Detection of free-living amoebae in domestic cats with and without naturally-acquired keratitis.

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly. In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance.

Vermamoeba vermiformis CDC-19 draft genome sequence reveals considerable gene trafficking including with candidate phyla radiation and giant viruses.

Vermamoeba vermiformis is a predominant free-living amoeba in human environments and amongst the most common amoebae that can cause severe infections in humans. It is a niche for numerous amoeba-resisting microorganisms such as bacteria and giant viruses. Differences in the susceptibility to these giant viruses have been observed. V. vermiformis and amoeba-resisting microorganisms share a sympatric lifestyle that can promote exchanges of genetic material. This work analyzed the first draft genome sequence of a V. vermiformis strain (CDC-19) through comparative genomic, transcriptomic and phylogenetic analyses. The genome of V. vermiformis is 59.5 megabase pairs in size, and 22,483 genes were predicted. A high proportion (10% (n = 2,295)) of putative genes encoded proteins showed the highest sequence homology with a bacterial sequence. The expression of these genes was demonstrated for some bacterial homologous genes. In addition, for 30 genes, we detected best BLAST hits with members of the Candidate Phyla Radiation. Moreover, 185 genes (0.8%) best matched with giant viruses, mostly those related to the subfamily Klosneuvirinae (101 genes), in particular Bodo saltans virus (69 genes). Lateral sequence transfers between V. vermiformis and amoeba-resisting microorganisms were strengthened by Sanger sequencing, transcriptomic and phylogenetic analyses. This work provides important insights and genetic data for further studies about this amoeba and its interactions with microorganisms.

Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea.

The free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies.

Combinatorial selection in amoebal hosts drives the evolution of the human pathogen Legionella pneumophila.

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.

Species composition of testate amoebae in Lake Monte Alegre, Ribeirão Preto, SP, Brazil / Composição de amebas testáceas no lago Monte Alegre, Ribeirão Preto, SP, Brasil

We aimed to perform the first survey of testate amoebae community composition in Lake Monte Alegre at São Paulo state, and contribute to mitigate the taxonomic impediment regarding thediversity of testate amoebae in Brazil. Samplings were performed in 20 sampling sites within the lake, inthe limnetic and littoral regions in April 2015, using a 58 μm-meshplankton net. We identified 20 taxa oftestate amoebae belonging to four families: Arcellidae (8 taxa), Centropyxidae (4 taxa), Difflugiidae (7 taxa) and Lesquereusiidae (2 taxa). The littoral region showed the highest number of taxa (n = 20), whereas inthe limnetic region 11 taxa were registered. Therefore, our results evidenced the importance of testateamoebae in aquatic systems, and that further research, taxonomic and/or ecological, in those environmentsshould include these organisms in their investigation. Moreover, we suggest that future research with greater sampling effort is necessary to expand the identification of possible cryptic species in this environment.

O objetivo deste estudo foi realizar o primeiro levantamento da composição da comunidade de amebas testáceas no lago Monte Alegre no Estado de São Paulo e, assim, contribuir para mitigar oimpedimento taxonômico relacionado à diversidade de amebas testáceas no Brasil. As amostragens foramrealizadas em 20 pontos do lago, nas regiões limnética e litorânea, no mês de abril de 2015, com rede deplâncton de 58 μm de abertura de malha. Foram identificados 20 táxons de amebas testáceas, divididos emquatro famílias: Arcellidae (8 táxons), Centropyxidae (4 táxons), Difflugiidae (7 táxons) e Lesquereusiidae(2 táxons). A região litorânea apresentou o maior número de táxons (n = 20), enquanto que na regiãolimnética foram registrados 11 táxons. Diante disso, é possível indicar que os nossos resultados evidenciama importância das amebas testáceas nos sistemas aquáticos e que novos estudos, taxonômicos e/ou ecológicos, nesses ambientes devem incluir estes organismos em suas investigações. Além disso, sugeremsefuturos estudos com maior esforço amostral para ampliar a identificação de possíveis espécies crípticas noambiente.

