Behind the Organic Veil: Assessing the Impact of Chemical Deflocculation on Organic Content Reduction and Lacustrine Arcellinida (Testate Amoebae) Analysis.

Arcellinida (testate lobose amoebae) are widely used as bio-indicators of lacustrine environmental change. Too much obscuring organic material in a gridded wet Petri dish preparation makes it difficult to observe all specimens present and slows quantification as the organic material has to be carefully worked through with a dissection probe. Chemical deflocculation using soda ash (Na2CO3·H2O), potassium hydroxide (KOH), or sodium hexametaphosphate ((NaPO3)6) has previously been shown to disaggregate and reduce organic content in lake sediments, but to date, no attempt has been made to comparatively evaluate the efficiency of these deflocculants in disaggregating organic content and their impact on Arcellinida analysis in lacustrine sediments. Here, we assess the effectiveness of soda ash, potassium hydroxide, and sodium hexametaphosphate treatments on removing organic content and the impact of those digestions on Arcellinida preservation in 126 sample aliquots subdivided from three sediment samples (YK-20, YK-25, and YK-57) collected from three lakes near Yellowknife, Northwest Territories, Canada. Following treatment, cluster analysis and Bray-Curtis dissimilarity matrix (BCDM) were utilized to determine whether treatments resulted in dissolution-driven changes in Arcellinida assemblage composition. Observed Arcellinida tests in aliquots increased drastically after treatment of organic-rich samples (47.5-452.7% in organic-rich aliquots and by 14.8% in aliquots with less organic matter). The BCDM results revealed that treatment with 5% KOH resulted in the highest reduction in observed organic content without significantly affecting Arcellinida assemblage structure, while soda ash and sodium hexametaphosphate treatments resulted in marginal organic matter reduction and caused severe damage to the arcellinidan tests.

Intra-Lake Arcellinida (Testate Lobose Amoebae) Response to Winter De-icing Contamination in an Eastern Canada Road-Side "Salt Belt" Lake.

Salt contamination of lakes, due to the application of winter de-icing salts on roads, presents a significant environmental challenge in the "salt belt" region of eastern North America. The research reported here presents the first deployment of a previously published proxy tool based on Arcellinida (testate lobose amoebae) for monitoring road salt contamination. The research was conducted at Silver Lake in Eastern Ontario, a 4-km-long lake with the heavily traveled Trans-Canada Highway (HWY 7) transiting the entire southern shore. The lake showed elevated conductivity (297-310 µS/cm) and sub-brackish conditions (0.14-0.15 ppt). Sodium levels were also elevated near the roadside (median Na = 1020 ppm). Cluster analysis and nonmetric multidimensional scaling results revealed four distinct Arcellinida assemblages: "Stressed Cool Water Assemblage (SCWA)," "Deep Cold Water Assemblage (DCWA)," both from below the 8-m thermocline, and the shallower water "Shallow Water Assemblage 1 (SWA-1)" and "Shallow Water Assemblage 2 (SWA-2)". Redundancy analysis showed a minor response of Arcellinida to road salt contamination in shallower areas of the lake, with confounding variables significantly impacting assemblage distribution, particularly beneath the thermocline (e.g., water temperature, water depth, sediment runoff from catchment [Ti], sediment geochemistry [Ca, S]). The results of this study indicate that the trophic structure of the lake has to date only been modestly impacted by the cumulative nature of road salt contamination. Nonetheless, the Silver Lake results should be considered of concern and warrant continued arcellinidan biomonitoring to gauge the ongoing and long-term effects of road salt on its ecosystem.

Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream.

