Salt-Enrichment Impact on Biomass Production in a Natural Population of Peatland Dwelling Arcellinida and Euglyphida (Testate Amoebae).

Unicellular free-living microbial eukaryotes of the order Arcellinida (Tubulinea; Amoebozoa) and Euglyphida (Cercozoa; SAR), commonly termed testate amoebae, colonise almost every freshwater ecosystem on Earth. Patterns in the distribution and productivity of these organisms are strongly linked to abiotic conditions-particularly moisture availability and temperature-however, the ecological impacts of changes in salinity remain poorly documented. Here, we examine how variable salt concentrations affect a natural community of Arcellinida and Euglyphida on a freshwater sub-Antarctic peatland. We principally report that deposition of wind-blown oceanic salt-spray aerosols onto the peatland surface corresponds to a strong reduction in biomass and to an alteration in the taxonomic composition of communities in favour of generalist taxa. Our results suggest novel applications of this response as a sensitive tool to monitor salinisation of coastal soils and to detect salinity changes within peatland palaeoclimate archives. Specifically, we suggest that these relationships could be used to reconstruct millennial scale variability in salt-spray deposition-a proxy for changes in wind-conditions-from sub-fossil communities of Arcellinida and Euglyphida preserved in exposed coastal peatlands.

Effect of temperature and colonization of Legionella pneumophila and Vermamoeba vermiformis on bacterial community composition of copper drinking water biofilms.

It is unclear how the water-based pathogen, Legionella pneumophila (Lp), and associated free-living amoeba (FLA) hosts change or are changed by the microbial composition of drinking water (DW) biofilm communities. Thus, this study characterized the bacterial community structure over a 7-month period within mature (> 600-day-old) copper DW biofilms in reactors simulating premise plumbing and assessed the impact of temperature and introduction of Lp and its FLA host, Vermamoeba vermiformis (Vv), co-cultures (LpVv). Sequence and quantitative PCR (qPCR) analyses indicated a correlation between LpVv introduction and increases in Legionella spp. levels at room temperature (RT), while at 37°C, Lp became the dominant Legionella spp. qPCR analysis suggested Vv presence may not be directly associated with Lp biofilm growth at RT and 37°C, but may contribute to or be associated with non-Lp legionellae persistence at RT. Two-way PERMANOVA and PCoA revealed that temperature was a major driver of microbiome diversity. Biofilm community composition also changed over the seven-month period and could be associated with significant shifts in dissolved oxygen, alkalinity and various metals in the influent DW. Hence, temperature, biofilm age, DW quality and transient intrusions/amplification of pathogens and FLA hosts may significantly impact biofilm microbiomes and modulate pathogen levels over extended periods.

Kaumoebavirus, a New Virus That Clusters with Faustoviruses and Asfarviridae.

In this study, we report the isolation of a new giant virus found in sewage water from the southern area of Jeddah (Saudi Arabia), with morphological and genomic resemblance to Faustoviruses. This new giant virus, named Kaumoebavirus, was obtained from co-culture with Vermamoeba vermiformis, an amoeboid protozoa considered to be of special interest to human health and the environment. This new virus has ~250 nm icosahedral capsids and a 350,731 bp DNA genome length. The genome of Kaumoebavirus has a coding density of 86%, corresponding to 465 genes. Most of these genes (59%) are closely related to genes from members of the proposed order Megavirales, and the best matches to its proteins with other members of the Megavirales are Faustoviruses (43%) and Asfarviruses (23%). Unsurprisingly, phylogenetic reconstruction places Kaumoebavirus as a distant relative of Faustoviruses and Asfarviruses.

Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia.

The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites.

Risk Assessment for the Spread of Serratia marcescens Within Dental-Unit Waterline Systems Using Vermamoeba vermiformis.

Vermamoeba vermiformis is associated with the biofilm ecology of dental-unit waterlines (DUWLs). This study investigated whether V. vermiformis is able to act as a vector for potentially pathogenic bacteria and so aid their dispersal within DUWL systems. Clinical dental water was initially examined for Legionella species by inoculating it onto Legionella selective-medium plates. The molecular identity/profile of the glassy colonies obtained indicated none of these isolates were Legionella species. During this work bacterial colonies were identified as a non-pigmented Serratia marcescens. As the water was from a clinical DUWL which had been treated with Alpron™, this prompted the question as to whether S. marcescens had developed resistance to the biocide. Exposure to Alpron™ indicated that this dental biocide was effective, under laboratory conditions, against S. marcescens at up to 1 × 10(8) colony forming units/millilitre (cfu/ml). V. vermiformis was cultured for 8 weeks on cells of S. marcescens and Escherichia coli. Subsequent electron microscopy showed that V. vermiformis grew equally well on S. marcescens and E. coli (P = 0.0001). Failure to detect the presence of S. marcescens within the encysted amoebae suggests that V. vermiformis is unlikely to act as a vector supporting the growth of this newly isolated, nosocomial bacterium.

