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An outbreak of nodular gill disease (NGD) in farmed rainbow trout Oncorhynchus mykiss (Walbaum, 1792) was recorded in Ladoga Lake (Karelia, north-western Russia) in Spring 2020. The disease was characterised by typical clinical signs including asphyxiation, distended opercula, loss of activity and swimming upside down under the water surface. Maximum monthly mortality was 15.2%. The histological examination of the gills showed deformation and clubbing of lamellae, epithelial hypertrophy and hyperplasia, lamellar fusion and fusion of filaments. Granulomas were located within the epithelial layer and/or rose above its surface. Light microscopic in vivo observations of the mucus smears from the affected gills revealed numerous amoeboid protists demonstrating a flattened body when adhering to the substratum, and blunt, radiating pseudopodia when afloat. Based on these morphological characters, these amoebae could be assigned to the Discosea (Amoebozoa), and analyses of their small subunit rRNA gene sequences showed that they belonged to the genus Vannella Bovee, 1965. The results reported herein support the designation of a new species, V. mustalahtiana sp. nov. Despite having been isolated from the gills of a freshwater fish, the species belongs to a clade of Vannella comprising mostly species isolated from marine and brackish water habitats. These findings may be essential for the aetiology and treatment of the disease.

As a result of the study, in the steppe zone of Ukraine (Odessa, Mykolaiv, Kirovohrad region), 12 species of naked amoebas were identified, which according to the modern Eukaryot system belong to three molecular clusters Tubulinea Smirnov et al., 2005, Discosea Cavalier-Smith, 2004., Discoba Simpson and Hampl et al., 2009. This species Vahlkampfia sp. (1), Vahlkampfia sp. (2), Deuteramoeba mycophaga Page, 1988, Saccamoeba stagnicola Page, 1974, Vexillifera sp., Vannella sp. Ripellaplatypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007, Cochliopodium sp. (1), Mayorella sp., Thecamoeba striata Penard, 1890, Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Acanthamoeba sp. (1). In the studied steppe soils, the most common were Vahlkampfia sp. (2), S. stenopodia, Vahlkampfia sp. (1), Vexillifera sp., Cochliopodium sp. (1); the least common – R. platypodia, D. mycophaga, T. striata, Mayorella sp. As a result of the cluster analysis, it was found that the largest share of common species is observed between Mykolaiv and Kirovograd regions (0.71) and Odessa and Kirovograd regions (0.53); the smallest is between the Odessa and Mykolaiv regions (0.43). According to the results of cluster analysis, the faunistic complexes of soil species of amoebae of the steppe region of Ukraine are united into two clusters: one of them being complexes characteristic of the Odessa region, and the other complexes of the Mykolaiv and Kirovograd regions. According to the results of nonparametric multidimensional scaling, it is established that the species complex of soil amoebae in the Kirovograd and Mykolaiv regions is determined by the increased soil temperature and acidity, compared to the Odessa region. As for moisture, this factor has little effect on the species complexes amoebae steppe region of Ukraine.

Gill parasitic infections challenge farming of rainbow trout (Oncorhynchus mykiss, Walbaum) in freshwater facilities. Apart from flagellates (Ichthyobodo, (Pinto) and ciliates (Ichthyophthirius (Fouquet), Ambiphrya (Raabe), Apiosoma (Blanchard), Trichodinella (Sramek-Husek) and Trichodina (Ehrenberg)), we have shown that amoebae are prevalent in Danish trout farms. Gills were isolated from farmed rainbow trout in six fish farms (conventional and organic earth pond and recirculated systems) and placed on non-nutrient agar (NNA) moistened with modified Neff's amoeba saline (AS) (15°C). Gill amoebae from all examined fish colonized the agar and were identified based on morphological criteria showing species within the genera Trinema (Dujardin) (family Trinematidae), Vannella (Bovee) (family Vannellidae). In addition, hartmannellid amoebae were recorded. We established a monoculture of Vannella sp., confirmed the genus identity by PCR and sequencing and performed an in vitro determination of antiparasitic effects (dose-response studies) of various compounds including sodium chloride (NaCl), hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant. All amoebae were killed in concentrations of 16.90 mg/ml (garlic), 17.90 mg/ml (oregano), NaCl (7.5 mg/ml), hydrogen peroxide (100 µg/ml), peracetic acid (0.03 µg/ml), formaldehyde (25 µg/ml) and the Pseudomonas H6 surfactant (250 µg/ml).

