↗
PubMed2005

Multiple symbiotes in larval black flies (Diptera: Simuliidae): associations of trichomycete fungi with ichthyosporeans, mermithid nematodes, and microsporidia.

Studies of the interactions between two or more species of symbiotic microorganisms in a single host animal have lagged behind those involving interactions between hymenopteran or dipteran parasitoids. We screened the larvae of four species of black flies (Cnephia ornithophilia, Simulium furculatum, Simulium innoxium, and Simulium vandalicum) to determine if the colonization and prevalence of trichomycete fungi were influenced by ichthyosporeans, mermithid nematodes, microsporidia, or other species of trichomycetes. Differences in the layering of the peritrophic matrix of microsporidian-infected larvae, compared with that of uninfected larvae, were associated with greater levels of colonization by the midgut-inhabiting trichomycete Harpella melusinae. Colonization levels of different species of trichomycetes were significantly correlated with one another in only 2 of 11 cases. Aside from the consistent attachment of the trichomycete Simuliomyces microsporus to the ichthyosporean Paramoebidium chattoni, we found little evidence that prevalence and colonization levels of trichomycetes were influenced by other symbiotes, including other trichomycetes. This trend might reflect the physical separation of trichomycetes, which are gut specific, from other symbiotes. In addition, any combinations of symbiotes that were wholly incompatible would not have been observed in our field-collected material.

Amoebida↗
PubMed2006

[Intra-amoebal development of Legionella pneumophila and the potential role of amoebae in the transmission of Legionnaires' disease].

Legionnaires' disease is one of the major infectious risks related to hospital water systems. It is commonly accepted, that the disease is transmitted to man mostly by inhalation of water aerosols contaminated by Legionella pneumophila. The ability of L. pneumophila to multiply intracellularly within some amoebae better explains the ecology, the pathogenicity, and the virulence of this bacterium against human alveolar macrophages. The presence of these amoebae in water systems located where cases of Legionnaire's disease broke out, partly explains the difficulty in eradicating Legionella. Some studies also show that amoebae can play a major role in the transmission of the disease to man. Some other studies point out that inhaled amoebae could be involved in the pathogenesis of Legionnaire's disease. Future strategies to prevent the transmission of Legionella will probably have to include efficient treatments against amoebae.

Acanthamoeba↗
PubMed2006

Evaluation of bithionol as a bath treatment for amoebic gill disease caused by Neoparamoeba spp.

This study examined the toxicity of bithionol to Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in fresh- and seawater and the efficacy of bithionol as a 1h seawater bath treatment for amoebic gill disease (AGD). To examine toxicity, fish were bathed for 1, 3 and 6h in bithionol, an anti-protozoal at 0, 1, 5, 10, 25 and 35mgL(-1) with toxicity determined by time to morbidity. Efficacy was examined by bathing AGD-affected Atlantic salmon and rainbow trout for 1h at bithionol concentrations of 1-25mgL(-1). Efficacy was determined by examining gill amoeba counts and identifying percent lesioned gill filaments at 1 and 24h after bath exposure to bithionol. For both species, bithionol was determined to be toxic at 25 and 35mgL(-1) exhibiting median lethal times (LT50s) ranging from 21 to 84min. Morbidity occurred in the 5 and 10mgL(-1) treatments, however, due to sampling regime there were not enough fish available to calculate LT50s. Only bithionol at 1mgL(-1) was considered non-toxic with no signs of morbidity. Bithionol was more toxic in seawater than freshwater and had no acute effects on gill Na+/K+ ATPase and succinic dehydrogenase, or plasma osmolality and chloride concentration. Bithionol at 1mgL(-1) reduced percent lesioned gill filaments in Atlantic salmon and rainbow trout by 33 and 27 per cent, respectively, compared to the seawater control. Similarly, numbers of amoeba were reduced by 33 and 43 per cent for Atlantic salmon and rainbow trout, respectively, when compared to the seawater control. Furthermore, bithionol reduced percent lesioned gill filaments as much as did the current industry standard of freshwater. This study demonstrated that a 1h seawater bath containing 1mgL(-1) bithionol could be an improvement to the current method of treatment for AGD-affected Atlantic salmon and rainbow trout.

Amebiasis↗
PubMed1996

Amebic keratitis in a wearer of disposable contact lenses due to a mixed Vahlkampfia and Hartmannella infection.

PURPOSE: To support the hypothesis that Acanthamoeba is not a unique cause of amebic keratitis, we report a case of amebic keratitis in which viable Acanthamoeba could not be isolated from corneal tissue. Vahlkampfia and Hartmannella, two other genera of free-living ameba, were isolated, however, using prolonged culture. METHODS: A 24-year-old wearer of soft contact lenses had keratitis. Extensive histologic and microbiologic investigations were performed on corneal scrape, biopsy, and keratoplasty tissue. Contact lenses, storage case, and the home water supply, where contact lens hygiene was practiced, were examined for the presence of micro-organisms. RESULTS: No viruses, pathogenic bacteria, or fungi were detected from corneal tissue samples. Amebae were observed using light and electron microscopy, but these could not be unequivocally classified using immunocytochemical staining. Viable Vahlkampfia and Hartmannella, but no Acanthamoeba, were isolated from the corneal biopsy sample. Indirect immunofluorescence with a range of polyclonal rabbit antisera raised against axenically cultivated stains of the three amebal genera was unhelpful because of cross-reactivity. A diverse range of micro-organisms was present within the storage case, including the three amebal species. Amebic cysts also were associated with the contact lens. CONCLUSION: A mixed non-Acanthamoeba amebic keratitis has been identified in a wearer of soft contact lenses where lack of storage case hygiene provided the opportunity for the free-living protozoa Vahlkampfia and Hartmannella to be introduced to the ocular surface. When Acanthamoeba-like keratitis occurs, but where Acanthamoeba cannot be isolated using conventional laboratory culture methods, alternate means should be used to identify other amebae that may be present. Polyclonal immunofluorescent antibody staining was unreliable for generic identification of pathogenic free-living amebae in corneal tissue.