Phylogeny and Redescription of the Testate Amoeba Diaphoropodon archeri (Chlamydophryidae, Thecofilosea, Cercozoa), De Saedeleer 1934, and Annotations on the Polyphyly of Testate Amoebae with Agglutinated Tests in the Cercozoa.

The genus Diaphoropodon, Archer 1869, comprises filose amoebae with agglutinated tests made of quartz grains, diatom frustules and other particulate materials. The key trait of the genus is a hyaline theca covered with numerous 5- to 10-µm-long, hairlike rods. Based on SSU rDNA phylogeny, we show that Diaphoropodon groups closely to Lecythium, a testate amoeba genus with a flexible but naked theca. Electron microscopic images reveal that the rods of Diaphoropodon are not perforating the test but lie randomly distributed on the surface of the amoeba. Comparing fairly naked cells from our cultures with cells from the environment leads to the conclusion that these rods play a role in agglutinating the material on the test.

Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

Free-living protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living protozoa.

Associations with free-living protozoa (FLP) have been implicated in the persistence of foodborne pathogenic bacteria in food-related environments. To date however no information is available on the presence of FLP in the gastrointestinal tract (GIT) of pigs, which represents an important reservoir for zoonotic foodborne bacteria and hence a potential location for associations with FLP. This is at least partly due to the lack of adequate protocols to recover FLP from intestinal content and feces. In the present study different protocols to recover FLP from the porcine GIT and feces were tested. The most effective protocols were then applied to explore the presence of live FLP in the pig GIT and feces. A filtration based protocol was identified as the most suitable method to recover viable FLP from the porcine GIT and feces. Cultivable FLP were recovered from different parts of the GIT, suggesting at least a transient presence of FLP in this habitat. Free-living amoebae species (Acanthamoeba spp., Hyperamoeba sp., Vannella sp., Vermamoeba vermiformis, hartmannellids and vahlkampfiids) but also ciliates (Colpoda sp. and Tetrahymena/Glaucoma lookalike) and flagellates (cercomonads, bodonids and glissomonads) were recovered and cultured from pig intestinal content. Acanthamoeba hatchetti and Filamoeba sinensis were isolated for the first time from pig intestinal content. Despite high gastric acidity, non-cyst forming amoeba species were also detected which suggests survival of their trophozoites in the animal GIT.

Current and future perspectives on the systematics, taxonomy and nomenclature of testate amoebae.

Testate amoebae are a polyphyletic assemblage of at least three major, unrelated taxonomic groups of unicellular amoeboid eukaryotes exhibiting a test. The focus on testate amoebae in scientific research has greatly increased in the past 20 years: from an average of about 5 papers a year in the mid-1990s to the current rate of more than 50 papers published yearly. The application range of these organisms is rapidly expanding as well: from the traditional fields of environmental monitoring and paleoecology, to forensic sciences and ecotoxicology studies. These developments are nevertheless strongly dependent on reliable taxonomy and nomenclature. However, scientometric data reveal that despite an ever-increasing necessity for the use of names (the product of taxonomy), the corresponding effort has not been achieved for improving testate amoebae systematics. As a consequence, inaccurate taxonomy yields to misinterpretations in the diversity of the organisms and to potentially incorrect conclusions. These and related problems are discussed in this study, highlighting the outcome of poor taxonomic expertise in accurate classification and phylogeny of testate amoebae, and the consequences derived from it. Additionally, this study is aimed to discuss the current status of testate amoebae classification, and to present all nomenclature and taxonomic changes in higher and lower taxonomic levels of testate amoebae, as a result of recent molecular reconstructions. Finally, we conclude with a list of the needs and suggestions toward a unified and modernized taxonomy of testate amoebae.