16 species of naked amoebae are recorded in the eplithic biotopes of Zhytomyr region, Ukraine: Saccamoeba sp. (1), Saccamoeba sp. (2), Saccamoeba sp. (3), Cashia limacoides Page, 1974, Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Thecamoeba sp., Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Mayorella cantabrigiensis Page, 1983, Mayorella sp., Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007, Acanthamoeba sp. (1), Cochliopodium sp., Vahlkampfia sp. (1). The frequency of occurrence in samples is highest for Thecamoeba sp., Mayorella sp., Vahlkampfia sp. (1), Korotnevella sp., Vannella sp., and is lowest for Saccamoeba sp. (1), C. limacoides, M. cantabrigiensis, Cochliopodium sp., S. stenopodia, R. platypodia, T. striata, Saccamoeba sp. (2). The species and morphotype diversity of naked amoebae of the epilithic mosses and lichens is influenced by the humidity and the height above soil of the substrate. The species and morphotype diversity of naked amoebae decreases with the increase in height above soil and the decrease of humidity of substrate. We recorded naked amoebae of 9 morphotypes: monotactic, dactylopodial, fan-shaped, lens-like, lingulate, mayorellian, striate, acanthopodial, and eruptive. Amoebae of the lens-like and fan-shaped morphotypes disappear with the increase of height of substrate above soil.

In water bodies of the Zakarpattia region and the surrounding areas we found 19 species of naked amoebae related to amoebae 3 classes, 8 families and 12 genera according to modern system. This species are: D. mycophaga, S. stagnicola, Saccamoeba sp. (2), Rhizamoeba sp. (1), Rhizamoeba sp. (2), K. stella, Vexillifera sp., V. lata, Vannella sp., Cochliopodium sp. (1), M. cantabrigiensis, M. vespertilioides, Mayorella sp. (1), T. striata, T. terricola, S. stenopodia, Acanthamoeba sp. (1), Vahlkampfia sp. (1), Vahlkampfia sp. (2). We found 11 species belong to the morphotypes: monotactic, lingulate, striate, rugose, lens-like, fan-shaped, mayorellian, dactylopodial, acanthopodial, branched, eruptive. Rhe distribution of naked amoebae and their morphotypes in waters of the Zakarpattia region is influenced by hydrophysical and hydrochemical factors of the environment (temperature, active reaction of water environment, the concentration of dissolved oxygen in the water and concentration of dissolved organic substances). It was found that 7 species of naked amoebae (Vexillifera sp., V. lata, Vannella sp., Cochliopodium sp. (1), Acanthamoeba sp. (1), Vahlkampfia sp. (1), Vahlkampfia sp. (2)) withstand the full range of temperatures (from + 16 ° C to + 18 ° C); four species of amoebae were found in the range of values of the active reaction of the water environment from 5.8 to 7.7 (Rhizamoeba sp. (1), Cochliopodium sp. (1), Vahlkampfia sp. (1), Vahlkampfia sp. (2)), and these species form a group of evrione species; the entire range of values of dissolved oxygen in the water can withstand the species of Vahlkampfia sp. (2) and it can be considered euryoxidic; T. striata prefers a significant content of water-soluble organic substances (from 8.54 mg O2/l to 14.20 mg O2/l).

Free-Living Amoebae (FLA) are widely distributed protozoa, which contain some groups considered as pathogenic microorganisms. These members are able to produce several opportunistic diseases including epithelial disorders, such as keratitis and fatal encephalitis. Even though they have been reported in numerous sources, such as soils, dust and water, there is no legislation related to the presence of these protozoa in soil-related environments worldwide. Therefore, there are no established prevention or disinfection protocols to advise the population regarding FLA infections or eliminate these microorganisms from human-related environments to date. Acanthamoeba spp. are the most common FLA isolated in soil samples, which is also the most common genera found in clinical cases. Thus, the aim of the present study was to evaluate the presence of potentially pathogenic FLA in human-related soil samples of Santiago Island, Cabo Verde. A total of 26 soil samples were seeded in non-nutrient agar plates (2%), incubated at 26 °C, and monitored daily to evaluate the presence of FLA. DNA was extracted from those plates on which there was suspected FLA growth, and PCR amplification of the 18S rRNA gene was carried out. A total of 17 from the 26 analysed samples were positive for FLA, where Acanthamoeba is the most abundant isolated genus (14/17; 82.4%), with the T4 genotype being the most common (13/14; 92.9%), followed by the T5 genotype, A. lenticulata (1/14; 7.1%). Moreover, Vermamoeba vermiformis, Stenamoeba dejonckheerei and Vannella pentlandi were isolated in three other samples. To the best of our knowledge, this is the first report of FLA presence in Cape Verde and the first report of V. vermiformis in beach sand worldwide.