Adult↗
PubMed2003

Temporal and spatial distribution of paramoebae in the water column--a pilot study.

Amoebic gill disease is the main disease affecting the salmonid industry in Tasmania, but no information on the distribution of the causative pathogen, Neoparamoeba pemaquidensis, in the aquatic environment is available. This pilot study aimed to determine temporal and spatial distributions of paramoebae species in the water column, using an immuno-dot blot technique. Water samples were collected from inside fish cages at various depths (0.5, 5.5 and 11.0 m) in both summer and winter, as well as various distances (0, 0.5, 240, 280, 750 and 1100 m) away from the sea cage and farming site. Paramoebae densities were estimated using the most probable number technique (MPN). Temperature, salinity, dissolved oxygen, turbidity, nitrite and nitrates, and bacterial counts were measured for each water sample. Data were analysed using a residual maximum likelihood test and significant associations between paramoebae densities and environmental factors were analysed. Results showed that densities were significantly higher in summer (P = 0.017), at 5.5 m depth (P = 0.029), and reduced to the lowest density at 1100 m away from the cage sites (P = 0.008). Bacterial counts, turbidity and temperature were found to be significantly associated with paramoebae densities.

Amoebida↗
PubMed2003

Cytopathogenicity of acanthamoeba, vahlkampfia and hartmannella: quantative & qualitative in vitro studies on keratocytes.

OBJECTIVES: To compare the cytopathogenicity of Vahlkampfia and Hartmannella clinical isolates with a type culture of Acanthamoeba castellanii. METHODS: The cytopathic effect produced during 24 h co-incubation with cultured keratocytes was assessed at set time intervals. Formal quantative studies involved image analysis of the area of cells remaining after 6 h. The mechanism of cytopathogenicity was elucidated using time-lapse video, light and scanning electron microscopy. The ability to produce cell damage in the absence of physical contact was studied using the transwell apparatus. The role of apotosis was also investigated. RESULTS: All three isolates produced near destruction of the keratocyte monolayer within 24 h, although initial cell destruction was more rapid with Acanthamoeba. For all three genera, the mechanism of cell damage involved physical attack and trogocytosis: cytopathic products were also implicated as cell damage was produced in the absence of physical contact, but apoptosis was not demonstrated. CONCLUSIONS: While the results do not prove that Vahlkampfia and Hartmannella are pathogens, they provide important evidence supporting the thesis that they cause keratitis by demonstrating that their ability to produce a cytopathic effect on keratocytes in vitro is similar in magnitude and mechanism to that of the known pathogen Acanthamoeba castellanii. The mechanisms by which small free-living amoebae produce cell damage is poorly understood. The ability of genera of amoebae other than Acanthamoeba to produce corneal infection remains controversial. In this study, the cytopathogenicity of Vahlkampfia and Hartmannella isolated from a case human keratitis are compared both quantitatively and qualitatively with that to the known pathogen Acanthamoeba castellanii. The results suggest that representatives of each of the 3 genera produce a similar degree of cytopathic effect on keratocytes after 24h of co-incubation and that a combination of physical and chemical factors are responsible.

Acanthamoeba↗
PubMed2002

Vertical distribution and abundance of gymnamoebae (Rhizopoda) in bottom sediments of the brackish water Nivå Bay (Baltic Sea, The Sound).

The sandy sediments of Nivå Bay (Baltic Sea, The Sound, Denmark) are often covered with the mats of sulphur bacteria and are temporarily anoxic. The vertical distribution and abundance of naked amoebae species in three sediment cores from this bay were studied. Amoebae were most abundant and diverse in the upper 1 cm of sediment, and their number and diversity decreased with increasing depth into the sediment. Amoebae were recovered from both upper oxygenated and deep anoxic layers of sediments. The species composition and abundance of amoebae was very heterogeneous, even at spatial scales of several centimeters, suggesting the existence of microhabitats selectively occupied by particular species. All species found were recorded from aerobic cultures and some of these amoebae occur in both the aerobic and anaerobic layers of the sediment. Minimal possible number of amoebae in the sediments, estimated for the first time as areal abundance integrated for depth was: core 1 -597 cm(-2); core 2 -1,110 cm(-2); core 3 -1,430 cm(-2). These abundances are probably best regarded as "potential" abundances of amoebae hidden in the sediments, as the question of the ratio between active and resting amoebae remains open.

Amoebida↗
PubMed2003

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: two new amitochondrial heterolobosean amoebae from anoxic environments.

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Amoebida↗
PubMed1992

Culture isolation of Acanthamoeba species and leptomyxid amebas from patients with amebic meningoencephalitis, including two patients with AIDS.

Acanthamoeba species and leptomyxid organisms are free-living amebas that cause meningoencephalitis, primarily in immunocompromised patients. We report the isolation and culture of Acanthamoeba species and leptomyxid amebas from four patients with fatal amebic meningoencephalitis. Acanthamoeba species were cultured from brain abscess specimens from three immunocompromised patients (including two patients with AIDS). In the case of the fourth patient, who had no identifiable immunodeficiency, leptomyxid amebas were cultured from a specimen from a subcutaneous nodule and were identified in amebic granulomas in brain tissue by the indirect immunofluorescence test. Persons with advanced infection due to the human immunodeficiency virus may be at increased risk for amebic meningoencephalitis, but the diagnosis should be considered in the differential diagnosis of any immunocompromised patient with cerebral abscesses.

AIDS-Related Opportunistic Infections↗