Testate Amoebae (Amebozoa: Arcellinida) in Tropical Lakes of Central Mexico / Amebas testadas (Amebozoa, Arcellinida) en lagos tropicales del centro de México

AbstractTestate amoebae are common single-celled eukaryotic organisms in aquatic ecosystems. Despite their important role in these ecosystems, and their potential as bioindicators and paleoindicators, they remain poorly studied in Mexico. The major objectives of this study were to: 1) increase knowledge of testate amoebae in Mexico's tropical lakes, and 2) create a catalog of high-quality scanning electron micrographs that can be used for future ecological and paleoenvironmental studies. We collected surface-sediment samples from 29 lakes, located in the Transmexican Volcanic Belt, one at each lake during June and October 2011, and March 2013. Sediments were collected with an Ekman grab and preserved in anhydrous ethanol. Sub-samples were observed under a stereomicroscope and morphometric data for each species were recorded. Total diameter and aperture diameter were measured on circular tests. Irregularly shaped tests were measured for length and width of the aperture and for the size of the whole test. If a specimen possessed spines, the length of one randomly selected spine was measured. The best-preserved specimen of each taxon was photographed with an optical microscope and a scanning electron microscope (SEM). We found 41 taxa of testate amoebae belonging to the genera: Arcella, Argynnia, Centropyxis, Cucurbitella, Cyclopyxis, Cyphoderia, Difflugia, Euglypha, Lesquereusia, Pentagonia, Pseudodifflugia and Scutiglypha. Twelve species not previously reported for Mexico were recorded, along with 13 varieties. The average number of taxa recorded in each lake was eight, and the highest taxonomic richness was 18. The taxon found in the greatest number of lakes was Centropyxis aculeata var. aculeata. Taxonomic richness varied among lakes in the same region. This could reflect lake-specific differences in environmental conditions, underscoring the need for more detailed studies that include collection of data on physical and chemical variables in the lakes. Our results highlighted the need of further studies for the distribution patterns and ecology of lacustrine testate amoebae. Rev. Biol. Trop. 64 (1): 377-397. Epub 2016 March 01.

ResumenLas amebas testadas son organismos eucariontes unicelulares frecuentes en ecosistemas acuáticos. A pesar de su importante rol en estos ecosistemas, y su potencial como bioindicadores y paleoindicadores, son poco estudiados en México. Los principales objetivos de este estudio fueron: 1) incrementar el conocimiento de las amebas testadas en lagos tropicales de México y 2) crear un catálogo de microfotografías electrónicas de barrido de alta calidad que pueda ser usado en futuros estudios ecológicos y paleoambientales. Recolectamos muestras de sedimentos superficiales de 29 lagos, ubicados en la Faja Volcánica Transmexicana, una vez en cada lago durante junio y octubre 2011 y marzo 2013. Los sedimentos se recolectaron con una draga tipo Ekman y se preservaron en etanol anhidro. Las sub-muestras se observaron al microscopio estereoscópico. Para cada especie fueron registrados sus datos morfométricos. En testas circulares fueron medidos el diámetro y la apertura total. En testas irregulares fueron medidos el largo y ancho de la apertura y el tamaño de toda la testa. Si un ejemplar poseía espinas, se seleccionó una al azar y se midió su longitud. El ejemplar mejor preservado de cada taxón fue fotografiado con un microscopio óptico y en microscopio electrónico de barrido (MEB). Encontramos 41 taxa de amebas testadas pertenecientes a los géneros Arcella, Argynnia, Centropyxis, Cucurbitella, Cyclopyxis, Cyphoderia, Difflugia, Euglypha, Lesquereusia, Pentagonia, Pseudodifflugia y Scutiglypha. Se registraron 12 especies y 13 variedades que no estaban reportadas previamente en México. El número promedio de taxa registrados en cada lago fue de ocho, y la riqueza taxonómica mayor fue de 18. El taxón encontrado en el mayor número de lagos fue Centropyxis aculeata var. aculeata. La riqueza taxonómica compartida por lagos de la misma región no fue muy similar. Esto puede reflejar diferencias en las condiciones ambientales, evidenciando la necesidad de elaborar estudios más detallados que incluyan la recolección de datos de las variables físico-químicas en los lagos. Nuestros resultados resaltan la necesidad de estudios posteriores acerca de los patrones de distribución y la ecología de las amebas testadas lacustres.

Molecular Detection of Legionella spp. and their associations with Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in a drinking water distribution system.