In microbiotopes of Ukraine we found 17 species of naked amoebae (moss, border “moss – soil”, soil). This species are: Vahlkampfia sp. (1), Vahlkampfia sp. (2), Willaertia sp., Deuteramoeba mycophaga (Pussard, Alabouvette & Pons, 1980) Page, 1988, Saccamoeba stagnicola Page, 1974, Hartmannella vermiformis Page, 1967, Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Glaeser, 1912, Cochliopodium sp., Mayorella cantabrigiensis Page, 1983, Mayorella sp., Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Acanthamoeba sp. (1), Filamoeba nolandi Page, 1967. On the border of biotopes "moss – soil", a specific variant of the population of naked amoebas does not form and an increase in its species composition is not observed, but those species that are characteristic of one of the neighboring microbiotopes remain. The soil fauna is characterized by amoeba Willaertia sp., D. mycophaga, Korotnevella sp., M. cantabrigiensis, F. nolandi. The specific composition of morphotypes of naked amoebas is not formed on the "moss – soil" boundary and neighboring microbiotopes are similar in composition to each other. The amoeba of politactic morphotype prefers soil, lingulate and mayorellian – soil and the border "moss – soil".

Amoebic gill disease (AGD) is an ectoparasitic condition affecting many teleost fish globally, and it is one of the main health issues impacting farmed Atlantic salmon, Salmo salar in Tasmania’s expanding aquaculture industry. To date, Neoparamoeba perurans is considered the only aetiological agent of AGD, based on laboratory trials that confirmed its pathogenicity, and its frequent presence on the gills of farmed Atlantic salmon with branchitis. However, the development of gill disease in salmonid aquaculture is complex and multifactorial and is not always closely associated with the presence and abundance of N. perurans. Moreover, multiple other amoeba species colonise the gills and their role in AGD is unknown.In this study we profiled the Amoebozoa community on the gills of AGD-affected and healthy farmed Atlantic salmon and performed in vivo challenge trials to investigate the possible role these accompanying amoebae play alongside N. perurans in AGD onset and severity. Significantly, it was shown that despite N. perurans being the primary aetiological agent, it is possible AGD has a multi-amoeba aetiology.Specifically, the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon was documented by culturing the gill community in vitro, then identifying amoebae using a combination of morphological and sequence-based taxonomic methods. In addition to N. perurans, 11 other Amoebozoa were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. This work highlighted that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.Drawing on this culture-based study, five new TaqMan quantitative PCR (qPCR) assays were developed and applied to more accurately determine the prevalence and abundance of multiple amoeba species colonising the gills of Atlantic salmon held at two Tasmanian farm sites over a one-year period. The presence of N. perurans was also assessed in parallel using a previously established qPCR method. N. perurans was the dominant species in the Amoebozoa community on gills, and its abundance positively correlated with the progression of gross gill pathology. Only sporadic detections of Pseudoparamoeba sp. and Vannellida species were observed across the sampling period at either farm site. Nolandella spp., however, were highly prevalent at one site at one sample time when N. perurans were not detected on gills presenting low levels of gross gill pathology.To investigate the pathogenic potential of Nolandella sp. and a more closely related amoeba to N. perurans, Pseudoparamoeba sp., in vivo challenges of naive Atlantic salmon were performed. Additionally, to elucidate how Nolandella sp. and Pseudoparamoeba sp. influence the onset or severity of N. perurans-induced AGD, the gill condition of fish challenged with N. perurans alone was compared to fish challenged with a mix of all three amoeba strains. Immersion challenge of all three species resulted in minor gill lesions, with the most severe epithelial hyperplasia documented in the N. perurans treatments, while lesions with infiltrating lymphocytes were the predominate pathology observed in fish challenged by Nolandella sp. and Pseudoparamoeba sp. The presence of individual Nolandella or Pseudoparamoeba cells were not linked with lesion sites, so the precise cause of pathology remains inconclusive. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of N. perurans-induced branchitis.Overall this investigation supports N. perurans being the primary agent of AGD, yet also shows that other species of amoebae which colonise the gills of Tasmanian Atlantic salmon can dominate the gill community and may be capable of causing some gill pathology under specific conditions. Thus, the involvement of non-N. perurans amoebae in AGD should not yet be discounted. The increasing list of putative gill pathogens, and the complexity of disease expression, provides supportive rationale to the consideration of gill disease in the context of dysbiosis of microbial community structure, rather than a pathological response to a single agent.