Occurrence of the lobose testate amoeba Pseudonebela africana (Amoebozoa, Arcellinida) in the Brazilian "cerrado".

Flagship species are defined as microbial eukaryote species with characteristic morphologies and restricted geographic distributions. These are proposed as ideal systems to elucidate patterns of geographical distribution in microbial eukaryotes. Here we present new records of the putative flagship species Pseudonebela africana, a lobose testate amoeba (Arcellinida) characterized by a cross or clover-shaped aperture and geographic distribution previously believed to be restricted to Africa. We have sampled P. africana from 5 separate ponds in the Central and Southwest Brazillian "cerrado", and characterized individuals both by light and electron microscopy. We provide a brief description to facilitate further studies on this poorly understood taxon, and show that light microscopy is sufficient for identification, an important feature for ecological and biogeographical studies.

"Slime molds" among the Tubulinea (Amoebozoa): molecular systematics and taxonomy of Copromyxa.

Copromyxa protea is a dung-inhabiting amoeboid organism that aggregates to form simple macroscopic fruiting structures, sorocarps, which are composed of a single cell type. In a recent effort to find the phylogenetic positions of the less well-known sorocarpic protists considered to be "cellular slime molds," or aggregatively fruiting amoebae, we isolated C. protea and sequenced the nuclear-encoded small subunit ribosomal RNA gene from four samples collected from cattle farms in the central USA. Phylogenetic analyses of these data place C. protea in the eukaryotic supergroup Amoebozoa together with the Tubulinea, in which there has been no previous report of an aggregative fruiting habit. This is consistent with the morphology of the trophozoites. In fact, Copromyxa protea is found to be very closely related to Hartmannella cantabrigiensis and to a since lost amoeba isolate, Hartmannella sp. 4/3Da/10. This new grouping of Copromyxa+H. cantabrigiensis is sister to Glaeseria, which together are sister to the Amoebidae (Amoeba+Chaos). We suggest renaming, H. cantabrigiensis as C. cantabrigiensis and designate isolate 4/3Da/10 as C. protea. Future work is needed to see if these newly assigned members of the genus Copromyxa also show evidence of an ability to fruit.

The life history of Flabellula baltica Smirnov (Gymnamoebae, Rhizopoda): adaptations to a spatially and temporally heterogeneous environment.

The polymorphic life history of the marine naked amoeba Flabellula baltica was studied. It can be interpreted in terms of adaptations to an environment that is patchy in time and space and it represents trade-off between longevity during starvation and the ability to initiate multiplication soon after food resource become available. The life history also represents bet hedging in that different cells within a clonal culture may respond in different ways when food is depleted.

Screening Swiss water bodies for potentially pathogenic free-living amoebae.

Free-ling amoebae (FLA) including Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia pedata, can cause opportunistic infections leading to severe brain pathologies. Human infections with pathogenic FLA have been increasingly documented in many countries. In Switzerland, thus far, the occurrence and distribution of potentially pathogenic FLA has not been investigated. Swiss water biotopes, including swimming pools, lakes, rivers and ponds, have now been screened for the presence of FLA, and assessment of their pathogenicity potential for a mammalian host has been undertaken. Thus, a total of 17 isolates were recovered by in vitro cultivation from these different aquatic sources. Characterization by sequence analysis of Acanthamoeba spp.-specific and 'FLA-specific PCR products amplified from 18s rDNA based on morphological traits, thermotolerance, and cytotoxicity towards murine fibroblasts yielded the following findings: Echinamoeba cf. exundans (3 isolates), Hartmannella spp. (3), Vannella spp. (4), Protacanthamoebica cf. bohemica (1), Acanthamoeba cf. castellanii (1) and Naegleria spp. (5). B. mandrillaris and N. fowleri did not range amongst these isolates. None of the isolates exhibited pronounced cytotoxicity and all failed to grow at 42 degrees C; therefore, they do not present any potential for CNS pathogenicity for humans.

Balamuthia mandrillaris: staining properties of cysts and trophozoites and the effect of 2,6-dichlorobenzonitrile and calcofluor white on encystment.