AIMS: This study investigated waterborne opportunistic pathogens (OPs) including potential hosts, and evaluated the use of Legionella spp. for indicating microbial water quality for OPs within a full-scale operating drinking water distribution system (DWDS). METHODS AND RESULTS: To investigate the occurrence of specific microbial pathogens within a major city DWDS we examined large volume (90 l drinking water) ultrafiltration (UF) concentrates collected from six sites between February, 2012 and June, 2013. The detection frequency and concentration estimates by qPCR were: Legionella spp. (57%/85 cell equivalent, CE l(-1) ), Mycobacterium spp. (88%/324 CE l(-1) ), Pseudomonas aeruginosa (24%/2 CE l(-1) ), Vermamoeba vermiformis (24%/2 CE l(-1) ) and Acanthamoeba spp. (42%/5 cyst equivalent, CE l(-1) ). There was no detection of the following microorganisms: human faecal indicator Bacteroides (HF183), Salmonella enterica, Campylobacter spp., Escherichia coli O157:H7, Giardia intestinalis, Cryptosporidium spp. or Naegleria fowleri. There were significant correlations between the qPCR signals of Legionella spp. and Mycobacterium spp., and their potential hosts V. vermiformis and Acanthamoeba spp. Sequencing of Legionella spp. demonstrated limited diversity, with most sequences coming from two dominant groups, of which the larger dominant group was an unidentified species. Other known species including Legionella pneumophila were detected, but at low frequency. The densities of Legionella spp. and Mycobacterium spp. were generally higher (17 and 324 folds, respectively) for distal sites relative to the entry point to the DWDS. CONCLUSIONS: Legionella spp. occurred, had significant growth and were strongly associated with free-living amoebae (FLA) and Mycobacterium spp., suggesting that Legionella spp. could provide a useful DWDS monitoring role to indicate potential conditions for non-faecal OPs. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide insight into microbial pathogen detection that may aid in the monitoring of microbial water quality within DWDS prior to customer exposures.

Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

Ophthalmology hospital wards contamination to pathogenic free living Amoebae in Iran.

The present study was conducted to determine the occurrence of potentially pathogenic free-living amoeba in ophthalmology wards in reference hospitals in Iran. Since an increasing number of Acanthamoeba Keratitis cases after eye surgery and eye trauma have been recently observed in this country, it could be possible that the disinfection procedures undertaken in the clinical setting may not have a good hygiene and disinfection procedures, hence the aim of this study. Therefore, 42 dust and biofilm samples were collected from different areas of ophthalmology wards and checked for the presence of FLA using morphological criteria, PCR based analysis and DNA sequencing. Of the 42 samples from dust and biofilm sources, 18(42.86%) isolates were found to contain FLA and 12(92.3%) isolates belonged to Acanthamoeba T4 genotype. Isolation of the pathogenic genotype T4 from medical instruments, including slit lamp in corneal wards, may be a threat for patients undergoing eye surgery in these wards. Other FLA isolated in this study included Acanthamoeba genotype T5, Vahlkampfia sp, Naegleria australiensis, Vermamoeba vermiformis and Echinamoeba exudans. To our knowledge, this is the first report of the presence of potentially pathogenic FLA in ophthalmology wards in Iran. Improved disinfection methods and monitoring of hospitals ward are thus necessary in this area in order to minimize the risk of infection in patients.

Distribution of free-living amoebae in a treatment system of textile industrial wastewater.