Amoebae of the order Vannellida (Amoebozoa, Discosea) have a fairly recognizable spatulate, fan-shaped or semi-circular outlines and a wide area of frontal hyaloplasm. They can be easily distinguished from the other groups of lobose amoebae even by light microscopy. The dorsal side of these amoebae is usually smooth and rarely bears ridges or folds, which are never numerous or regular. We have isolated an unusual species of vannellid amoebae, called Vannella primoblina n. sp. from a terrestrial substrate. It has well-developed dorsal relief consisting of regularly appearing folds and ridges. This amoeba superficially resembles members of the genus Thecamoeba. However, molecular analysis showed that this strain belongs to the genus Vannella. This finding indicates that dorsal folds may also be a characteristic of some species of vannellid amoebae and probably are a functional detail of the cell morphology rather than an apomorphy of Thecamoebida lineage. Overall outlines of the cell and the presence of the expanded frontal hyaline area remains the most reliable characters used to differentiate vannellid amoebae from other gymnamoebae lineages.

Free-living amoebae (FLA) are ubiquitous protists. Pathogenic FLA such as N. fowleri can be found in hot springs in Guadeloupe, soil being the origin of this contamination. Herein, we analyzed the diversity and distribution of FLA in soil using a targeted metataxonomic analysis. Soil samples (n = 107) were collected from 40 sites. DNA was extracted directly from soil samples or from FLA cultivated at different temperatures (30, 37 and 44 &deg, C). Metabarcoding studies were then conducted through FLA 18SrDNA amplicons sequencing, amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against SILVA database using QIIME2 and SHAMAN pipelines. Vermamoeba were detected in DNA extracted directly from the soil, but to detect other FLA an amoebal enrichment step was necessary. V. vermiformis was by far the most represented species of FLA, being detected throughout the islands. Although Naegleria were mainly found in Basse-Terre region, N. fowleri was also detected in Grand Terre and Les Saintes Islands. Acanthamoeba were mainly found in areas where temperature is approx. 30 &deg, C. Vannella and Vahlkampfia were randomly found in Guadeloupe islands. FLA detected in Guadeloupe include both pathogenic genera and genera that can putatively harbor microbial pathogens, therefore posing a potential threat to human health.

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.

The result of research conducted in the water bodies of Lviv region identified 21 species of naked amoebae that belong to 3 classes, 12 families and 13 genera. The are species: Deuteramoeba mycophaga (Pussard, Alabouvette, Lemaitre & Pons, 1980) Page, 1988, Saccamoeba stagnicola Page, 1974, Saccamoeba limax (Dujardin, 1841) Page, 1974, Saccamoeba limna Bovee, 1972, Hartmannella cantabrigiensis Page, 1974, Echinamoeba exundans Page, 1975, Rhizamoeba flabellate Goodey, 1914, Vexillifera sp., Korotnevella sp., Vannella lata Page, 1988, Vannella cirrifera (Frenzel, 1892) Page, 1988, Cochliopodium sp., Mayorella cantabrigiensis Page, 1983, Mayorella bigemma Schaeffer, 1926, Mayorella leidyi Bovee, 1970, Mayorella sp. (1), Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Thecamoeba quadrilineata (Carter, 1856) Lepsi, 1960, Vahlkampfia sp. (1), Acanthamoeba sp. (2), Acanthamoeba sp. (1). Comparing the specific lists of naked amoebae in different regions of Ukraine, we have found that the specific composition of these opposites in L’viv region is most similar to Zhytomyr region (55 %), and the least similar to Sumy region (36 %). According to the results of cluster analysis, the faunal complexes of amoebae in Ukraine are united into two clusters. In the first cluster there are complexes of Kiev, Lviv, Zhytomyr and Rivne regions, and in the second one – Sumy region. The active response of aquatic environment, concentration of dissolved oxygen in the water and organic substances have different effects on the distribution of amoebae in the water bodies of Lviv region. Allocated 6 ecological groups of protozoa: evian, stenon, eriksen, lanoxin, those marked within the narrow range of permanganate oxidation values and within the broad one. New to the fauna of Ukraine were amoeba S. limna , R. flabellata , E. exundans , V. cirrifera , Acanthamoeba sp. (2). We found two amoebae of the genus Acanthamoeba Volkonsky, 1931, which are clearly distinguished from each other according to the morphological characteristics ( Acanthamoeba sp. (1) and Acanthamoeba sp. (2)). However, for final species identification of amoebae of the genus Acanthamoeba conduct additional studies involving data on the optimal conditions of cultivation, virulence and analysis of enzymes.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