Fecal indicator bacteria (FIB) are the basis for water quality regulations and are considered proxies for waterborne pathogens when conducting human health risk assessments. The direct detection of pathogens in water and simultaneous identification of the source of fecal contamination are possible with microarrays, circumventing the drawbacks to FIB approaches. A multigene target microarray was used to assess the prevalence of waterborne pathogens in a fecally impaired mixed-use watershed. The results indicate that fecal coliforms have improved substantially in the watershed since its listing as a 303(d) impaired stream in 2002 and are now near United States recreational water criterion standards. However, waterborne pathogens are still prevalent in the watershed, as viruses (bocavirus, hepatitis E and A viruses, norovirus, and enterovirus G), bacteria (Campylobacter spp., Clostridium spp., enterohemorrhagic and enterotoxigenic Escherichia coli, uropathogenic E. coli, Enterococcus faecalis, Helicobacter spp., Salmonella spp., and Vibrio spp.), and eukaryotes (Acanthamoeba spp., Entamoeba histolytica, and Naegleria fowleri) were detected. A comparison of the stream microbial ecology with that of sewage, cattle, and swine fecal samples revealed that human sources of fecal contamination dominate in the watershed. The methodology presented is applicable to a wide range of impaired streams for the identification of human health risk due to waterborne pathogens and for the identification of areas for remediation efforts.IMPORTANCE The direct detection of waterborne pathogens in water overcomes many of the limitations of the fecal indicator paradigm. Furthermore, the identification of the source of fecal impairment aids in identifying areas for remediation efforts. Multitarget gene microarrays are shown to simultaneously identify waterborne pathogens and aid in determining the sources of impairment, enabling further focused investigations. This study shows the use of this methodology in a historically impaired watershed in which total maximum daily load reductions have been successfully implemented to reduce risk. The results suggest that while the fecal indicators have been reduced more than 96% and are nearing recreational water criterion levels, pathogens are still detectable in the watershed. Microbial source tracking results show that additional remediation efforts are needed to reduce the impact of human sewage in the watershed.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran.

The present study tested 80 samples of municipal, geothermal and recreational water samples for the occurrence of waterborne free living amoebae (FLA) including Acanthamoeba, Balamuthia mandrillaris, Vahlkampfiids and Vermamoeba in Semnan province, North half of Iran. Four sets of primers including JDP1,2 primers, ITS1,2 primers (Vahlkampfiids), 16S rRNABal primers (Balamuthia mandrillaris) and NA1,2 primers (Vermamoeba) were used to confirm the morphological identification. From the 80 water samples tested in the present study, 16 (20%) were positive for the outgrowth of free living amoebae based on the morphological page key. Out of the 34 municipal water samples, 7 (20.6%) were positive for outgrowth of Free living amoeba, belonging to Vermamoeba, Naegleria and Acanthamoeba using molecular tools. Three out of the six investigated hot springs were also contaminated with Naegleria spp. Sequencing of the ITS1,2 region of the Vahlkampfiid isolates revealed the highest homology with N. gruberi (2 isolates), N. australiensis (1 isolate) and N. pagei (3 isolates). This is the first report of N. gruberi in the country. Using morphological and molecular analysis, Balamuthia mandrillaris was undetected in all the water samples. The present study further confirmed the occurrence of potentially pathogenic waterborne free living amoebae in habitats with high human activity. It is of utmost importance that more studies are conducted to evaluate the niches of B. mandrillaris and N. fowleri in Iran and worldwide. Such investigations regarding the relevance of FLA as a hazard to humans, should be brought to the notice of the health authorities.

Nebela jiuhuensis nov. sp. (Amoebozoa; Arcellinida; Hyalospheniidae): A New Member of the Nebela saccifera - equicalceus - ansata Group Described from Sphagnum Peatlands in South-Central China.

Hyalospheniids are among the most common and conspicuous testate amoebae in high-latitude peatlands and forest humus. These testate amoebae were widely studied as bioindicators and are increasingly used as models in microbial biogeography. However, data on their diversity and ecology are still very unevenly distributed geographically: notably, data are lacking for low-latitude peatlands. We describe here a new species, Nebela jiuhuensis, from peatlands near the Middle Yangtze River reach of south-central China with characteristic morphology. The test (shell) has hollow horn-like lateral extensions also found in N. saccifera, N. equicalceus (=N. hippocrepis), and N. ansata, three large species restricted mostly to Sphagnum peatlands of Eastern North America. Mitochondrial cytochrome oxidase (COI) data confirm that N. jiuhuensis is closely related to the morphologically very similar North American species N. saccifera and more distantly to N. ansata within the N. penardiana group. These species are all found in wet mosses growing in poor fens. Earlier reports of morphologically similar specimens found in South Korea peatlands suggest that N. jiuhuensis may be distributed in comparable peatlands in Eastern Asia (China and Korea). The discovery of such a conspicuous new species in Chinese peatlands suggests that many new testate amoebae species are yet to be discovered, including potential regional endemics. Furthermore, human activities (e.g., drainage, agriculture, and pollution) have reduced the known habitat of N. jiuhuensis, which can thus be considered as locally endangered. We, therefore, suggest that this very conspicuous micro-organism with a probably limited geographical distribution and specific habitat requirement should be considered as a flagship species for microbial biogeography as well as local environmental conservation and management.