Free-living amoebae have been found in soil, air and a variety of aquatic environments, but few studies have been conducted on industrial wastewater and none on wastewater from the textile industry. The aim of this study was to determine the presence and distribution of free-living amoebae in a biological treatment system that treats textile industrial wastewater. Samples were taken from input, aeration tank, sedimentation tank and output. Samples were centrifuged at 1200g for 15min, the sediment was seeded on non-nutritive agar with Enterobacter aerogenes (NNE) and the plates were incubated at 30 and 37°C. Free-living amoebae were present in all stages of the treatment system. The highest number of amoebic isolates was found in the aeration tank and no seasonal distribution was observed during the year. A total of 14 amoeba genera were isolated: Acanthamoeba, Echinamoeba, Korotnevella, Mayorella, Naegleria, Platyamoeba, Saccamoeba, Stachyamoeba, Thecamoeba, Vahlkampfia, Vannella, Vermamoeba, Vexillifera and Willaertia. The most frequently found amoebae were Acanthamoeba and Vermamoeba which were found in all treatment system stages. The constant presence and diversity of free-living amoebae in the treatment system were important findings due to the characteristics of the wastewater from the textile plant in terms of the residue content from colorants, fixers, carriers, surfactants, etc., used in fabric dyeing and finishing processes. The factors that determined the presence and distribution of amoebae in the activated sludge system were their capacity to form cysts, which allowed them to resist adverse conditions; food availability; an average temperature of 27-33°C; dissolved oxygen in average concentrations above 2mg/L, and pH in a range of 5.9-7.1.

Distributions and biomass of benthic ciliates, foraminifera and amoeboid protists in marine, brackish, and freshwater sediments.

The quantitative importance of ciliates, foraminifers, and amoebae was investigated in marine, brackish, and freshwater sediments from 15 littoral stations. Total protozoan communities were usually dominated by ciliates in term of abundance, while amoebae often dominated in terms of biomass. Applying the biomass-metabolic rate equation, ciliates, amoebae, and foraminifera were estimated to contribute 66% of the total abundance and 33% of the biomass, but up to 55% of the combined metabolic rate to the micro- and meiobenthos in the 15 sediments. Statistical analyses using ciliate data demonstrated: (1) species composition and community structures represented significant differences between freshwater and marine/brackish sediments, and subsequently between temperate and arctic sampling sites; (2) the occurrence of dominant ciliates and their allocation to feeding types indicated that herbivory was the most common feeding strategy in these sediments; (3) multivariate analyses showed all of the tested environmental factors (temperature, salinity, silt/clay, carbon, nitrogen, and chlorophyll a) to be important to varying degrees, but especially the combination of salinity, temperature, and silt/clay. Multiple factor effects or comprehensive influences might be important in regulating the distribution of protozoa in sediments. The importance of protozoa in sediment systems and the potential ecological significance of cysts are discussed.

Survey on diversity of marine/saline anaerobic Heterolobosea (Excavata: Discoba) with description of seven new species.

Diversity of the anaerobic Heterolobosea (Excavata: Discoba) is only poorly understood, especially in marine environments. We have isolated and cultured 16 strains of anaerobic heteroloboseid amoebae and flagellates from brackish, marine and saline anoxic habitats worldwide. Phylogenetic analyses of SSU rDNA sequences and light-microscopic observations showed that all the strains belong to the family Psalteriomonadidae, the main anaerobic lineage of Heterolobosea, and that they represent eight species from the genera Monopylocystis, Harpagon and Pseudoharpagon. Seven species are newly isolated and described here as Monopylocystis minor n. sp., Monopylocystis robusta n. sp., Monopylocystis elegans n. sp., Monopylocystis disparata n. sp., Harpagon salinus n. sp., Pseudoharpagon longus n. sp. and Pseudoharpagon tertius n. sp. Amoebae, cysts and the ultrastructure of the genus Pseudoharpagon are presented for the first time.

Structure of the testate amoebae community in different habitats in a neotropical floodplain / Estrutura da comunidade de amebas testáceas em diferentes hábitats em uma planície de inundação neotropical

This study evaluated the differences in composition, abundance and morphology of testate amoebae among different habitats of the same aquatic environment (plankton, aquatic macrophyte and sediment) in the Upper Paraná River floodplain. Triplicate samplings were undertaken monthly at each habitat from April 2007 to March 2008. The structure of the community of testate amoebae was different among the habitats. The species typical for each habitat, according to Indval, were classified by their shell morphology. Arcella species together with Difflugia gramen and Difflugia pseudogramem were more abundant for plankton. Trinema and Phryganella stood out by their abundance and frequency in aquatic macrophytes. Centropyxis was an indicator of sediment. The results indicated a higher frequency of hemispherical and spherical shells in plankton and spherical and elongated shells in aquatic macrophytes. In the sediment, there was a high frequency of elongated species. Our results support the hypothesis that the community of testate amoebae has different structures among the habitats, refuting the idea that the organization of this community in plankton is guided by random events like the resuspension of organisms from the sediment and their displacement from marginal vegetation.