Vannella samoroda n. sp. (Amoebozoa, Vannellida) was isolated from the mouth of the Malaya Samoroda river flowing into Elton, the largest European hypersaline lake (Russia). Among all rivers of the area, it has the highest salt content (ca. 110‰). Amoebae maintained in seawater medium with ca. 77‰ salts concentration had a set of morphological characters typical of Vannella spp.: rounded, fan-shaped, or spatulate locomotive form, floating form with bent, blunt-ended hyaline pseudopodia, and a cell coat consisting of regularly packed palisade elements and scarce simple filaments. Phylogenetic analyses based on SSU rRNA and cytochrome C oxidase subunit 1 genes show that the amoeba is most closely related to Vannella ebro Smirnov, 2001, but represents a distinct species. The clade of V. ebro and V. samoroda branches among marine species of Vannella. The studied species is the first member of the genus Vannella from a continental saline habitat described using molecular data. Interestingly, it has a broad range of salinity tolerance: cells reproduce above 18‰, while survival of a few cells regularly occurs even in highly diluted Prescott and James medium. The normal culture restores itself when PJ medium is substituted with 77‰ seawater medium even after months of experimental incubation.

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.

International audience; Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events and behave as facultative intracellular pathogen of oyster haemocytes. Herein, we wondered whether V. tasmaniensis LGP32 resistance to phagocytosis is specific to oyster immune cells or contributes to resistance to other phagocytes, like marine amoebae. To address this question, we developed an integrative study, from the first description of amoeba diversity in oyster farms to the characterization of LGP32 interactions with amoebae. An isolate of the Vannella genus, Vannella sp. AP1411, which was collected from oyster farms, is ubiquitous, and belongs to one clade of Vannella that could be found associated with Vibrionaceae. LGP32 was shown to be resistant to grazing by Vannella sp. AP1411 and this phenotype depends on some previously identified virulence factors: secreted metalloprotease Vsm and copper efflux p-ATPase CopA, which act at different steps during amoeba-vibrio interactions, whereas some other virulence factors were not involved. Altogether, our work indicates that some virulence factors can be involved in multi-host interactions of V. tasmaniensis ranging from protozoans to metazoans, potentially favouring their opportunistic behaviour.

The case study targeted to determine the aetiology of nodular gill disease (NGD) of farmed rainbow trout. The methods included microscopical examination of gill material in fresh, culturing of isolated organisms, histology, transmission electron microscopy and molecular biology identification. The results revealed an intravital colonization of fish gills by the testate amoeba Rhogostoma minus Belar, 1921. Rhogostoma infection was found in all fish examined microscopically (15/15); in contrast, naked amoebae related to fully developed NGD lesions were found in minority of these fish (5/15). They belonged to four genera, Acanthamoeba, Vermamoeba, Naegleria and Vannella. Results presented in this study contribute to the mosaic of findings that contrary to amoebic gill disease of marine fish turn attention to the possibility of the heterogeneous, multi-amoeba-species and multifactorial aetiology of NGD.

Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events. Among them, V. tasmaniensis LGP32 behaves as a facultative intracellular pathogen of oyster hemocytes, a property rather unusual in vibrios. Herein, we asked whether LGP32 resistance to phagocytosis could result from coincidental selection of virulence factors during interactions with heterotrophic protists, such as amoeba, in the environment. To answer that question, we developed an integrative study, from the first description of amoeba diversity in oyster-farming areas to the characterization of LGP32 interactions with amoebae of the Vannella genus that were found abundant in the oyster environment. LGP32 was shown to be resistant to grazing by amoebae and this phenotype was dependent on previously identified virulence factors: the secreted metalloprotease Vsm and the copper efflux p-ATPase CopA. Using dedicated in vitro assays, our results showed that these virulence factors act at different steps during amoeba-vibrio interactions than they do in oysters-vibrio interactions. Hence, the virulence factors of LGP32 are key determinants of biotic interactions with multiple hosts ranging from protozoans to metazoans, suggesting that the selective pressure exerted by amoebae in marine coastal environments favor coincidental selection of virulence factors.

