Repertoire of free-living protozoa in contact lens solutions.

BACKGROUND: The repertoire of free-living protozoa in contact lens solutions is poorly known despite the fact that such protozoa may act as direct pathogens and may harbor intra-cellular pathogens. METHODS: Between 2009 and 2014, the contact lens solutions collected from patients presenting at our Ophthalmology Department for clinically suspected keratitis, were cultured on non-nutrient agar examined by microscope for the presence of free-living protozoa. All protozoa were identified by 18S rRNA gene sequencing. RESULTS: A total of 20 of 233 (8.6 %) contact lens solution specimens collected from 16 patients were cultured. Acanthamoeba amoeba in 16 solutions (80 %) collected from 12 patients and Colpoda steini, Cercozoa sp., Protostelium sp. and a eukaryotic more closely related to Vermamoeba sp., were each isolated in one solution. Cercozoa sp., Colpoda sp., Protostelium sp. and Vermamoeba sp. are reported for the first time as contaminating contact lens solutions. CONCLUSION: The repertoire of protozoa in contact lens solutions is larger than previously known.

Prevalence of pathogenic free-living amoeba and other protozoa in natural and communal piped tap water from Queen Elizabeth protected area, Uganda.

BACKGROUND: Pathogenic water dwelling protozoa such as Acanthamoeba spp., Hartmannella spp., Naegleria spp., Cryptosporidium spp. and Giardia spp. are often responsible for devastating illnesses especially in children and immunocompromised individuals, yet their presence and prevalence in certain environment in sub-Saharan Africa is still unknown to most researchers, public health officials and medical practitioners. The objective of this study was to establish the presence and prevalence of pathogenic free-living amoeba (FLA), Cryptosporidium and Giardia in Queen Elizabeth Protected Area (QEPA). METHODS: Samples were collected from communal taps and natural water sites in QEPA. Physical water parameters were measured in situ. The samples were processed to detect the presence of FLA trophozoites by xenic cultivation, Cryptosporidium oocysts by Ziehl-Neelsen stain and Giardia cysts by Zinc Sulphate floatation technique. Parasites were observed microscopically, identified, counted and recorded. For FLA, genomic DNA was extracted for amplification and sequencing. RESULTS: Both natural and tap water sources were contaminated with FLA, Cryptosporidium spp. and Giardia spp. All protozoan parasites were more abundant in the colder rainy season except for Harmannella spp. and Naegleria spp. which occurred more in the warmer months. The prevalence of all parasites was higher in tap water than in natural water samples. There was a strong negative correlation between the presence of Acanthamoeba spp., Hartmannella spp., Cryptosporidium spp. and Giardia spp. with Dissolved Oxygen (DO) (P < 0.05). The presence of Cryptosporidium spp. showed a significant positive correlation (P < 0.05) with conductivity, pH and Total Dissolved Solids (TDS); whereas the presence of Giardia spp. had only a strong positive correlation with TDS. Molecular genotyping of FLA produced 7 Acanthamoeba, 5 Echinamoeba, 2 Hartmannella, 1 Bodomorpha, 1 Nuclearia and 1 Cercomonas partial sequences. CONCLUSIONS: All water collection sites were found to be contaminated with pathogenic protozoa that could possibly be the cause of a number of silent morbidities and mortalities among rural households in QEPA. This implies that water used by communities in QEPA is of poor quality and predisposes them to a variety of protozoan infections including the FLA whose public health importance was never reported, thus necessitating adoption of proper water safety measures.

Arcellacea (testate lobose amoebae) as pH indicators in a pyrite mine-acidified lake, Northeastern Ontario, Canada.

Arcellacea (testate lobose amoebae) were examined in 24 sediment-water interface samples collected over two late August field seasons in 2010 and 2011, from James and Granite lakes, Temagami Region, Northeastern Ontario. The work was carried out to quantitatively test species-environment relationships in a lake system known to be characterized by a significant pH gradient, partially the result of contamination from the early twentieth century Northland Pyrite Mine Co., located on the shoreline in the southern basin of James Lake. Redundancy analysis confirmed that arcellacean assemblage structure was most strongly controlled by pH, explaining 14.06 % (p < 0.002) of the total variance. Q- and R-mode cluster analysis supported by detrended correspondence analysis yielded two major faunal assemblages. The Oligotrophic Assemblage (1) had a Shannon Diversity Index (SDI) ranging up to 2.45, typical of healthy boreal lakes. This assemblage characterized samples collected from higher pH stations within James and Granite lakes away from the immediate area of the mine site, while the Low pH Assemblage 2010 (2a) and Low pH Assemblage 2011 (2b) groupings were from the very low pH environments of James Lake adjacent to the former mine site. Both low diversity assemblages (SDI ranging from 0.62 to 1.22) were characterized by Arcella vulgaris, a species known to thrive in hostile lacustrine environments. Differing depositional conditions during August 2010, a probable result of different prevailing wind patterns that summer, led to allochthonous specimens of the seasonally planktic Cucurbitella tricuspis dominating the Low pH Assemblage 2010 (2a) fauna.