Este estudo avaliou as diferenças na composição, abundância e morfologia das amebas testáceas entre diferentes hábitats de um mesmo ambiente aquático (plâncton, macrófitas aquáticas e sedimento) da planície de inundação do alto rio Paraná. As amostras foram coletadas mensalmente no período de abril de 2007 a março de 2008. A estrutura da comunidade de amebas testáceas foi diferente entre os hábitats. As espécies típicas para cada hábitat, de acordo com o Indval, foram classificadas pela morfologia da teca. Espécies de Arcella, Difflugia gramen e Difflugia pseudogramem foram mais abundantes para o plâncton. Trinema e Phryganella destacaram-se pela alta abundância e frequência nas macrófitas aquáticas. Centropyxis foi considerado indicador do sedimento. Os resultados indicaram uma alta frequência de tecas esféricas e hemisféricas no plâncton e de tecas alongadas nas macrófitas aquáticas. No sedimento foi registrada uma maior frequência de espécies alongadas. Nossos resultados suportam a hipótese que a comunidade de amebas testáceas possui estrutura diferente entre os hábitats, refutando a ideia que a comunidade presente no plâncton é guiada por processos estocásticos como a ressuspensão dos organismos do sedimento e simples carreamento da vegetação marginal.

Potentially patoogenic free-living amoebae in sme flood-affected areas during 2011 chiang mai flood / Amebas potencialmente patogenicas de vida livre em algumas areas afetadas durante a inundacao de 2011 em Chiang Mai

SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA. .

RESUMO A pesquisa foi feita para investigar a presença de amebas de vida livre (FLA) durante a inundação em Chiang Mai, Tailândia, ano de 2011. A partir de diferentes áreas de inundação sete amostras de água foram coletadas e testadas para a presença de amebas usando métodos moleculares e de cultura. Através da cultura monoxênica, FLA foi detectada em todas as amostras após incubação a 37 °C. As FLA crescendo a 37 °C foram identificadas morfologicamente como Acanthamoeba spp, Naegleria spp e algumas amebas não determinadas. Somente três amostras (42,8%) definidas como FLA termotolerantes continuaram a crescer a 42 °C. Por métodos moleculares duas FLA termotolerantes tiveram 99% de identidade com a Acanthamoeba sp e 98% de identidade com Hartmannella vermiformis enquanto as duas FLA termotolerantes foram identificadas como Echinamoeba exundans (100% de identidade) e Hartmannella sp (99% de identidade). Este primeiro relato da ocorrência de FLA em águas durante inundações informa ao público que ele deve estar atento de FLA potencialmente patogênica. .

Isolation and identification of free-living amoebae from tap water in Sivas, Turkey.

The present work focuses on a local survey of free-living amoebae (FLA) that cause opportunistic and nonopportunistic infections in humans. Determining the prevalence of FLA in water sources can shine a light on the need to prevent FLA related illnesses. A total of 150 samples of tap water were collected from six districts of Sivas province. The samples were filtered and seeded on nonnutrient agar containing Escherichia coli spread. Thirty-three (22%) out of 150 samples were found to be positive for FLA. The FLA were identified by morphology and by PCR using 18S rDNA gene. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of three different species, Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. Naegleria fowleri, Balamuthia mandrillaris, or Sappinia sp. was not isolated during the study. All A. castellanii and A. polyphaga sequence types were found to be genotype T4 that contains most of the pathogenic Acanthamoeba strains. The results indicated the occurrence and distribution of FLA species in tap water in these localities of Sivas, Turkey. Furthermore, the presence of temperature tolerant Acanthamoeba genotype T4 in tap water in the region must be taken into account for health risks.