Free-living amoebae (FLA) are protozoa that are widely distributed in nature. They are important in the cycling of nutrients in aquatic food chains, but their distribution in natural aquatic environments is not well known. We conducted a survey to determine the presence and distribution of FLA and their relation to some physicochemical parameters in streams of the Mexico Basin in Central Mexico. Thirty-two sites from 18 streams were sampled. Samples were centrifuged and cultured onto NNA-media to isolate amoebae. Identifications were based on morphology. The pathogenicity of Acanthamoeba isolates was tested. Oxygen saturation, temperature, pH, specific conductance, water flow, dissolved reactive phosphorus, nitrites, nitrates, ammonia, dissolved inorganic nitrogen and coliforms were determined. One hundred-and-twenty FLA representing 18 genera were identified. The most frequent genera were Vannella, Rosculus and Acanthamoeba. The frequency of potentially pathogenic FLA was low and only 3 Acanthamoeba isolates were invasive in mice. The highest species richness of FLA was found in streams located into agriculture activity areas and those close to small villages that discharge wastewater into them. Water temperatures were always below 17 °C. Oxygen saturation and pH were within the limits for the growth of most FLA. The presence of Acanthamoeba and Naegleria was low; nevertheless, they include potentially pathogenic species and can act as vectors and reservoirs for microbial pathogens and can produce human infections.

To clarify the structure of microbial food webs in groundwater, knowledge about the protist diversity and feeding strategies is essential. We applied cultivation-dependent approaches and molecular methods for further understanding of protist diversity in groundwater. Groundwater was sampled from a karstified aquifer located in the Thuringian Basin (Thuringia, Germany). Cultivable protist abundance estimated up to 8,000 cells/L. Eleven flagellates, 10 naked amoebae, and one ciliate morpho-species were detected in groundwater enrichment cultures. Most of the flagellates morpho-species, typically

The goal of this study was to characterize microbial eukaryotes over a 12-month period to provide insight into the occurrence of potential bacterial predators and hosts in premise plumbing. Nearly 6,300 partial 18S rRNA gene sequences from 24 hot (36.9-39.0 °C) and cold (6.8-29.1 °C) drinking water samples were analyzed and classified into major eukaryotic groups. Each major group, consisting of free-living amoebae (FLA)/protozoa, algae, copepods, dinoflagellates, fungi, nematodes, and unique uncultured eukaryotic sequences, showed limited diversity dominated by a few distinct populations, which may be characteristic of oligotrophic environments. Changes in the relative abundance of predators such as nematodes, copepods, and FLA appear to be related to temperature and seasonal changes in water quality. Sequences nearly identical to FLA such as Hartmannella vermiformis, Echinamoeba thermarmum, Pseudoparamoeba pagei, Protacanthamoeba bohemica, Platyamoeba sp., and Vannella sp. were obtained. In addition to FLA, various copepods, rotifers, and nematodes have been reported to internalize viral and bacterial pathogens within drinking water systems thus potentially serving as transport hosts; implications of which are discussed further. Increasing the knowledge of eukaryotic occurrence and their relationship with potential pathogens should aid in assessing microbial risk associated with various eukaryotic organisms in drinking water.

Amoebae grazing can be an important loss factor for blooms of the common cyanobacterium Microcystis. Some Microcystis strains seem to be protected against amoebae grazing, but it is unclear whether this is achieved by their colony morphology or biochemically. These factors were investigated in grazing experiments using two Microcystis-grazing amoebae (Korotnevella sp. and Vannella sp.) and two Microcystis strains with differing colony morphology (aeruginosa and viridis morphotype) and different sensitivity to amoebae grazing. Amoebae did not increase in density and failed to reduce the growth rate of cultures of the amoebae insensitive viridis strain, irrespective of whether the Microcystis strain was colonial or unicellular. This suggests that the extended mucilage matrix surrounding viridis colonies is not the main defence mechanism against amoebae grazing. At the same time, the growth rate of both unicellular and colonial cultures of the amoebae-sensitive aeruginosa strain was heavily reduced by the growing amoebae. The addition of filtered viridis-conditioned medium to aeruginosa cultures significantly decreased both amoebae growth and its effect on aeruginosa growth rates, which indicates that extracellular compounds constitutively produced by viridis are at least partially responsible for their insensitivity to amoebae grazing. These results demonstrate the potential importance of chemical interactions between lower trophic levels (protists) for Microcystis bloom dynamics.

We studied amoebae associated with nodular gill disease (NGD) outbreaks in rainbow trout Oncorhynchus mykiss (Walbaum) in fish farms in South-Western Germany. Gills of 12 diseased rainbow trout were examined in fresh, by isolation attempts, histologically and using in situ hybridisation (ISH). A total of nine amoeba strains of the genera Acanthamoeba (1), Hartmannella (2), Naegleria (1), Protacanthamoeba (1) and Vannella (4) were isolated and determined using light microscopical, ultrastructural and molecular methods. Specific molecular probes designed from the SSU rDNA sequences of individual amoeba strains were used for non-radioactive ISH in histological sections. Association of Naegleria sp. with NGD and a direct ISH proof of Naegleria trophozoites attached to hyperplastic gill epithelium are novel findings, expanding the number of possible agents of NGD and supporting the hypothesis on multicausal aetiology of this disease.