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans. Furthermore, cysts, but not trophozoites, fluoresced when stained with calcofluor white. Calcofluor white and DCB, a cellulose synthesis inhibitor, inhibited B. mandrillaris encystment. This is the first report suggesting possible glycan biosynthesis in B. mandrillaris encystment, and this pathwaymay provide a potentially useful drug target and help improve treatment.

Balamuthia mandrillaris resistance to hostile conditions.

The resistance of Balamuthia mandrillaris to physical, chemical and radiological conditions was tested. Following treatments, viability was determined by culturing amoebae on human brain microvascular endothelial cells for up to 12 days. B. mandrillaris cysts were resistant to repeated freeze-thawing (five times), temperatures of up to 70 degrees C, 0.5 % SDS, 25 p.p.m. chlorine, 10 microg pentamidine isethionate ml(-1) and 200 mJ UV irradiation cm(-2).

Amoeba at attention: phylogenetic affinity of Sappinia pedata.

The genus Sappinia, a taxon of free-living amoebae with trophozoites that typically have two closely appressed nuclei, contains two named species, Sappinia pedata, the type species, and S. diploidea. The amoebae of both species are essentially identical according to the literature. The two species are distinguished by S. pedata having a standing amoeba stage, incorrectly interpreted as a cyst, and S. diploidea having sessile, bicellular cysts. Using four isolates of S. pedata collected from around the world, we present detailed light micrographic illustrations of all stages of its life cycle. We confirm that the standing amoeba lacks a cell wall. In two isolates of S. pedata, there are bicellular cysts indistinguishable from those of S. diploidea. Using sequence data from the nuclear small subunit ribosomal RNA gene, we conclude that S. pedata and the published neotype of S. diploidea are congeneric but not conspecific. The genus branches within Thecamoebidae. Sequencing of the actin gene confirms the inclusion of Sappinia in Thecamoebidae. Resolving the taxonomy of Sappinia is gaining importance because it has recently been attributed as an opportunistic human pathogen.

Mechanism of intrusion of a microspordian-like organism into the nucleus of host amoebae (Vannella sp.) isolated from a keratitis patient.

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.

High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1.

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris.

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.

Interaction of Pasteurella multocida with free-living amoebae.

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.

In vitro interactions between Neoparamoeba sp. and Atlantic salmon epithelial cells.

Neoparamoeba sp., including the putative aetiological agent of amoebic gill disease in cultured fish (N. pemaquidensis), were incubated in vitro with an Atlantic salmon gill epithelium (RGE-2) cell line. Proliferation by the amoeba population was dependent upon culture osmolarity; no growth occurred at 330 mm x kg(-1) but a sixfold increase was observed at 1000 mm x kg(-1). At 780 mm x kg(-1) there was a fourfold increase in the amoeba population but a concurrent decrease in RGE-2 cell density that was significantly greater than that caused by the high culture osmolarity alone. This apparent cytopathic effect (CPE) developed rapidly and resulted in complete cytolysis of the monolayer in 5 days. CPE occurred in multiple foci and presented as cell vacuolation, rounding and clumping, and the rapid clearance of large areas of the cell monolayer. The possibility that CPE is because of the presence of Neoparamoeba sp. derived cytolytic products is discussed in the context of the pathology of the disease in vivo and the occurrence of secreted cytopathogenic compounds in other amoeba species.

Spatial distribution of gymnamoebae (Rhizopoda, Lobosea) in brackish-water sediments at the scale of centimeters and millimeters.

In order to study micro-spatial distribution of amoebae, an intact slice of sandy sediment from the brackish-water Nivå Bay (Baltic Sea, The Sound), 40 x 24 mm in size and 2 mm in thickness was gently sectioned into cubes, 2 x 2 x 2 mm in size. Each cube was inoculated into enrichment media to reveal the biodiversity of amoebae. Seventeen species of amoebae were recovered. The 2-D map of amoebae species distribution in the slice, consisting of 240 2 x 2 mm cells was drawn and analyzed. Results show heterogeneous distribution of amoebae at the scale of centimeters and millimeters and confirm the idea of the presence of microhabitats, selectively occupied by amoebae species. Three types of distribution patterns were found: random, aggregated and equally spaced. Microelectrode studies indicated that amoebae distribution was not related to the dissolved oxygen content in the sediment. The studied slice of sediment contained several pronounced "hotspots" of amoebae biodiversity, where up to four species co-occur in the same area. Seven species of amoebae numbered 1-4 specimens in the studied slice (i.e. there was 0.5-2 cell ml(-1)). Analysis of the amoebae distribution map shows the high probability of undersampling rare amoebae species during faunistic studies.