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.

Impact of drinking water conditions and copper materials on downstream biofilm microbial communities and Legionella pneumophila colonization.

AIMS: This study examined the impact of pipe materials and introduced Legionella pneumophila on downstream Leg. pneumophila colonization and microbial community structures under conditions of low flow and low chlorine residual. METHODS AND RESULTS: CDC biofilm(™) reactors containing either unplasticized polyvinylchloride (uPVC) or copper (Cu) coupons were used to develop mature biofilms on Norprene(™) tubing effluent lines to simulate possible in-premise biofilm conditions. The microbial communities were characterized through 16S and 18S rRNA gene clone libraries and Leg. pneumophila colonization was determined via specific qPCR assays. The Cu significantly decreased downstream microbial diversity, approximately halved bacterial and eukaryotic abundance, with some groups only detected in uPVC-reactor tubing biofilms. However, some probable amoeba-resisting bacteria (ARB) like Mycobacterium spp. and Rhodobacteraceae were significantly more abundant in the Cu than uPVC-reactor tubing biofilms. In particular, Leg. pneumophila only persisted (postinoculation) within the Cu-reactor tubing biofilms, and the controlled low chlorine residue and water flow conditions led to a general high abundance of possible free-living protozoa in all tubing biofilms. The higher relative abundance of ARB-like sequences from Cu-coupons vs uPVC may have been promoted by amoebal selection and subsequent ARB protection from Cu inhibitory effects. CONCLUSIONS: Copper pipe and low flow conditions had significant impact on downstream biofilm microbial structures (on plastic pipe) and the ability for Leg. pneumophila colonization post an introduction event. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that compares the effects of copper and uPVC materials on downstream biofilm communities grown on a third (Norprene(™)) surface material. The downstream biofilms contained a high abundance of free-living amoebae and ARB, which may have been driven by a lack of residual disinfectant and periodic stagnant conditions. Given the prevalence of Cu-piping in buildings, there may be increased risk from drinking water exposures to ARB following growth on pipe/fixture biofilms within premise drinking water systems.

Multiplex Real-Time Polymerase Chain Reaction Assay To Detect Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris in Different Water Sources.

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

Description and phylogenetic relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida).

Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

Balamuthia mandrillaris, agent of amebic encephalitis: detection of serum antibodies and antigenic similarity of isolates by enzyme immunoassay.

We report the development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Balamuthia mandrillaris, a free-living ameba that is an etiologic agent of granulomatous amebic encephalitis (GAE). As part of the California Encephalitis Project (CEP), we have tested serum and cerebrospinal fluid (CSF) samples from a subgroup of 130 hospitalized encephalitis patients (out of approximately 430 samples) over a 16-month period. Case criteria were based on clinical, laboratory, and occupational/recreational histories. All serum samples initially underwent screening by immunofluorescent antibody (IFA) staining with results ranging from no detectable ameba antibodies to titers of 1:256. In addition to the 130 samples tested prospectively, sera and/or CSF from 11 previously confirmed cases of balamuthiasis, six healthy individuals, and earlier CEP submissions with high IFA antibody titers were also tested retrospectively. Among the 130 samples, two cases of balamuthiasis were identified by ELISA and confirmed by the polymerase chain reaction (PCR). The availability of sera from human and animal cases and from varied geographic areas allowed comparisons of serologic similarities of the different Balamuthia strains and human sera. All sera, whether from human or other mammals, reacted with all strains of Balamuthia, as they did with Balamuthia amebae from different geographic areas. Enzyme-linked immunosorbent assay results were consistent with the IFA results. Differences between readings were likely due to cross-reactivity between Balamuthia antigens and unidentified antibodies in serum.