Morphological identification of naked lobose amoebae has always been a problem, hence the development of reliable molecular tools for species distinction is a priority for amoebae systematics. Previous studies based on SSU rDNA sequences provided a backbone for the phylogeny of Amoebozoa but were of little help for the species distinctions in this group. On one hand, the SSU rDNA sequences were rather conserved between closely related species; on the other hand, the intra-strain polymorphism of the SSU gene obscured species identification. In the present study, a 3' fragment of the SSU, a complete ITS1-5.8S-ITS2 block and a 5' fragment of COI gene were cloned and sequenced for six Vannella morphospecies, of which V. simplex was represented by six different isolates. SSU rDNA and ITS were found to be inappropriate for species differentiation, while distinctive and homogenous COI sequences were obtained for each well-defined morphospecies. Moreover, a number of distinct COI genotypes have been identified among V. simplex isolates. This suggests that COI may be a good candidate for DNA barcoding of amoebae, but further studies are necessary to confirm the accurateness of the COI gene as a barcode in other gymnamoebae, and to understand the taxonomic meaning of COI variations.

The effect of Dexiostoma (filter feeder), Vannella, Chilodonella (raptorial feeders), Spumella , and Neobodo (direct interception feeders) on the morphology of multispecies bacterial biofilms was investigated in small flow cells. The filter feeder Dexiostoma campylum did not alter biofilm volume and porosity but stimulated the formation of larger microcolonies compared with ungrazed biofilms. In contrast, the raptorial feeder Vannella sp. efficiently grazed bacteria from the biofilm surface, leading to smaller microcolonies and lower maximal and basal layer thickness compared with ungrazed biofilms. Microcolony formation was not stimulated in the presence of the sessile Spumella sp. Chilodonella uncinata rasped bacteria from the outer surface leading to mushroom-shaped microcolonies. In the presence of C. uncinata and Spumella sp., the biofilm volume was 2.5–6.3 times lower compared with ungrazed biofilms. However, the biofilm porosity and the ratio of biofilm surface area to biofilm volume were 1.5–3.7 and 1.2–1.8 times higher, respectively. Thus, exchange of nutrients and gases between the biofilm and its surrounding fluid should also be improved in deeper biofilm layers, hence accelerating microbial growth.

The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini , and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

Within the range of the Bulgarian Antarctic Program (1987-2010) some parasitological investigations on the coccidian parasites of 3 penguin species from South Shetland Islands (Livingston and St George Islands) were conducted, namely: Pygoscelis antarctica, P. papua and P. adeliae . Three coccidian species were found, one of which was described as a new species: Eimeria pygosceli Golemansky, 2003. It was isolated from the fecal samples of all 3 penguin species, living at South Shetland Islands. The other not identified coccidian’s were of the genera Eimeria and Isopspora. (Table 1 ). The great number of coccidian’s oocysts in some of the examined birds (from 80 – 220 oocysts in one microscopic field) suggest for their pathogenic role on the penguin’s population. As a result of our studies on some soil, moss and freshwater habitats from Livingston Island a total of 48 rhizopods were found. Three of them were naked amoebae of the genera Hartmanella, Thecamoeba and Vannella and the other 45 species were testate amoebae of 17 genera. Data on their occurrence frequency (pF) and dominance frequency (DF) were given (Table 2). The research on the marine interstitial testate amoebae from the supralittoral of the Antarctic region of Chile and Livingston Island shows the presence of 17 species of psammobiotic and psammophilous testate amoebae (Table 3).