Evolution and diversity of dictyostelid social amoebae.

Dictyostelid social amoebae are a large and ancient group of soil microbes with an unusual multicellular stage in their life cycle. Taxonomically, they belong to the eukaryotic supergroup Amoebozoa, the sister group to Opisthokonta (animals + fungi). Roughly half of the ~150 known dictyostelid species were discovered during the last five years and probably many more remain to be found. The traditional classification system of Dictyostelia was completely overturned by cladistic analyses and molecular phylogenies of the past six years. As a result, it now appears that, instead of three major divisions there are eight, none of which correspond to traditional higher-level taxa. In addition to the widely studied Dictyostelium discoideum, there are now efforts to develop model organisms and complete genome sequences for each major group. Thus Dictyostelia is becoming an excellent model for both practical, medically related research and for studying basic principles in cell-cell communication and developmental evolution. In this review we summarize the latest information about their life cycle, taxonomy, evolutionary history, genome projects and practical importance.

Amoebae and Legionella pneumophila in saline environments.

Amoeboid protists that harbor bacterial pathogens are of significant interest as potential reservoirs of disease-causing organisms in the environment, but little is known about them in marine and other saline environments. We enriched amoeba cultures from sediments from four sites in the New England estuarine system of Mt. Hope Bay, Massachusetts and from sediments from six sites in the Great Salt Lake, Utah. Cultures of amoebae were enriched using both minimal- and non-nutrient agar plates, made with fresh water, brackish water or saltwater. Recovered amoeba cultures were assayed for the presence of Legionella species using nested polymerase chain reactions (PCR) and primers specific for the genus. Positive samples were then screened with nested amplification using primers specific for the macrophage infectivity potentiator surface protein (mip) gene from L. pneumophila. Forty-eight percent (185 out of 388) of isolated amoeba cultures were positive for the presence of Legionella species. Legionella pneumophila was detected by PCR in 4% of the amoeba cultures (17 out of 388), and most of these amoebae were growing on marine media. Our results show that amoebae capable of growing in saline environments may harbor not only a diverse collection of Legionella species, but also species potentially pathogenic to humans.

Molecular confirmation of Sappinia pedata as a causative agent of amoebic encephalitis.

Pathogenic free-living amoebae, such as Acanthamoeba species, Balamuthia mandrillaris, and Naegleria fowleri, are known to cause infections of the central nervous system in human and other animals. In 2001, a case of human encephalitis was reported that was caused by another amoeba with morphological features suggestive of Sappinia. The amoeba originally identified as Sappinia diploidea was identified, most likely as S. pedata, by use of newly developed real-time polymerase chain reaction assays. This amoeba had previously been found only in environmental sources, such as soil and tree bark. The results illustrate the potential for other free-living amoebae, which are not normally associated with human disease, to cause occasional infections.

Microeukaryote diversity in a marine methanol-fed fluidized denitrification system.

The marine methanol-fed fluidized denitrification system operated by the Montreal Biodome includes carriers on which a denitrifying biofilm has developed. Previous observations showed a high abundance of microeukaryotes living in and around the biofilm. These eukaryotes may influence the system's denitrification efficiency. The composition of the microeukaryote population was determined. Microscopic observations showed at least 20 different morphologies that included large numbers of ciliates. Molecular analyses of an 18S ribosomal RNA (rDNA) gene library revealed 31 different phylotypes. Alveolobiontes were the most abundant phylotypes and made up 75% of the 159 screened clones. Other eukaryotic groups, including Stramenopiles, Fungi, Amoebozoa, and nematodes, were also present. From 18S rDNA specific sequences, one of the Amoebozoa-affiliated phylotypes was visualized by fluorescence in situ hybridization. It had a rod-like irregular shape and measured less than 5 mum in length. We determined the impact of protozoans on the denitrifying activity. In a laboratory-scale batch culture assays, the denitrifying biofilm was treated with cycloheximide and nystatin that eliminated the protozoans. No difference in the denitrification rate was found. However, planktonic bacteria were more abundant in the treated culture medium.