Disseminated Balamuthia mandrillaris amoeba infection in an AIDS patient from Brazil.

This report describes a 32-year-old male AIDS patient. He presented with a clinical picture characterized by severe headache, blurred vision, and fever that had lasted for 10 days. At admission, no remarkable neurologic abnormalities were observed. Cranial tomography showed a ring-enhanced lesion with edema and a mass effect in the right occipital lobe. The initial diagnosis was toxoplasmosis, and treatment of this was administered. However, 5 days later, the patient's clinical status worsened and he died. The necropsy showed necrotizing and hemorrhagic encephalitis, with trophozoites similar to an amoeba species. Furthermore, the kidneys, adrenal glands, thyroid gland, and liver were also involved. The amoeba Balamuthia mandrillaris was identified by an immunofluorescence test.

Characterization of Neoparamoeba pemaquidensis strains: PCR-RFLP of the internal transcribed spacer region from the amoeba and endosymbiont.

Neoparamoeba pemaquidensis continues to be an ongoing problem for commercial finfish aquaculture and has also sporadically been associated with mass mortalities of commercially relevant marine invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of the biology as it applies to host range, pathogenicity, tissue tropism, and geographic distribution is severely lacking. This may stem from the inability of current diagnostic tests based on morphology, immunology, and molecular biology to differentiate strains at the subspecies level. In the present study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the internal transcribed spacer (ITS) region that can accurately differentiate amoeba strains of N. pemaquidensis. The investigation focused on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont, Ichthyobodo necator related organism (IRO), ITS region as an alternative marker. The combination of host amoeba and endosymbiont ITS PCR-RFLP analyses was successfully used to correctly identify and characterize an N. pemaquidensis isolate from an outbreak of amoebic gill disease in Atlantic salmon Salmo salar from the west coast of North America (Washington State, USA).

Cochliopodium gallicum n. sp. (Himatismenida), an amoeba bearing unique scales, from cyanobacterial mats in the Camargue (France).

Cochliopodium gallicum n. sp., isolated from cyanobacterial mats in the Camargue (France) is the smallest marine species of Cochliopodium to date. Its unusual tectum consists of flat plate-shaped scales with honeycomb-like centres, underlain by a layer of filamentous structures connected to each other in the basal and apical parts. The tectum is very fine and can be easily lost under inappropriate EM fixation. In its light-microscopical features, this species resembles Ovalopodium carrikeri Sawyer, 1980, a himatismenid that is believed to possess a scaleless, fuzzy or hairy "glycocalyx". We suggest that O. carrikeri might have been a similar species that lost scales under fixation. Our finding makes desirable a re-investigation of the genus Ovalopodium.

"Minute" species of Cochliopodium (Himatismenida): Description of three new fresh- and brackish-water species with a new diagnosis for Cochliopodium minus Page, 1976.

Only two species of Cochliopodium below 20mum in size are currently known. These are the recently described marine Cochliopodium gallicum Kudryavtsev and Smirnov, 2006 and a freshwater C. minutum West, 1901, an incompletely described species that has never been re-isolated since its initial description. In this paper, the descriptions of three independent species of similar size below 20microm, C. minutoidum, C. kieliense, and C. maeoticum are presented. In the light microscope they all resemble the description of C. minutum, C. minutoidum being most similar to this species. However, the available description of C. minutum is inadequate to permit the assignment of any strain to this species with confidence. Therefore, a status of nomen dubium is proposed for C. minutum. Furthermore, C. minutoidum appears to be very similar to a fish endobiont identified by Dyková et al. (1998. Cochliopodium minus, a scale-bearing amoeba isolated from organs of perch Perca fluviatilis. Dis. Aquat. Org. 34, 205-210) as C. minus. Direct comparison of my strain of C. minutoidum with the type strain of C. minus suggests that it is not identical to C. minus. The obtained data enabled me to add the ultrastructural features to the diagnosis of C. minus for more precise identification of this species.