Free-living amoebae (FLA) occur ubiquitously in many aquatic habitats and humid soils as well as in "artificial" water samples. In addition to their role as pathogens, FLA are known to serve as natural hosts and vehicles of transmission for various intracellular organisms. An otherwise healthy 24-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of corneal trauma. No acanthamoebae could be determined by culture methods. A Vannella strain (called VanAun0) isolated from corneal scrapings showed intracellular aggregating organisms. Within 1-2 days, the host amoebae ruptured, and numerous coccoid organisms (called Kaun1) were released. We succeeded in detecting the mechanisms of infection and intrusion of this eukaryotic organism, growing within the nucleus of the FLA, by light and electron microscopy. It could be shown that the spores at the cell membrane of strain KAun1 resemble Microsporidia and were taken up into the Amoeba by phagocytosis after adhesion of the spores and food cup formation (infective phase). The spores were transported into the cytoplasm of the vannellae in food vacuoles. Phase contrast microscopy revealed early stages of the parasites moving through the cytoplasm into the nucleus of the host amoeba. Electron microscopy showed the proliferation of polymorphic stages within the karyoplasm. The life cycle of these microsporidian-like organisms ended up with a sporogenic phase in which a terminal differentiation took place and numerous spores were released by rupture of the host cell wall. With the rupture of the host amoeba's cell membrane, the cycle started again from the beginning, the released infectious spores being ingested by other host amoebae. In particular, the morphology of the organelles made visible by electron microscopy finally allowed us to classify the endocytobionts as a microsporidan-like organism. Infection of Vannella sp. with the microsporidia-like organism strain KAun1 is a suitable model for studying the host-parasite relations of organisms using their hosts as so-called Trojan horses.

Several representatives of the so-called free-living amoebae (FLA) are of medical relevance, not only as facultative pathogens but also as vehicles for pathogenic bacteria. Some FLA can survive and even grow under extreme environmental conditions. Bat guano is an exceptional habitat, the conditions becoming gradually more extreme with aging. In the current study, samples of bat guano of different ages from five caves in Slovenia were screened for the presence of FLA. FLA were isolated from almost all guano samples, including guano with a pH of 3.5. Only the two samples that had been drawn from >20-year-old guano were negative for FLA. Generally, FLA diversity correlated to high concentrations of cultivable bacteria (∼10(8) CFU/g) and fungi (∼10(5) CFU/g). Interestingly, the absence of FLA in seasoned guanos was mirrored by the presence of dictyostelid slime moulds. The isolated amoebae were identified as belonging to the genera Acanthamoeba, Copromyxa, Naegleria, Sappinia, Tetramitus, Thecamoeba, Vahlkampfia, Vannella and Vermamoeba. To the best of our knowledge, this is the first study on the diversity of FLA in guano.

Interactions with free-living protozoa (FLP) have been implicated in the persistence of pathogenic bacteria on food products. In order to assess the potential involvement of FLP in this contamination, detailed knowledge on their occurrence, abundance and diversity on food products is required. In the present study, enrichment and cultivation methods were used to inventory and quantify FLP on eight types of commercial vegetable sprouts (alfalfa, beetroot, cress, green pea, leek, mung bean, red cabbage and rosabi). In parallel, total aerobic bacteria and Escherichia coli counts were performed. The vegetable sprouts harbored diverse communities of FLP, with Tetrahymena (ciliate), Bodo saltans and cercomonads (flagellates), and Acanthamoeba and Vannella (amoebae) as the dominant taxa. Protozoan community composition and abundance significantly differed between the sprout types. Beetroot harbored the most abundant and diverse FLP communities, with many unique species such as Korotnevella sp., Vannella sp., Chilodonella sp., Podophrya sp. and Sphaerophrya sp. In contrast, mung bean sprouts were species-poor and had low FLP numbers. Sampling month and company had no significant influence, suggesting that seasonal and local factors are of minor importance. Likewise, no significant relationship between protozoan community composition and bacterial load was observed.

Four species of gymnamoebae isolated from a sandy shore were subjected to hydrodynamic disturbance within microcosms. Increasing the level of disturbance increased the generation times of all four isolates. Generation times of a small Platyamoeba sp. and a limax amoeba were least affected by increasing disturbance. The greatest increase in generation times was observed in populations of two large (>15 μm) Mayorella sp. and Vannella sp. This study shows that hydrodynamic disturbance may be an important community structuring force within natural marine amoebae communities.

Twenty strains of flattened amoebae including 17 isolated from fish were characterised morphologically both at light microscopical and ultrastructural levels and assigned to either the genus Vannella Bovee, 1965 or the genus Platyamoeba Page, 1969. Sequence-based phylogenetic analyses of SSU rRNA genes from a data set representing a total of 29 strains of flattened amoebae strongly indicated that morphological features discriminating between these genera do not reflect phylogenetic relationships of representative strains. Contrary to a previous study, strains of this expanded assemblage formed clusters that did not reflect their environmental origin. Monophyletic groups were of mixed origins and contained freshwater as well as marine strains of both genera isolated in geographically distant localities of various continents. These findings were supported by results of phylogenetic analyses of selected strains based on ITS sequences. However, topologies of acquired ITS trees were not congruent with results inferred from SSU rRNA analyses.