Sampling gene diversity across the supergroup Amoebozoa: large EST data sets from Acanthamoeba castellanii, Hartmannella vermiformis, Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp.

From comparative analysis of EST data for five taxa within the eukaryotic supergroup Amoebozoa, including two free-living amoebae (Acanthamoeba castellanii, Hartmannella vermiformis) and three slime molds (Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp.), we obtained new broad-range perspectives on the evolution and biosynthetic capacity of this assemblage. Together with genome sequences for the amoebozoans Dictyostelium discoideum and Entamoeba histolytica, and including partial genome sequence available for A. castellanii, we used the EST data to identify genes that appear to be exclusive to the supergroup, and to specific clades therein. Many of these genes are likely involved in cell-cell communication or differentiation. In examining on a broad scale a number of characters that previously have been considered in simpler cross-species comparisons, typically between Dictyostelium and Entamoeba, we find that Amoebozoa as a whole exhibits striking variation in the number and distribution of biosynthetic pathways, for example, ones for certain critical stress-response molecules, including trehalose and mannitol. Finally, we report additional compelling cases of lateral gene transfer within Amoebozoa, further emphasizing that although this process has influenced genome evolution in all examined amoebozoan taxa, it has done so to a variable extent.

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.

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.

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.

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.

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.

Laboratory and field-based studies of abundances, small-scale patchiness, and diversity of gymnamoebae in soils of varying porosity and organic content: evidence of microbiocoenoses.

Soil samples (varying in granularity) from four natural sites were cultured in microcosms to determine small-scale patchiness in abundance and diversity of gymnamoebae. Eighty grams of the same thoroughly mixed soil, either moistened with distilled water (- nutrients) or supplemented with an equivalent vol. of organically enriched water (+ nutrients), were placed in covered glass jars and incubated for 14 d (25 degrees C). Abundances (number/gram soil) were assessed in each of 3 core samples (5-10 mm apart). Assay precision was estimated to be +/- 4%. Abundances were similar in the 3 closely-spaced samples, but occasional samples had higher abundances, probably representing localized enriched sites ("nutrient hot spots"). Diversity within the triplicate, closely spaced samples varied substantially. Mean abundance and diversity of amoebae were consistently higher in organically enriched soil and in soil of increasing granularity. Field samples collected directly from two of the sites showed similar patterns of abundance and diversity as found in the experimental studies, indicating substantial small-scale compartmentalization of soil protist communities. These data provide evidence of soil eukaryotic microbiocoenoses and indicate that soil microfauna may encounter wide variations in resources and prey communities as they migrate within small distances of several millimeters or less.

SSU rRNA-based phylogenetic position of the genera Amoeba and Chaos (Lobosea, Gymnamoebia): the origin of gymnamoebae revisited.

Naked lobose amoebae (gymnamoebae) are among the most abundant group of protists present in all aquatic and terrestrial biotopes. Yet, because of lack of informative morphological characters, the origin and evolutionary history of gymnamoebae are poorly known. The first molecular studies revealed multiple origins for the amoeboid lineages and an extraordinary diversity of amoebae species. Molecular data, however, exist only for a few species of the numerous taxa belonging to this group. Here, we present the small-subunit (SSU) rDNA sequences of four species of typical large gymnamoebae: Amoeba proteus, Amoeba leningradensis, Chaos nobile, and Chaos carolinense. Sequence analysis suggests that the four species are closely related to the species of genera Saccamoeba, Leptomyxa, Rhizamoeba, Paraflabellula, Hartmannella, and Echinamoeba. All of them form a relatively well-supported clade, which corresponds to the subclass Gymnamoebia, in agreement with morphology-based taxonomy. The other gymnamoebae cluster in small groups or branch separately. Their relationships change depending on the type of analysis and the model of nucleotide substitution. All gymnamoebae branch together in Neighbor-Joining analysis with corrections for among-site rate heterogeneity and proportion of invariable sites. This clade, however, is not statistically supported by SSU rRNA gene sequences and further analysis of protein sequence data will be necessary to test the monophyly of gymnamoebae.