Concentration effects of Winogradskyella sp. on the incidence and severity of amoebic gill disease.

To study the concentration effects of the bacterium Winogradskyella sp. on amoebic gill disease (AGD), Atlantic salmon Salmo salar were pre-exposed to 2 different doses (10(8) or 10(10) cells 1(-1)) of Winogradskyella sp. before being challenged with Neoparamoeba spp. Exposure of fish to Winogradskyella sp. caused a significant increase in the percentage of AGD-affected filaments compared with controls challenged with Neoparamoeba only; however, these percentages did not increase significantly with an increase in bacterial concentration. The results show that the presence of Winogradskyella sp. on salmonid gills can increase the severity of AGD.

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.

Distribution of Neoparamoeba sp. in sediments around marine finfish farming sites in Tasmania.

Marine sediment samples collected from various sites at 2 Atlantic salmon farms in Tasmania were analysed for the presence of Neoparamoeba sp., an amoeba associated with amoebic gill disease (AGD) in farmed Atlantic salmon. Environmental variables of the sediment layer at each site, including redox potential and sulphide concentration, were measured and the general biological condition assessed by video observation. Sediments and environmental data were collected on 4 occasions at each site over a 12 mo period. Neoparamoeba sp. was detected in populations of amoebae recovered by culture from all sites and in 50% of all sediment samples taken. There was evidence of a seasonal influence on the presence of the amoeba, but this was different at each farm. No Neoparamoeba sp. was recovered from any sites at Farm 1 during the winter of 2002 whereas at Farm 2 this was the case for the summer of 2003. There appeared to be no relationship between the presence of Neoparamoeba sp., salmon farming activities and environmental parameters.

Vertical distribution of gymnamoebae (rhizopoda, lobosea) in the top layer of brackish-water sediments.

The paper reports on the distribution of amoebae in a vertical slice of sandy sediment from the brackish-water Nivå Bay (Baltic Sea, The Sound). The 24 x 20 mm slice 2 mm in thickness was gently cut from the top of sediment to a depth of 20 mm using coverslips, and further sectioned into 2 x 2 x 2 mm cubes. Each cube was inoculated into enrichment media to reveal the biodiversity of amoebae. The map of amoebae species distribution in the slice was drawn and analyzed. The results show patchy distributions of many amoebae species. Amoebae tend to form a band of maximum diversity within the depth interval 2-16 mm. Two mini-cores of sediment located close to the butt-ends of the slice were collected using cut 1 ml syringes. They were sliced and inoculated to reveal the biodiversity of amoebae at different depths. The distribution patterns of amoebae in the mini-cores were similar to the ones obtained in the slice, and show evident patchiness of some species. It seems that abundant species mostly form irregular patches 1-2 cm across, whereas many species are rare and appear in few number of specimens or in patches smaller than 2 mm.

Balamuthia mandrillaris meningoencephalitis: the first case in southeast Asia.

We present a case of 23-year-old man with acute meningoencephalitis, accompanied by inflammation of a nasal ulcer. He had been healthy until six months prior to admission to the hospital when he had a motorcycle accident. A star-shaped wound at his nose was incurred after falling into a swamp. A computed tomogram of the brain showed two nonenhancing hypodense lesions at the left caudate nucleus and the right parietal lobe, ependymitis and leptomeningeal enhancement. A skin biopsy showed chronic noncaseous granulomatous inflammation without demonstrated microorganisms. The patient did not respond to the empirical treatment with cloxacillin, ceftriaxone, and amphotericin B, and eventually died on the thirteenth day of hospitalization. At autopsy, hematoxylin and eosin-stained brain sections showed a chronic necrotizing inflammation with numerous amebic trophozoites and rare cysts. Definitive identification of Balamuthia mandrillaris was made by fluorescent immunohistochemical analysis. There were 10 Naegleria fowleri primary amebic meningoencephalitis, eight Acanthamoeba granulomatous amebic encephalitis, and three Acanthamoeba meningitis in Thailand. To our knowledge, this case is the first reported case of B. mandrillaris in Southeast Asia.